Registration Dossier

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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in all strains tested (OECD Test Guideline 471) (Hashima Laboratory, 2005a).

Cytogenicity in mammalian cells: positive in CHL/IU cells with activation (OECD Test Guideline 473) (Hashima Laboratory, 2005b).


Mutagenicity in mammalian cells: negative in CHO cells (OECD
Test Guideline 476) (Hüls AG, 1996b).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Principles of method if other than guideline:
Details of metabolic activation not available from translated report.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and 5,6-benzoflafove induced rat liver S9
Test concentrations with justification for top dose:
0, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
- Solvent used: Dimethyl sulfoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
Remarks:
TA 98, 100, WP2 uvrA without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 with activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-amimoanthracene
Remarks:
all strains with activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; preincubation

NUMBER OF REPLICATIONS: 3 plates per dose, experiment repeated

DETERMINATION OF CYTOTOXICITY
- Method: other: reduction in number of revertants
Rationale for test conditions:
Dosage: Without S9 mix: 0, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate (TA98, TA100, TA1535, TA1537); 0, 312.5, 625, 1250, 2500, and 5000 µg/plate (WP2uvrA) With S9 mix: 0, 312.5, 625, 1250, 2500 and 5000  µg/plate (TA98, TA100, TA1535, WP2uvrA); 
0, 156.3, 312.5, 625, 1250, 2500 and 5000  µg/plate (TA1537)

Metabolic activation: Phenobarbital and 5,6-benzoflavone induced rat liver S9; full detail of S9 mix are included in the Japanese report though they are not given in the English translation
Key result
Species / strain:
bacteria, other: Salmonella typhimurium TA100, TA1535, TA1537, TA98; Escherichia coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: at 5000 µg/plate for TA100, TA1535, TA1537 and TA98 without S9 mix, and at 5000 µg/plate for TA1537 with S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1 Experiment 1: without activation (mean of 3 plates)

Concentration µg/plate  TA 100  TA1535  E. coli WP2  TA98  TA1537
 0  105  9  34  17  10
 156.3  113  8   NR  19  8
 312.5  125  8  40  17  10
 625  112  7  32  12  6
 1250  123  14  34  14  7
 2500  115  13  38  18  8
 5000  86  7  31  23  9
 Positive control  521  558  143  425  263
  Cytotoxic*  yes  yes  no  yes  yes

* yes indicates bacterial growth inhibition observed

NT indicates not tested

Table 2 Experiment 1: with activation (mean of 3 plates)

Concentration  µg/plate    TA 100  TA 1535  E coli WP2  TA 98  TA 1537
 0  124  44  42  24  11
 156.3  NT  NT  NT  NT  17
 312.5  1225  12  41  22  15
 625  142  9  46  25  15
 1250  149  8  42  27  9
 2500  148  8  49  35  17
 5000  153  12  41  38  14
Positive control   9007  331  868  405  135
  Cytotoxic*  no  no  no  no  yes

* yes indicates bacterial growth inhibition observed

NT indicates not tested

Conclusions:
Trimethoxy(vinyl)silane has been tested in a valid study, conducted according to OECD TG 471 and in compliance with GLP, using the preincubation method. No increase in the number of revertants was observed in any of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 at any concentration up to 5000 µg/plate with or without metabolic activation. The results of the repeat experiment agreed with those of the first experiment. It is concluded that the test substance is negative for the induction of mutations under the conditions of the test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese hamster lung (CHL/IU) cells
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and 5,6-benzoflavone induced rat live S9
Test concentrations with justification for top dose:
0, 93.8, 187.5, 375, 750, 1500 µg/mL
Vehicle / solvent:
- DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without activation
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
with activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period:
- Exposure duration: 6 and 24 hours
- Expression time (cells in growth medium): 24 hours


SPINDLE INHIBITOR (cytogenetic assays): no data

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no data
- Other:

OTHER:
Key result
Species / strain:
mammalian cell line, other: Chinese hamster lung (CHL/IU) cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 1500 µg/mL
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Structural chromosomal aberrations were induced at 750 and 1500 ug/mL with the 6 hr short treatment method and S9 mix (31.5 and 58.0 %, respectively). Polyploidy was not evident. Lowest concentration producing cytogenetic effects in vitro: With metabolic activation (6 hr short treatment method): 750 µg/ml (clastogenicity)
Ethenyltrimethoxysilane induced chromosomal aberrations in the presence of metabolic activation under the conditions of this study. The positive control agents, Mitomycin C and dimethyl nitrosamine, both produced highly significant increases in chromosome aberrations.

Table 1: Results of chromosome analysis, 6 h treatment, with activation

Treatment

 Solvent 

Control

Positive Control

93.8 µg/ml

187.5 µg/ml

375 µg/ml

 750 µg/ml

 1500 µg/ml

Survival ratio (%)

100

79

97

100

99

94

92

 

Mean

Chromatid aberrations

gaps

0

0

0

0

0

0

0

breaks

0

49

0

3

1

21

47

interchanges

1

110

1

1

8

60

110

Chromosome aberrations

gaps

0

0

0

0

0

0

0

breaks

0

0

0

0

0

0

0

interchanges

0

0

0

0

1

1

0

Incidence cells with aberrations (-gaps) (%)

0.5

64.5

0.5

2.0

5.0

31.5

58.0

Polyploidy

no

no

no

no

no

no

no

Endo reduplication

NR

NR

NR

NR

NR

NR

NR

 NR not reported

 

Table 2: Results of chromosome analysis, 6 h treatment, without activation

Treatment

 Solvent 

Control

Positive Control

93.8 µg/ml

187.5 µg/ml

375 µg/ml

 750 µg/ml

 1500 µg/ml

Survival ratio

100

86

97

98

98

94

98

 

Mean

Chromatid aberrations

gaps

0

0

0

0

0

0

0

breaks

0

50

0

1

1

1

1

interchanges

0

75

1

0

1

0

1

Chromosome aberrations

gaps

0

0

0

0

0

0

0

breaks

0

0

0

0

0

0

interchanges

0

0

0

0

0

0

0

Incidence cells with aberrations (-gaps) (%)

0

0.5

0.5

1.0

0.5

0.5

50.0

Polyploidy

no

no

no

no

no

no

no

Endo reduplication

NR

NR

NR

NR

NR

NR

NR

NR not reported

Table 3: Results of chromosome analysis, 24 h treatment, without activation

Treatment

 Solvent 

Control

Positive Control

93.8 µg/ml

187.5 µg/ml

375 µg/ml

 750 µg/ml

 1500 µg/ml

Survival ratio

100

84

97

98

98

100

95

 

Mean

Chromatid aberrations

gaps

0

0

0

0

0

0

0

breaks

1

34

0

0

1

0

0

interchanges

1

66

0

0

0

0

0

Chromosome aberrations

gaps

0

0

0

0

0

0

0

breaks

0

0

0

0

0

0

0

interchanges

0

0

0

0

0

1

0

Incidence cells with aberrations (-gaps) (%)

1.0

41.5

0

0

0.5

0.5

0

Polyploidy

no

no

no

no

no

no

no

Endo reduplication

NR

NR

NR

NR

NR

NR

NR

NR not reported

Conclusions:
Trimethoxy(vinyl)silane has been tested in a valid study, conducted according to OECD TG 473 and in compliance with GLP for the induction of chromosome aberrations in mammalian cells.The test substance produced a dose related increase in chromosome aberrations in the presence of metabolic activation. No induction of polyploidy was observed. It is concluded that the test subsance is positive for the induction of chromosome aberrations in Chinese hamster lung cells under the conditions of this test. The reviewer has seen a detailed summary in English, but has not seen the full Japanese study report.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-08-19 to 1996-10-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1984
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
with S9: 18, 60, 180, 600, and 1800 µg/ml; without S9: 10, 30, 100, 300 and 1000 µg/ml
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(20)-methylcholanthrene
Remarks:
with activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration:4 h with and without S9
- Expression time (cells in growth medium): 20 hours
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): H10 medium

NUMBER OF REPLICATIONS: experiment was repeated. 3 plates per culture were used for evaluation of mutagenicity

NUMBER OF CELLS EVALUATED: 3 x 200 cells per culture seeded to plates. Stained with Giesma before counting

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency



Evaluation criteria:
Positive if causes statistically significant dose related increase in mutant frequency at concentrations resulting in greater than 20% cell survival, reaching a value above the maximum spontaneous mutant frequency.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: >1800 µg/plate without S9; some cytotoxicity noted at all concentrations with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
Results of preliminary study: In the absence of S9 mix, treatment with Dynasylan VTMO concentrations up to 1800µg/ml had no significant influence on the cloning efficiency, while at the next higher dose (3000  µg/ml) no viable cells were left.  In the presence of S9, all treatments with Dynasylan VTMO resulted in cytotoxicity. 

COMPARISON WITH HISTORICAL CONTROL DATA: solvent controls were low (0-4 0-20 mutants/10E6 cells) in comparison with historical controls (0-20 0-20 mutants/10E6 cells)

ADDITIONAL INFORMATION ON CYTOTOXICITY:

In the two independent experiments of the main study, the mutant frequencies of the negative controls (culture medium) were consistent with historical control data for this test system (i.e. 0-20 mutants/10E6 cells).  Treatment of CHO
cells with the positive controls, 3-methylcholanthrene (with S9 mix) or ethyl methane sulfonate (without S9 mix) resulted
in highly significant increases of the mutant frequencies of the HPRT locus, thus demonstrating the sensitivity of the
test system and the activity of the S9 mix.

In the main study, treatment of CHO cells with Dynasylan VTMO in each of two independent experiments did not result
in biologically significant increases of the mutant frequency of the HPRT locus.  Statistically significant
increases (as compared to the low concurrent negative controls of 0 to 4 mutants/10E6 viable cells) were observed
with a number of test compound concentrations.  However, these were within the range of historical laboratory
negative controls and did not show a dose-response relationship.  These mutant frequencies were considered to
be the result of normal assay variation and not indicative of a mutagenic effect of Dynasylan VTMO.

Conclusions:
Trimethoxy(vinyl)silane has been tested in a valid study, conducted according to OECD TG 476 and in compliance with GLP. No statistically significant dose related increases in the number of revertants was observed either with or without activation. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Comet assay in mouse (nose-only inhalation): Negative (OECD Test Guideline 489) (Charles River, 2018).

Micronucleus assay in mouse (ip administration): Negative (OECD Test Guideline 474) (Bushy Run Research Center, 1985).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Remarks:
The restrictions were that only 1000 immature erythrocytes were scored for incidence of micronucleated immature erythrocytes: the current guideline requires 2000 to be scored.
Qualifier:
according to guideline
Guideline:
other: EPA Health effects guidelines 560/6-83-001
Principles of method if other than guideline:
Method: other: BRRC Standard Operating Procedures 7.2.18A, 7.2.19A and 7.2.20A
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hilltop laboratory animals
- Age at study initiation: 4 weeks
- Weight at study initiation: 24.0-28.9 g (male); 18.2-22.4 g (female)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: 5 mice per sex per cage in shoe-box type plastic cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5-6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): monitored, not recorded
- Humidity (%): monitored, not recorded
- Air changes (per hr): no information
- Photoperiod (hrs dark / hrs light): 12 hours


IN-LIFE DATES: From March 26 1985 to April 5 1985:
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil;

- Justification for choice of solvent/vehicle: instability in water
- Concentration of test material in vehicle:
Details on exposure:
Intraperitoneal injection
Duration of treatment / exposure:
30, 48 and 72 hours
Frequency of treatment:
Single treatment
Post exposure period:
up to 72 hours
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
700 mg/kg bw/day
Dose / conc.:
1 125 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- triethylenemelamine
- Justification for choice of positive control(s): standard positive control
- Route of administration: ip injection
- Doses / concentrations: 500 µg/kg bw
Tissues and cell types examined:
Peripheral erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on LD 50

DETAILS OF SLIDE PREPARATION:Stained with Giesma

METHOD OF ANALYSIS: micronuclei were identified as darkly stained spherical inclusion in PCEs; PCEs were identified by pale blue staining of cytoplasm

OTHER: PCE/NCE ratio was calculated for approximately 1000 cells as a measure of cytotoxicity.
Evaluation criteria:
A positive result would be interpreted by a statistically significant (p < 0.05) increase above vehicle control with indication of dose response.
Statistics:
Fisher's exact test, males and female data combined as no statistically significant difference between them.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
decrease in PCE/NCE ratio at 1125 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500-2000 mg/kg
- Solubility: no information
- Clinical signs of toxicity in test animals: no information
- Evidence of cytotoxicity in tissue analyzed: slight decrease in PCE/NCE ratio
- Rationale for exposure: based on LD50 determined in an acute toxicity study
- Harvest times:
- Other:


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no evidence for induction of micronuclei
- Ratio of PCE/NCE (for Micronucleus assay): ratio was slightly decreased at the highest dose level
- Appropriateness of dose levels and route: appropriate dose and route
- Statistical evaluation: appropriate evaluation

Table 1 Results of toxicity assay: males

Dose mg/kg bw

No of animals tested

No of animals dead

% mortatlity

Mean PCE/NCE ratio

Percent of control

2000

5

4

80%

42.5

91.6%

1414

5

3

60%

1000

5

1

20%

707

5

0

0%

500

5

0

0%

Solvent control

5

0

0%

46.4

100%

 

Table 2 Results of toxicity assay: females

Dose mg/kg bw

No of animals tested

No of animals dead

% mortatlity

Mean PCE/NCE ratio

Percent of control

2000

5

3

60%

39.3

80.0%

1414

5

3

60%

1000

5

2

40%

707

5

0

0%

500

5

0

0%

Solvent control

5

0

0%

49.0

100%

 

Table 3 Micronucleus frequency and PCE/NCE ratio (mean of 5 animals for each sex)

Dose mg/kg bw

Sampling Timehours

Sex

Mean PCE/NCE ratio

Percent of control

Total micronuclei*

Mean+/- SD

% PCE with MN

350

30

M

46.6

92.4

18

3.6+/-2.19

0.36

F

41.0

12

2.4+/-1.52

0.24

700

M

38.8

88.6

18

3.6+/-1.52

0.36

F

45.2

21

4.2+/-1.30

0.42

1125

M

48.8

103.0

22

3.7+/-2.50

0.37

F

49.0

23

3.3+/-1.11

0.33

Vehicle control

M

55.0

100

15

3.0+/-2.0

0.30

F

42.8

14

2.8+/-0.84

0.28

Positive control

M

26.2

54.2

126

25.2+/-8.56

2.52

F

25.2

149

29.5+/-16.02

2.98

350

48

M

44.4

110.2

21

4.2+/-1.92

0.42

F

42.4

13

2.6+/-1.14

0.26

700

M

34.0

93.9

12

2.4+/-1.67

0.24

F

40.0

7

1.4+/-0.55

0.14

1125

M

40.8

106.3

22

3.7+/-1.75

0.37

F

43.0

20

3.3+/-2.16

0.33

Vehicle control

M

39.4

100

12

2.4+/-1.52

0.24

F

45.4

14

2.8+/-2.17

0.28

Positive control

M

10.4

27.2

174

34.8+/-3.48

3.48

F

11.0

176

35.2+/-9.63

3.52

350

72

M

46.2

92.0

16

3.32+/-1.64

0.32

F

41.0

7

1.4+/-1.14

0.14

700

M

43.0

83.3

9

1.8+/-0.84

0.18

F

36.0

6

1.2+/-1.10

0.12

1125

M

29.3

62.7

14

2.3+/-1.51

0.23

F

30.0

9

1.5+/-1.38

0.15

Vehicle control

M

50.2

100

22

4.4+/-1.14

0.44

F

47.0

15

3.0+/-1.73

0.30

Positive control

M

13.0

29.3

25

6.2+/-3.59

0.62

F

15.0

14

3.5+/-3.70

0.35

*in 1000 PCE


Conclusions:
Trimethoxy(vinyl)silane has been tested in a reliable and valid micronucleus assay according to a protocol that is similar to the current OECD TG 474 and in compliance with GLP. No statistically significance increase in the frequency of micronucleated polychromatic erythrocytes was observed in peripheral erythrocytes of mice treated with the test substance by ip injection. There was indication of slight toxicity to bone marrow at the highest dose in female mice, which is evidence that the test substance had reached the target cells. It is concluded that trimethoxy(vinyl)silane is negative for the induction of micronuclei under the conditions of this test.
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, Section 4, Health Effects. No.412, 28-day (subacute) Inhalation Toxicity Study
Version / remarks:
2017
Deviations:
yes
Remarks:
the study design was based on the study objectives (assessment of DNA damage), OECD 489 and OECD 412
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Sprague-Dawley
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.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany OR Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: 7 weeks old
- Weight at study initiation: between 212 and 267 g
- 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

ENVIRONMENTAL CONDITIONS
- 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
Route of administration:
inhalation
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- 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.

TEST ATMOSPHERE
- Brief description of analytical method used: Analysed concentrations of trimethoxy(vinyl)silane in the exposure atmospheres were determined using a gas chromatograph (GC). 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.
- Samples taken from breathing zone: yes. Samples were collected from the approximate animals-breathing zone of the exposure system via one-eighth inch heated stainless steel tubing.
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
Dose / conc.:
342 ppm (nominal)
Dose / conc.:
670 ppm (nominal)
Dose / conc.:
1 444 ppm (nominal)
Dose / conc.:
314 ppm (analytical)
Dose / conc.:
650 ppm (analytical)
Dose / conc.:
1 314 ppm (analytical)
No. of animals per sex per dose:
5 animals per sex per group
Control animals:
yes
Positive control(s):
ethylmethanesulphonate
- 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.
- Vehicle(s)/solvent(s) for positive control used: 0.9% Sodium Chloride Injection, USP
- Lot/batch no. (if required): 77-166-DK
- The vehicle, 0.9% sodium chloride injection, was dispensed daily for the preparation of the positive control formulations.
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, trimethoxy(vinyl)silane 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, trimethoxy(vinyl)silane 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.
Statistics:
Statistical analysis was performed only for % tail DNA.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Lung Tissue: The presence of ‘clouds’ in the test substance groups was = 1.0%, which was lower than the % of clouds in the negative control group (2.0%). Group variances for mean of medians of the % Tail DNA in the negative control group and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data. No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent negative control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05). No dose dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01). The positive control, EMS, induced a statistically significant increase in the % Tail DNA in lung cells as compared to the negative control group (Student’s t test, p = 0.05). In the negative control group, % Tail DNA was within the historical vehicle control range for the lung.

- Liver Tissue: The presence of ‘clouds’ in the test substance groups was = 1.6%, which was comparable with the % of clouds in the negative control group (1.4%). Group variances for mean of medians of the % Tail DNA in the negative control group and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data. No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent negative control group (ANOVA followed by Dunnett’s post hoc analysis, p > 0.05). A dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p < 0.01). The positive control, EMS, induced a statistically significant increase in the % Tail DNA in liver cells as compared to the negative control group (Student’s t test, p = 0.05). In the negative control group, % Tail DNA was within the historical vehicle control range for the liver.

- Bone Marrow Tissue: The presence of ‘clouds’ in the low test substance group was 0.4% which was lower than the negative control group and the mid and high test substance groups were = 1.4%, which are comparable with the % of clouds in the negative control group (1.2%). Group variances for mean of medians of the % Tail DNA in the negative control group and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data. No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent negative control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05). No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01). The positive control, EMS, induced a statistically significant increase in the % Tail DNA in bone marrow cells as compared to the negative control group (Student’s t test, p = 0.05). In the negative control group, % Tail DNA was within the historical vehicle control range for the bone marrow.

These results in lung, liver, and bone marrow cells show that the negative control % Tail DNA was within the Test Site’s historical range and the positive control had a statistically significant increase in % Tail DNA compared to the negative control. Thus, all criteria for a valid test were met for lung, liver, and bone marrow.
Conclusions:
Trimethoxy(vinyl)silane has been tested in a reliable and valid comet assay according to the appropriate OECD Test Guideline 489 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 trimethoxy(vinyl)silane is negative for the induction of DNA damage under the conditions of this test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Information is available from reliable studies for all the required in vitro endpoints. Where there was more than one result for an endpoint the most reliable study available was chosen as key study. Negative results were obtained in all bacterial and mammalian mutagenicity assays. Positive results were obtained with activation in both the in vitro cytogenicity studies available. A negative result was obtained in an in vivo micronucleus assay.

Trimethoxy(vinyl)silane has been tested in a valid study, conducted according to OECD Test Guideline 471 and in compliance with GLP, using the preincubation method. No increase in the number of revertants was observed in any of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 at any concentration up to 5000 µg/plate with or without metabolic activation. Appropriate positive and solvent controls were included and gave expected results. The results of the repeat experiment agreed with those of the first experiment. It is concluded that the test substance is negative for the induction of mutations under the conditions of the test (Hashima Laboratory, 2005a). The result is supported by negative results in another study of bacterial mutagenicity, conducted according to OECD Test Guideline 471 and in compliance with GLP (Hüls AG, 1996a).

Trimethoxy(vinyl)silane has been tested in a valid study, conducted according to OECD Test Guideline 473 and in compliance with GLP for the induction of chromosome aberrations in mammalian cells. The test substance produced a dose related increase in chromosome aberrations in the presence of metabolic activation. No induction of polyploidy was observed. It is concluded that the test substance is positive for the induction of chromosome aberrations in Chinese hamster lung cells under the conditions of this test. The reviewer has seen a detailed summary in English but has not seen the full Japanese study report (Hashima Laboratory, 2005b).

A similar result was obtained in a supporting study, conducted according to Health Effects Test Guidelines "HG-Chromo-In Vitro", EPA (adopted August, 1982) and in compliance with GLP, in which trimethoxy(vinyl)silane produced significant dose dependent increases in chromosome aberrations in the presence of metabolic activation in Chinese hamster ovary cells, and less significant increases in the absence of metabolic activation (Bushy Run Research Center, 1985a). It was concluded that the test substance is positive for the induction of chromosome aberrations in mammalian cells under the conditions of the test.

Trimethoxy(vinyl)silane has been tested in a valid study, conducted according to OECD Test Guideline 476 and in compliance with GLP. No statistically significant dose related increases in the number of revertants was observed either with or without activation. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study (Huls, 1996b).

Trimethoxy(vinyl)silane has been tested in a reliable and valid micronucleus assay according to a protocol that was similar to the current OECD Test Guideline 474 and in compliance with GLP. No statistically significance increase in the frequency of micronucleated polychromatic erythrocytes was observed in peripheral erythrocytes of mice treated with the test substance by intraperitoneal (ip) injection. There was indication of slight toxicity to bone marrow at the highest dose in female mice, which is evidence that the test substance had reached the target cells. It is concluded that trimethoxy(vinyl)silane is negative for the induction of micronuclei under the conditions of this test (Bushy Run Research Center, 1985b). Trimethoxy(vinyl)silane hydrolyses very rapidly at physiological pH (half-life of 0.1 hours) and this is the main breakdown process the substance would undergo, so it is considered that the intraperitoneal route would have resulted in exposure to relevant substance-related products.

Trimethoxy(vinyl)silane has been tested in a reliable and valid comet assay according to the appropriate OECD Test Guideline 489 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 trimethoxy(vinyl)silane is negative for the induction of DNA damage under the conditions of this test (Charles River, 2018).


Justification for classification or non-classification

The available information for the substance indicates that when tested in vitro, trimethoxy(vinyl)silane (CAS 2768-02-7) does not induce mutations in bacterial or mammalian cells. The substance does cause chromosomal aberrations in vitro in the presence of metabolic activation, but this potential for genetic toxicity was not confirmed in in vivo studies, so it is concluded that trimethoxy(vinyl)silane is not genotoxic.

Based on the available data, trimethoxy(vinyl)silane does not require classification for genetic toxicity according to Regulation (EC) No 1272/2008.