Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial mutagenicity: R/A from CAS 23432-64-6 and CAS 23432-65-7: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 102: negative with and without metabolic activation (OECD 471)

Mammalian cytogenicity (Chromosome Aberration): negative with and without metabolic activation (OECD 473)

Mammalian mutagenicity (mouse lymphoma assay): positive with metabolic activation (OECD 476)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-08-13 to 2014-09-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Results of dose range finding test are in contrast to the results observed in the main study.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Type and identity of media:
- Basic medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamine
- Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium)
- Exposure medium: for 3 hour exposure: cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium); for 24 hour exposure:
cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
- Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/ml trifluorothymidine (TFT)
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague Dawley rats treated with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
Test concentrations with justification for top dose:
Experiment 1:
- 1.7, 5.4, 17, 52, 164, 512, 1600, 2373 µg/ml with and without metabolic activation (3 h)

Experiment 2:
- 1.7, 5.4, 17, 52, 164, 512, 1600, 2373 µg/ml without metabolic activation (24 h)
Vehicle / solvent:
- Vehicle solvent used: DMSO; 1% (v/v)
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - Methyl methanesulfonate: 15 µg/ml (3 h) and 5 µg/ml (24 h) in DMSO without S9-mix - Cyclophosphamide: 7.5 µg/ml in HBSS (3, 24 h) with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- in suspension

DURATION:
1st experiment: 3 h exposure with and without S9 mix
2nd experiment: 24 h exposure without S9 mix
- Expression time (cells in growth medium): 2 days after the end of the treatment, cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtiter plates containing TFT selective medium. The microtitre plates were incubated for 11 or 12 days.
- Selection time: 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14-15 days

SELECTION AGENT (mutation assays):
- 5 µg/ml trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- two

DETERMINATION OF CYTOTOXICITY:
- Method: cloning efficiency and relative total growth

OTHER:
- Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations.
Evaluation criteria:
The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10E6 cells
- A dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥ 40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
Statistics:
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
at > 52 µg/ml with metabolic activation (3 h)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was only observed in the presence of S9-mix (at > 512 µg/ml), resulting in a relative total growth of 40%.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
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 examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 52 to 2373 µg/ml in the absence and presence of S9-mix with a 3 hour treatment period and at 10 to 475 µg/ml in the absence of S9-mix with a 24 hour treatment period. No toxicity in the suspension growth was observed up to and including the highest test substance concentration of 2373 μg/ml compared to the suspension growth of the solvent control both in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Experiment 1: Cytotoxic and mutagenic response of methyl-N- [3-(trimethoxysilyl)propyl]carbamate in the mouse lymphoma L5178Y test system

Dose (µg/ml)

RSG

(%)

CEday2

(%)

RSday2

(%)

RTG

(%)

Mutation frequency per 106 survivors

total

small

large

without metabolic activation

3 hours treatment

SC1

100

102

100

100

81

63

16

SC2

84

106

84

19

1.7

88

86

93

82

87

66

18

5.4

95

81

88

83

76

60

14

17

88

93

100

86

102

80

19

52

93

63

68

63

125

106

17

164

91

72

78

71

79

55

22

512

81

90

97

79

111

71

35

1600

84

79

85

72

107

83

21

2373

85

81

88

74

109

78

27

MMS

60

55

59

36

1084

792

184

with metabolic activation

3 hours treatment

SC1

100

81

100

100

62

27

33

SC2

69

88

32

53

1.7

88

83

110

97

75

34

39

5.4

83

97

128

107

89

47

38

17

102

105

140

143

75

31

41

52

135

99

132

178

82

37

41

164

90

89

118

105

130

69

54

512

74

77

102

76

379

182

143

1600

61

61

81

50

604

258

237

2373

52

58

76

40

714

366

212

CP

47

19

26

12

2839

1426

1033

Experiment 2: Cytotoxic and mutagenic response of methyl-N-[3-(trimethoxysilyl)propyl]carbamate in the mouse lymphoma L5178Y test system

Dose (µg/ml)

RSG

(%)

CEday2

(%)

RSday2

(%)

RTG

(%)

Mutation frequency per 106 survivors

total

small

large

without metabolic activation

24 hours treatment

SC1

100

89

100

100

62

24

36

SC2

90

77

32

42

1.7

87

78

87

76

57

15

41

5.4

91

88

98

89

68

18

48

17

95

85

95

90

80

21

57

52

103

79

89

91

83

19

62

164

99

93

104

102

69

21

47

512

94

105

118

110

53

17

35

1600

90

80

90

81

103

49

50

2373

89

81

91

81

80

33

45

MMS

88

77

86

76

687

234

367

Note: All calculations were made without rounding off

RSG= Relative Suspension Growth; CE= Cloning Efficiency: RS= Relative Survival; RTG= Relative Total Growth; SC= Solvent control= dried dimethyl sulfoxide; MMS= Methylmethanesulfonate; CP= Cyclophosphamide

Historical control data of the spontaneous mutation frequencies of the solvent controls for the mouse lymphoma assay

 

Mutation frequency per 106survivors

 

- S9-mix

+ S9-mix

 

3 hour treatment

24 hour treatment

 

Range

[50 – 119]

[50 – 150]

[50 – 167]

Mean

73

70

81

SD

16

19

25

n

152

132

283

Historical control data of the mutation frequencies of the positive controls for the mouse lymphoma assay

 

 

Mutation frequency per 106survivors

 

 

- S9-mix

+ S9-mix

 

 

3 hour treatment

24 hour treatment

 

Range

[501 – 3165]

[504 – 1445]

[593 – 4356]

Mean

902

750

1377

SD

367

198

593

n

78

67

141

SD = Standard deviation

n = Number of observations

Conclusions:
Interpretation of results: negative without metabolic activation
positive with metabolic activation after 3 h

The in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells performed according to OECD guideline 476 and in compliance with GLP, employing doses of up to 2373 µg/ml, revealed that the test substance does not show mutagenic potential without metabolic activation. In contrast, the test material is mutagenic under the given experimental conditions with metabolic activation. In summary, the mutagenic activity is confined only to incubations with metabolic activation.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-07-30 to 2014-10-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
Type and identity of media:
RPMI 1640 medium supplemented with
- 20% (v/v) fetal calf serum (FCS, heat-inactivated)
- Penicillin/streptomycin (50 U/ml and 50 µg/ml)
- L-glutamine (2 mM)
- Heparin (30 U/ml)
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented postmitochondrial fraction (S9 mix), prepared from the livers of Sprague Dawley rats treated with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
Test concentrations with justification for top dose:
Experiment 1:
- 512, 1600, 2373 μg/ml with and without metabolic activation (3 h)

Experiment 2:
- 512, 1600, 2373 μg/ml without metabolic activation (24 and 48 h)
Vehicle / solvent:
- Vehicle solvent used: DMSO; 1% (v/v)
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
Mitomycin C: 0.5 and 0.75 μg/ml (3 h), 0.2 and 0.3 µg/ml (24 h) and 0.1 and 0.15 µg/ml (48 h) without S9 - Cyclophosphamide: 10 µg/ml (3 h) with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- in medium

DURATION:
- Exposure duration: 3, 24 and 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 3h treatment: 24h; 24h treatment: 24h; 48 h treatment: 48 h

SPINDLE INHIBITOR:
- colchicine 0.5 µg/ml medium

STAIN:
- Giemsa 5% (v/v) in Sörensen buffer pH 6.8

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED:
- 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

METABOLIC ACTIVATION:
- As clear negative result were obtained in the presence of metabolic activation, the repetition of the experiment was not considered necessary. Therefore, long term treatment without metabolic activation was performed.
Evaluation criteria:
A test substance is considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.
Statistics:
χ² test, one-sided, p < 0.05
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
The highest concentration of 2373 µg/ml showed no precipitation in the culture medium. The pH and osmolarity of a concentration of 2373 µg/ml were 7.86 and 427 mOsm/kg respectively (compared to 7.94 and 426 mOsm/kg in the solvent control).

RANGE-FINDING/SCREENING STUDIES:
Lymphocytes were cultured for 48 h and thereafter exposed to 52, 164, 512, 1600 and 2373 µg/ml of methyl-N-[3-(trimethoxysilyl)propyl]carbamate for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A negative control was included at each exposure time. The highest tested concentration was the recommended concentration of 2373 µg/ml). Cytotoxicity of methyl-N-[3-(trimethoxysilyl)propyl]carbamate in the lymphocyte cultures was determined using the mitotic index. Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was the recommended 2373 µg/ml.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Chromosome aberrations in human lymphocyte cultures treated with methyl-N-[3-(trimethoxysilyl)propyl]carbamate in the absence/presence of S9-mix in the cytogenetic assay

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in %

with gaps

without gaps

Exposure period 3h, fixation time 24h, without S9 mix

DMSO

1.0% (v/v)

100

6

5.5

MMC

0.5

52

55

55

Test substance

512

96

0.5

0.5

1600

104

2

1.5

2373

83

2.5

2.5

Exposure period 3h, fixation time 24h, with S9 mix

DMSO

1.0% (v/v)

100

3.5

3.5

CP

10

70

55

55

Test substance

512

96

0.5

0.5

1600

136

3

3

2373

125

1

1

Exposure period 24h, fixation time 24h, without S9 mix

DMSO

1.0% (v/v)

100

0

0

MMC

0.5

64

52

51

Test substance

512

110

1

0.5

1600

99

0

0

2373

96

0

0

Exposure period 48h, fixation time 48h, without S9 mix

DMSO

1.0% (v/v)

100

1.5

0

MMC

0.5

73

75

69

Test substance

512

67

0.5

0

1600

58

1

1

2373

47

0.5

0.5

Conclusions:
Interpretation of results: negative

The chromosome aberration test in cultured peripheral human lymphocytes performed according to OECD guideline 473 and in compliance with GLP, employing doses of up to 2373 µg/ml, showed that the test substance does not induce chromosomal aberrations with and without metabolic activation. Therefore, the test material should be classified as non-clastogenic.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
weight of evidence
Justification for type of information:
refer to analogue justification document provided in IUCLID section 13
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Species / strain:
S. typhimurium, other: TA 1535, TA 97a, TA 98, TA 100 and TA 102
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:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative

The Salmonella/microsome test performed according to OECD guideline 471 and in compliance with GLP with two source substances methyl-N-[(trimethoxysilyl)methyl]carbamate (CAS 23432-64-6) and methyl-N-{[dimethoxy(methyl)silyl]methyl}carbamate (CAS 23432-65-7), employing doses of up to 5000 µg/plate, showed that the source substances do not produce bacteriotoxic effects. No indications of mutagenic effects of the source substances could be found at assessable doses of up to 5000 µg/plate in any of the Salmonella typhimurium strains used. The source substances were considered to be non-mutagenic without and with metabolic activation in the plate incorporation as well as in the pre-incubation method of the Salmonella/microsome test. As explained in the analogue justification, the differences between the target and the source substances are unlikely to lead to differences in bacterial mutagenicity.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Mammalian mutagenicity (comet assay): negative (OECD 489)

Link to relevant study records
Reference
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:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
July 29, 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approximately 7-9 weeks old
- Weight at study initiation: not reported
- Housing: housed 2-3 animals / sex / group / cage in IVC cages
- Diet: Altromin 1324 maintenance diet for rats and mice provided ad libitum
- Water: tap water provided ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no: MKCD1021 (pre-experiment); MKCG 3257 (main experiment)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulations were kept under magnetic stirring for approximately 10 minutes or until visual homogeneity was achieved.
Duration of treatment / exposure:
2 days
Frequency of treatment:
Daily
Dose / conc.:
400 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
5 males per dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
ethylmethanesulphonate
- Justification for choice of positive control(s): Selection was based on the results of the JaCVAM validation trial
- Route of administration: oral
- Doses / concentrations: 200 mg/kg bw
Tissues and cell types examined:
Cells from the liver, glandular stomach and duodenum were isolated, embedded in agarose, lysed and DNA allowed to migrate under electrophoresis conditions.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose levels were based on the results of a dose-range finding acute toxicity study.The maximum dose applied in the dose range-finding study was 2000 mg/kg bw. The maximum volume administered was 10 mL/kg bw and was compatible with physiological space available. Since only slight toxic effects were determined after the first and second administration of 2000 mg/kg bw in one animal of each sex, the same dose was applied in two additional animals of each sex. Lower doses were not tested.

TREATMENT AND SAMPLING TIMES: Animals of the vehicle control group and the three test item dose groups were treated orally by gavage daily for a period of 2 consecutive days. The animals of the positive control were treated once by oral gavage 4 h before sacrifice. During the period of administration, the animals were observed precisely each day for signs of toxicity. 4 h after last treatment, animals were deeply anaesthetized using ketamine/xylazine. The abdominal aorta was cut and the blood was released. Organs assigned to comet assay (liver, glandular stomach and duodenum) were removed, rinsed with cold mincing buffer to remove residual blood and stored in ice-cold mincing buffer on ice until further processing. The times to remove the tissues until the preparation of the slides were recorded in the raw data. A part of each analysed organ was preserved in 10% neutral-buffered formalin for histopathological evaluation.

DETAILS OF SLIDE PREPARATION: During the comet assay procedure individual cells were embedded in a thin agarose gel on a microscope slide. All cellular proteins were then removed from the cells by removing the cover slips and then incubating the slides overnight in chilled lysing solution. The DNA was allowed to unwind by incubating the slides in an alkaline (pH > 13) electrophoresis solution. Following the unwinding, the liberated DNA underwent electrophoresis, allowing the broken DNA fragments or damaged DNA to migrate away from the nucleus. This was accomplished by placing the slides in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with electrophoresis solution. Slides were placed in the electrophoresis chamber in a random order. After electrophoresis, the slides were neutralised by rinsing with neutralisation buffer three times and incubated again in ice-cold ethanol and air-dried afterwards. After dehydration, cells were stained by applying a gel red staining solution on top of the slides and covering with a cover slip.

METHOD OF ANALYSIS: 150 cells/animal/tissue were analysed, if available. Comet slides were analysed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and the Comet Software ‘Comet Assay IV’ (Perceptive Instrument, software version 2.1.2). The slides were coded so that the evaluator was not aware of which dose group was evaluated. Each slide was screened for cells in a meandering pattern in the unfrosted area of the slide by an evaluator. The calculation of the different parameters was done automatically by the Comet Software, but the set front, middle and back lines of the comet were adjusted manually if they are not set correctly automatically. All cells of the visual field were scored, except of e.g. overlapping cells, cells with an atypical nucleus, cells with a strong background or “hedgehogs” (cells that exhibit a microscopic image consisting of a small or non-existent head and a large diffuse tail, were considered to be heavily damaged cells). Therefore, cells were classified into three potential categories scorable, non-scorable and “hedgehog” (cells that exhibit a microscopic image consisting of a small or non-existent head and a large diffuse tail are considered to be heavily damaged cells). To avoid artefacts only scorable cells (defined round to oval nucleus) and at least 150 cells per sample on two slides (75 cells per slide) were scored. The %-tail intensity is the parameter for evaluation and interpretation of DNA damage, and was determined by the DNA staining intensity present in the tail region expressed as a percentage of the cell's total staining intensity including the nucleus.
Evaluation criteria:
The alkaline comet assay is considered acceptable if it meets the following criteria according to OECD guideline 489:
- the concurrent negative control data are considered acceptable for addition to the laboratory historical control database;
- the concurrent positive controls should induce responses that are compatible to those previously generated and included in the historical positive control database and produce a statistically significant increase compared with the concurrent negative control; and
- three doses and if available 150 cells per organ of each animal have been analysed

Increases in DNA damage in the presence of clear evidence for cytotoxicity during e.g. clinical observations should be interpreted with caution. A positive response should minimally yield a statistically significant increase in the %-tail DNA in at least one dose group at a single sampling time in comparison with the negative control value. The positive control should produce a positive response, and if not, the study data will not be acceptable.

The test material is considered to be clearly positive if:
- at least one of the test doses exhibits a statistically significant increase in tail intensity compared with the concurrent negative control, and
- this increase is dose-related when evaluated with an appropriate trend test, and
- any of these results are outside the distribution of the historical negative control data.

The test material is considered to be clearly negative if:
- none of the test concentrations exhibits a statistically significant increase in tail intensity compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data,
- direct or indirect evidence supports exposure of, or toxicity to, the target tissue(s).
Statistics:
All slides, including those of positive and vehicle controls were independently coded before microscopic analysis and subsequently scored blinded. The median %-tail DNA for each slide was determined and the mean of the median values was calculated for each of the tissue types from each animal.

For each tissue type, the mean of the individual animal means was then determined to give a group mean. Statistical analysis was done using a variety of approaches.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
High-dose animals showed moderate toxic effects such as reduction of spontaneous activity, prone position, half eyelid closure and ataxia directly after the first administration. The same symptoms were present in the mid-dose group were less pronounced.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the registration substance did not induce biologically relevant DNA-strand breaks in any of the tissues evaluated. The result indicates no adverse effect of the test item on the DNA of liver, glandular stomach and duodenum cells after oral administration in rats.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

No data on genetic toxicity (mutagenicity) in bacteria in vitro is available with methyl-N-[3-(trimethoxysilyl)propyl] carbamate (CAS 23432-62-4). Therefore, read across from the structurally analogues methyl-N-[(trimethoxysilyl)methyl] carbamate (CAS 23432-64-6) and methyl-N-{[dimethoxy(methyl)silyl]methyl}carbamate (CAS 23432-65-7) was applied. Refer to analogue justification document provided in IUCLID section 13

Genetic toxicity (mutagenicity) in bacteria in vitro

A reliable bacterial gene mutation study (Ames test) performed according to OECD 471 and in compliance with GLP with methyl-N-[(trimethoxysilyl)methyl]carbamate (CAS 23432-64-6) is available (BSL Bioservice, 2003d). The strains Salmonella typhimurium TA 1535, TA 97a, TA 98, TA 100 and TA 102 were tested according to the plate incorporation (experiment I) and pre-incubation (experiment II) procedure in the absence and presence of a metabolic activation system (phenobarbitone/β-naphthoflavone-induced rat liver S9-mix). Two independent experiments were conducted in three repetitions at each concentration from 31.6 to 5000 µg/plate (experiment I and II). No cytotoxicity was observed in a preliminary toxicity test with TA 98 and TA 100 after treatment with the test item up to 5000 µg/plate. No precipitation was recorded at any tested concentration during the course of the study. Appropriate solvent (DMSO) and positive controls were included and gave the expected results. No significant increase in the number of revertants was observed in any of the tester strains treated with the test substance with and without metabolic activation.

Another reliable genetic toxicity study addressing mutagenicity in bacteria in vitro with methyl-N-{[dimethoxy(methyl)silyl]methyl carbamate (CAS 23432-65-7) is available and was performed according to OECD 471 (Ames test) and in compliance with GLP (BSL Bioservice, 2004c). The strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were tested according to the plate incorporation (experiment I) and pre-incubation (experiment II) procedure in the absence and presence of a metabolic activation system (phenobarbitone/β-naphthoflavone-induced rat liver S9-mix). Two independent experiments were conducted in three repetitions at each concentration from 31.6 to 5000 µg/plate. No cytotoxicity and no precipitation of the test material were observed at any tested concentration during the course of the study. Appropriate negative, solvent (DMSO) and positive controls were included into the test and gave the expected results. No significant increase in the number of revertants was observed in any of the tester strains with and without metabolic activation.

Based on the above results the test material was considered to be not mutagenic to bacteria under the conditions of the tests.

In conclusion, based on the available data from 2 structural analogues it was concluded that methyl-N-[3-(trimethoxysilyl)propyl]carbamate (CAS 23432-62-4) is not mutagenic to bacteria.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

An in vitro chromosome aberration test in human lymphocytes was performed with methyl-N-[3-(trimethoxysilyl)propyl]carbamate (CAS 23432-62-4) according to OECD 473 and in compliance with GLP (WIL, 2014b, draft version). Human peripheral lymphocytes were cultured and treated with the test material or vehicle (1% DMSO) in the absence or presence of a metabolic activation system (phenobarbitone/β-naphthoflavone-induced rat liver S9-mix) in duplicates at concentrations of 512, 1600 and 2373 µg/ml for 3 hours in experiment I and 24 and 48 hours in the abscence of metabolic activation in experiment II. In experiment I fixation and staining of the cells were performed 24 hours after start of exposure with the test material. In the second experiment, fixation and staining was performed 24 or 48 hours after start of exposure with the test material. No cytotoxicity was observed in a preliminary toxicity test with cultured lymphocytes treated with 52, 164, 512, 1600 and 2373 µg/ml of the test material for 3, 24 and 48 h in the absence and 3 h in the presence of S9-mix. Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was the recommended 2373 µg/ml. Cytotoxicity was assessed by determination of the mitotic index. Appropriate solvent and positive controls were included in the test and gave the expected results. Neither precipitation nor cytotoxicity was recorded at any concentration during the course of the study. The test substance did not cause a statistically significant, dose-related increase in chromosome aberrations under the tested conditions. Based on the draft results of the study the test material is considered not to be clastogenic in this chromosome aberration test in vitro.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

An in vitro gene mutation test in Mouse lymphoma L5178Y cells was performed with methyl-N-[3-(trimethoxysilyl)propyl]carbamate (CAS 23432-62-4) according to OECD 476 and in compliance with GLP (WIL, 2015b). Mouse lymphoma cells were treated with the test material or vehicle (1% DMSO) in the absence or presence of a metabolic activation system (phenobarbitone/β-naphthoflavone-induced rat liver S9-mix) in duplicates at concentrations from 1.7 to 2373 µg/ml for 3 hours (-/+ metabolic activation, experiment I) and 24 hours (- metabolic activation, experiment II). After exposure with the test material mouse lymphoma cells were cultured in microtiter plates containing 5 µg/ml trifluorothymidine selective medium for additional 11 - 12 days. Fixation of the cells was performed 14 to 15 days after start of exposure with the test material. Appropriate solvent and positive controls were included in the test and gave the expected result. No cytotoxicity was observed in a preliminary toxicity test with L5178Y mouse lymphoma cells treated with concentrations from 52 to 2373 µg/ml (-/+ metabolic activation) for 3 hours and 10 to 475 µg/ml for 24 hours (- metabolic activation). Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the mutagenicity assays considering the highest dose level was the recommended 2373 µg/ml. No evidence of marked toxicity following exposure to the test item was recorded in the absence of metabolic activation in both experiments. Cytotoxicity was only observed in the presence of metabolic activation (3 hours, experiment I) at concentrations >512 µg/ml which is in contrast to the results of the previously performed preliminary toxicity test. The test substance did not cause a statistically significant, dose-related increase in mutant frequency in the absence of metabolic activation. In contrast, genotoxicity was observed following exposure to the test item at concentrations >52 µg/ml in the presence of metabolic activation (3 hours, experiment II).

In conclusion, methyl-N-[3-(trimethoxysilyl)propyl]carbamate is mutagenic in the TK mutation test system under the given experimental conditions. The mutagenic activity is confined only to incubations with metabolic activation. In order to further assess the positive result of the in vitro mouse lymphoma assay, testing of gene mutation in mammalian cells in vivo was further investigated.

Genetic toxicity (mutagenicity) in vivo

An in vivo comet assay was performed with methyl-N-[3-(trimethoxysilyl)propyl]carbamate (CAS 23432-62-4) according to OECD 489 and in compliance with GLP (BSL, 2019). This study was performed to assess the genotoxic potential of the registration substance by measuring the ability to induce DNA-strand breaks in different organs of rats (liver, glandular stomach and duodenum). Three dose levels (0.2 MTD: 400 mg/kg bw, 0.5 MTD: 1000 mg/kg bw and MTD: 2000 mg/kg bw) were used covering a range from the MTD to little or no toxicity. The animals treated with a dose of 0.2 MTD showed no clinical symptoms. After administration of
doses of 0.5 MTD and MTD mild to moderate signs of systemic toxicity were observed. The animals showed reduction of spontaneous activity, prone position, half eyelid closure and ataxia. The test item could be analytically detected in all plasma samples obtained from whole blood except the vehicle control group demonstrating systemic bioavailability after oral administration which is evidence that liver cells were exposed to the test item. The mean values noted for the dose groups which were treated with the test item were within the range of the concurrent vehicle control and within the historical control limits. No biologically relevant increase of tail intensity was found after treatment with the test item in any of the dose groups and organs evaluated compared to the vehicle controls. Ethyl methanesulfonate (200 mg/kg bw) administered orally was used as positive control, which induced a statistically significant increase in DNA damage for all evaluated organs.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the registration substance did not induce biologically relevant DNA-strand breaks in liver, glandular stomach and duodenum after oral administration in rats. Therefore, methyl-N-[3-(trimethoxysilyl)propyl]carbamate (CAS 23432-62-4) is considered to be non-DNA damaging under these experimental conditions in thein vivo mammalian Alkaline Comet Assay.







Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.