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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay: N,N’,N’’-tricyclohexyl-1-methylsilanetriamine was negative with and without activation in S. typhimurium strains TA 98, TA100, TA1535, TA1537 and TA 1538 (OECD TG 471).
Cytogenicity in mammalian cells: N,N’,N’’-tricyclohexyl-1-methylsilanetriamine was negative in Chinese hamster ovary cells (OECD TG 473).
Mutagenicity in mammalian cells: N,N’,N’’-tricyclohexyl-1-methylsilanetriamine was negative with and without activation in L5178Y mouse lymphoma cells (OECD TG 476).

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
Study period:
1989-05-10 to 1989-12-08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The restrictions were that the range of strains tested does not comply with the current guideline.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1981
Deviations:
yes
Remarks:
the range of strains tested does not include a strain to detect cross-linking mutations
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine operon
Species / strain / cell type:
other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Metabolic activation:
with and without
Metabolic activation system:
biphenyl polychlorides-activated rat liver S9
Test concentrations with justification for top dose:
0.1, 0.5, 1.0, 2.5, 5.0 μl/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: amino-2-anthracene
Remarks:
TA 98, TA 100, TA 1535, TA 1537, TA 1538, with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
TA 1535, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 100, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98, without metabolic activation
Details on test system and experimental conditions:
ACTIVATION: Cofactors were added to the S9 mix to reach the following concentrations: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 4 mM NADP in 100 mM potassium-phosphate-buffer pH 7.4. The concentration of S9 in the mix was 5%, and 0.5 ml of S9 mix were added to test substance, overlay agar and bacterial suspension giving a final concentration of approximately 1% S9 in the plates.

METHOD OF APPLICATION: Vigel-Bonner agar medium, plate incorporation

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium): no data

SELECTION AGENT (mutation assays): agar medium

NUMBER OF REPLICATIONS: triplicate plates, experiment repeated

DETERMINATION OF CYTOTOXICITY
- Method: reduction in number of revertants; condition of background lawn.
Evaluation criteria:
If the number of colonies apparent in presence of test article is greater than or equal to twice the number of colonies apparent in the negative control group, the result is considered to be positive.
Statistics:
Relevant statistics (st.dev, mean) were calculated
Key result
Species / strain:
other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
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

Table 1. Results for main study no 1, mean revertants, with and without metabolic activation

Concentration

μl/dish

TA 1535

TA 1537

TA 1538

TA 100

TA 98

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

0

10.7

8.3

10.3

10.0

43.0

41.3

101.0

95.3

28.0

24.0

0.1

9.3

9.0

12.7

10.3

39.3

38.7

120.3

86.7

34.0

23.7

0.5

7.3

9.7

8.0

10.7

39.3

34.7

93.0

103.0

38.0

20.3

1.0

12.3

8.0

9.7

13.0

33.0

32.7

87.3

88.3

35.0

21.3

2.5

8.7

6.7

9.0

11.7

37.7

21.0

110.7

87.3

31.7

22.7

5.0

7.7

1.0

9.7

1.3

27.0

2.3

112.7

86.0

32.7

22.0

SR

11.0

8.3

8.7

9.3

44.0

39.0

102.0

 

27.3

25.7

2 NF 0.001

 

 

 

 

 

 

 

 

 

551.5

2A

185.0

 

93.5

 

1258.0

 

1538.0

 

1135.5

 

MMS 0.1

 

 

 

 

 

 

 

308.5

 

 

EMS 10

 

1002.5

 

 

 

 

 

 

 

 

9 AA 0.05

 

 

 

331.9

 

 

 

 

 

 

2 NF 0.001

 

 

 

 

 

240.0

 

 

 

 

Table 2. Results for main study no 2, mean revertants, with and without metabolic activation

Concentration

μl/dish

TA 1535

TA 1537

TA 1538

TA 100

TA 98

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

0

11.7

11.3

7.7

11.3

45.7

31.3

107.0

109.0

24.3

30.0

0.1

12.0

8.3

11.0

8.7

43.3

28.7

95.0

90.7

27.3

21.3

0.5

9.7

13.3

7.7

9.0

35.0

30.7

105.3

115.3

28.3

32.7

1.0

12.0

11.0

8.7

6.7

37.0

30.0

119.7

129.7

30.0

22.3

2.5

11.0

17.0

10.3

6.0

32.0

0.7

128.0

96.7

19.0

10.0

5.0

6.0

7.0

6.3

0.7

16.3

0.0

101.7

52.0

8.0

0.0

SR

11.3

10.0

6.3

11.0

40.3

27.7

110.0

83.3

30.0

23.0

2 NF 0.001

 

 

 

 

 

 

 

 

 

408.5

2A

133.0

 

62.5

 

1193.0

 

1118.5

 

1106.6

 

MMS 0.1

 

 

 

 

 

 

 

293.5

 

 

EMS 10

 

1295.5

 

 

 

 

 

 

 

 

9 AA 0.05

 

 

 

216.5

 

 

 

 

 

 

2 NF 0.001

 

 

 

 

 

291.0

 

 

 

 

SR: spontaneous revertants

2 NF: 2 -nitrofluorene (mg/dish)

2A: 2 -aminoanthracene (mg/dish)

MMS: methyl metanesulphonate (mg/dish)

EMS: ethyl metanesulphonate (mg/dish)

9 -AA: 9 -aminoacridine (mg/dish)

Conclusions:
N,N’,N’’-Tricyclohexyl-1-methylsilanetriamine has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471 (1989), compliant with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or TA 3538 in the initial or the repeat experiments using the plate incorporation method up to limit concentrations. Appropriate positive and solvent (acetone) and controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria 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
Study period:
2015-06-02 to 2015-12-07
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)
Version / remarks:
2014-09-26
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: minimum essential medium (MEM)
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) S9 mix
Test concentrations with justification for top dose:
Experiment I:
without metabolic activation: 2, 5, 10, 20, 50, 100, 200 μg/mL
with metabolic activation: 50, 100, 200, 500, 1000, 2000 μg/mL

Experiment II:
without metabolic activation: 1, 2, 5, 10, 25, 50, 75, 100 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofurane (THF)
- Justification for choice of solvent/vehicle: According to the results of the solubility test, the test item was dissolved in tetrahydrofurane (THF).
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
tetrahydrofurane (THF)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
tetrahydrofurane (THF)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
ACTIVATION: Cofactors were added to the S9 mix to reach the following concentrations: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-phosphate-buffer pH 7.4.
METHOD OF APPLICATION: in medium MEM
DURATION
- Preincubation period: 2 days
- Exposure duration:
Experiment I: 4 hours, with and without metabolic activation
Experiment II: 21 hours, without metabolic activation
- Expression time (cells in growth medium): 21 hours
- Selection time (if incubation with a selection agent): hypotonic solution (0.4% KCl) for 15-20 min
- Fixation time (start of exposure up to fixation or harvest of cells): 21 hours

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 1000 cells/culture; 150 metaphases were scored per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; RICC

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data
Statistics:
Relative statistics used (mean, statistical significance p<0.05).
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:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY: No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

Table 1 Results of chromosomal aberration test with cultured mammalian cells (V79)

 

Dose

Group

Concentration

µg/mL

Relative Mitotic Index

(%)

RICC

(%)

Mean % Aberrant Cells

Historical Laboratory Negative Control Range

Precipitation

Including

Gaps

Excluding Gaps

Experiment I and II, without metabolic activation

Experiment I

4 hour treatment, 21 hour preparation interval

C

0

113

105

1.0

0.3

0.0% - 4.0 %

aberrant cells

-

S

0

100

100

1.0

0.3

-

3

10

107

93

0.7

0.3

-

4

20

116

93

1.3

0.0

-

5

50

103

92

0.3

0.3

+

EMS

900

107

72

11.3

9.0

-

 

Experiment II

21 hour treatment, 21 hour preparation interval

C

0

96

113

2.7

0.7

0.0 % - 4.0 %

aberrant cells

-

S

0

100

100

3.0

1.3

-

5

25

108

89

3.3

2.0

-

6

50

94

79

1.3

0.0

-

7

75

86

87

1.3

1.3

+

EMS

600

55

73

34.4

30.4

-

Experiment I, with metabolic activation

Experiment I

4 hour treatment, 21 hour preparation interval

C

0

89

103

2.3

0.7

0.0 % - 4.3 %

aberrant Cells

-

S

0

100

100

0.7

0.7

-

1

50

109

108

2.3

0.3

-

2

100

82

91

2.0

1.3

-

3

200

106

87

1.7

1.7

+

CPA

1.5

89

90

8.3

5.5

-

C: Negative Control (Culture Medium)

S: Solvent Control (THF)

EMS: Ethylmethanesulfonate

CPA: Cyclophosphamide

RICC: Relative Increase in Cell Count

+ : with precipitation

- : without precipitation

Conclusions:
N,N’,N’’-Tricyclohexyl-1-methylsilanetriamine has been tested in a valid study according to OECD 473 and under GLP, up to precipitating concentrations. No increase in the number of cells with aberrations was observed either with or without metabolic activation in Chinese hamster V79 cells. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of this study.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-06-02 to 2016-01-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD guideline 490 ("In vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene") 2015-07-28
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase (TK) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: medium without methotrexate but thymidine, hypoxanthine and glycine
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) S9 mix
Test concentrations with justification for top dose:
0.0025, 0.005, 0.010, 0.025, 0.05, 0.10, 0.25 and 0.50 mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: According to the results of the solubility test the test item was dissolved in tetrahydrofuran (THF).
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
tetrahydrofuran (THF)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
tetrahydrofuran (THF)
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
tetrahydrofuran (THF)
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METABOLIC ACTIVATION: Cofactors were added to the S9 mix to reach the concentrations below: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-phosphate-buffer pH 7.4.

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days at 37 °C
- Selection time (if incubation with a selection agent): 6 days
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): selective medium

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED: 2000 cells/well

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
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 106 cells
- A dose-dependent increase in mutant frequency is detected.
Statistics:
t-test
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:
valid
Positive controls validity:
valid

Table 1. Results Experiment I, with and without metabolic activation

 

Test Group

Conc.

 RCEa[%]

RTGb[%]

MFc[mutants/ 106cells]

IMFd[mutants/ 106cells]

GEFeexceeded

Statistical

Significant Increasef

Precipitate

 [mg/mL]

Exp I

without metabolic activation

 

C1

0

91.3

101.1

110.7

/

/

/

-

C2

109.8

129.3

/

/

/

-

S1

0

100.0

100.0

121.1

/

/

/

-

S2

/

/

/

-

4

0.0025

116.0

115.7

111.5

-9.6

-

-

-

5

0.005

95.8

93.2

123.9

2.7

-

-

-

6

0.010

111.8

115.3

104.7

-16.4

-

-

-

7

0.025

94.3

92.0

119.0

-2.1

-

-

-

8

0.05

89.9

85.0

125.1

3.9

-

-

-

9

0.10

85.8

78.8

127.4

6.2

-

-

-

10

0.25

94.3

74.8

100.5

-20.6

-

-

-

11

0.50

89.9

46.1

122.5

1.4

-

-

+

EMS

300 µg/mL

79.6

68.5

1029.8

908.7

+

+

-

MMS

10 µg/mL

67.8

58.3

798.9

677.7

+

+

-

 

Exp I

with metabolic activation

 

C1

0

108.1

110.1

126.6

/

/

/

-

C2

99.8

106.0

/

/

/

-

S1

0

100.0

100.0

118.3

/

/

/

-

S2

/

/

/

-

4

0.0025

108.1

114.3

108.4

-9.9

-

-

-

5

0.005

109.9

105.5

110.4

-7.8

-

-

-

6

0.010

85.9

91.7

117.3

-0.9

-

-

-

7

0.025

111.7

107.8

96.5

-21.7

-

-

-

8

0.05

92.5

89.5

122.0

3.7

-

-

-

9

0.10

93.9

92.6

107.8

-10.5

-

-

-

10

0.25

111.7

118.3

107.6

-10.7

-

-

-

11

0.50

101.4

91.2

83.4

-34.9

-

-

+

B[a]P

3.5 µg/mL

93.9

68.8

572.7

454.4

+

+

-

C:  Negative Controls

S:  Solvent Controls (THF 0.5% v/v)

a:   Relative Cloning Efficiency, RCE = [(CEdose group/ CEof corresponding controls) x 100

 Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

b:   Relative Total Growth, RTG = (RSG x RCE)/100

c:   Mutant Frequency,

MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

d:   Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

e:   Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded

f:   statistical significant increase in mutant frequency compared to solvent controls (Mann Whitney test , p<0.05).
+: significant; -not significant

EMS:   Ethylmethanesulfonate [300 µg/mL]

MMS:  Methylmethanesulfonate [10 µg/mL]

B[a]P:  Benzo[a]pyrene [3.5 µg/mL]

Conclusions:
N,N’,N’’-Tricyclohexyl-1-methylsilanetriamine has been tested in a valid study according to OECD 490 and under GLP. No statistically and biologically significant increase in the mutant frequency was observed with or without activation when tested up to precipitation concentrations in mouse lymphoma L5178Y cells. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal fraction of Phenobarbital and β-Naphthoflavone induced rat livers
Test concentrations with justification for top dose:
Experiment I, with and without metabolic activation:
- TA 98, TA 100: 31.6, 100, 316, 1000, 2500, 5000 µg/plate
- TA 1535, TA 1537, TA 102: 10, 31.6, 100, 316, 1000, 2500 µg/plate
Experiment II, with and without metabolic activation: 62.5, 125, 250, 500, 1000, 3000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: the solvent was compatible with the survival of the bacteria and S9 activity
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535, TA 100, without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
TA 1537, TA 98, without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 102, without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA1537, TA 98, TA 100, TA 102, with metabolic activation
Details on test system and experimental conditions:
ACTIVATION: The protein concentration in the S9 preparation was 36 mg/ml. The S9 mix contained the following co-factors: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 5 mM NADP in 100 mM sodium-orthophosphate-buffer, pH 7.4, and 8.5 parts of cofactor solution were added to 1.5 parts of S9. 0.5 ml S9 mix was added to 0.1 ml test solution and 2 ml of overlay agar, giving a final concentration of approximately 3% S9 in the plates.

METHOD OF APPLICATION: in agar: plate incorporation

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours

SELECTION AGENT (mutation assays): histidine deficient agar

NUMBER OF REPLICATIONS: triplicate plates were used, and an independent repeat experiment was conducted.

DETERMINATION OF CYTOTOXICITY
- Method: condition of bacterial lawn/reduction in number of revertants
Evaluation criteria:
A test item is considered mutagenic when: a dose related increase in the number of revertants occurs; a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
Statistics:
Relative statistics (standard deviation, mean) were calculates.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 316 µg/plate, TA 102 without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment I:
- TA 1535, TA 1537, TA 100, TA 98: 2500 µg/plate and higher, with and without metabolic activation
- TA 102: at 316 µg/plate and higher, without metabolic activation; at 2500 µg/plate, with metabolic activation
Experiment II:
- TA 1535, TA 1537, TA 100, TA 98: at 3000 µg/plate, with and without metabolic activation
- TA 102: at 1000 and higher µg/plate, without metabolic activation; at 3000 µg/plate, with metabolic activation

PRECIPITATION
No precipitation of test item was observed.

Table 1. Experiment I, plate incorporation, revertant colonies per plate (mean of three plates), with and without metabolic activation

Concentration

μg/plate

TA 1535

TA 1537

TA 100

TA 98

TA 102

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

A dest

15

18

24

16

139

100

25

27

237

293

Ethanol

16

20

24

15

120

122

37

30

237

237

31.6

19

19

28

18

139

97

31

41

219

212

100

22

14

21

19

140

111

22

37

175

251

316

27

16

21

17

116

105

27

41

165

249

1000

25

19

16

16

81

106

28

33

260

225

2500

16

15

21

13

59

72

14

25

235

234

5000

6

7

3

3

0

0

0

0

118

219

4-NOPD 10

 

 

 

 

 

 

 

798

 

 

4-NOPD 40

 

 

 

199

 

 

 

 

 

 

2-AA 2.5

190

 

147

 

1296

 

1269

 

 

 

2-AA 10

 

 

 

 

 

 

 

 

558

 

Sodium azide 10

 

1163

 

 

 

1121

 

 

 

 

MMS 1μL

 

 

 

 

 

 

 

 

 

2136

Table 2. Experiment II, plate incorporation, revertant colonies per plate (mean of three plates), with and without metabolic activation

Concentration

µg/plate

TA 1535

TA 1537

TA 100

TA 98

TA 102

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

A dest

14

14

18

14

118

109

37

19

214

311

Ethanol

20

18

15

11

117

119

45

28

216

273

62.5

18

14

22

12

119

95

41

29

212

254

125

15

12

17

14

123

109

41

33

207

284

250

17

13

23

14

114

118

34

25

214

291

500

17

19

17

13

122

116

33

29

187

268

1000

19

16

18

10

107

97

40

27

202

189

3000

2

9

7

0

71

40

22

16

98

190

4-NOPD 10

 

 

 

 

 

 

 

1339

 

 

4-NOPD 40

 

 

 

228

 

 

 

 

 

 

2-AA 2.5

233

 

239

 

2227

 

1756

 

 

 

2-AA 10

 

 

 

 

 

 

 

 

521

 

Sodium azide 10

 

1324

 

 

 

1348

 

 

 

 

MMS 1μL

 

 

 

 

 

 

 

 

 

1819

 

4 -NOPD: 4-nitro-o-phenylene-diamine

2 -AA: 2-aminoanthracene

MMS: methylmethanesulphonate

 

 

 

Conclusions:
1,1-Dimethyl-N,N'-bis(1-methylpropyl)silanediamine has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471, in compliance with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or TA 102 in the initial or the repeat experiments using the plate incorporation method up to limit concentrations. Appropriate positive, solvent and negative controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) S9 mix
Test concentrations with justification for top dose:
Experiment I:
10, 31.6, 100, 316, 1000, 2500, 5000 µg/plate

Experiment II:
150, 300, 500, 900, 1500, 3000, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and S9 activity.
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535, TA 100, without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
TA 1537, TA 98, without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 102, without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA 1537, TA 98, TA 100, TA 102, with metabolic activation
Details on test system and experimental conditions:
ACTIVATION: The protein concentration in the S9 mx was 36 mg/ml. The S9 mix contained the following co-factors: 8 mM MgCl₂, 33 mM KCl, 5 mM glucose-6-phosphate, 5 mM NADP in 100 mM sodium ortho-phosphate-buffer, pH 7.4
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): Vogel-Bonner Medium E agar plates

NUMBER OF REPLICATIONS: triplicate plates were used, and the experiment repeated. The initial experiment used the plate incorporation method, the repeat experiment used preincubation.

DETERMINATION OF CYTOTOXICITY
- Method: condition of bacterial lawn/ reduction in number of revertants
Evaluation criteria:
The results is considered positive when: a dose related increase in the number of revertants occurs and/or a reproducible biological relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
Statistics:
Relative statistics (standard deviation, mean) were calculated.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 1000 µg/plate (TA 1535 with metabolic activation)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment I:
- TA 1537, TA 100, TA 102, TA 98: toxic effect at 2500 µg/plate and higher, with and without metabolic activation
- TA 1535: toxic effect at 2500 µg/plate and higher, without metabolic activation; toxic at 1000 µg/plate, with metabolic activation
Experiment II:
- TA 98, TA 100, TA 102, TA 1535: toxic effect at 1500 µg/plate, without metabolic activation and 3000 µg/plate, with metabolic activation
- TA 1537: toxic effect at 3000 µg/plate with and without metabolic activation
PRECIPITATION
No precipitation was observed in the study.

Table 1. Experiment 1, plate incorporation, revertant colonies per plate (mean of three plates), with and without metabolic activation

Concentration

μg/plate

TA 1535

TA 1537

TA 100

TA 98

TA 102

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

A dest

11

11

14

12

110

98

31

28

177

286

Ethanol

16

13

13

15

101

102

36

37

205

266

10

21

16

14

16

126

110

38

28

203

276

31.6

16

15

14

14

130

123

36

31

215

245

100

15

15

19

12

109

131

42

40

206

261

316

14

18

15

14

105

100

31

27

194

200

1000

12

15

13

14

106

96

32

31

180

192

2500

0

9

4

2

75

0

23

0

73

89

5000

0

3

2

0

27

0

0

0

0

47

4-NOPD 10

 

 

 

 

 

 

856

 

 

2-AA 2.5

103

 

85

 

2306

 

1947

 

 

Sodium azide 10

 

1213

 

 

 

1131

 

 

 

 

MMS 1μL

 

 

 

 

 

 

 

 

 

1789

2-AA 10

 

 

 

 

 

 

 

 

742

 

4-NOPD 40

 

 

 

185

 

 

 

 

 

 

Table 2. Experiment 2, preincubation, revertant colonies per plate (mean of three plates), with and without metabolic activation

Concentration

µg/plate

TA 1535

TA 1537

TA 100

TA 98

TA 102

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

A dest

14

16

9

11

110

64

38

32

198

343

Ethanol

16

14

13

8

113

109

38

35

231

294

150

21

14

12

17

139

101

44

37

218

292

300

23

17

13

14

89

83

41

29

220

291

500

23

18

12

10

100

80

40

34

231

269

900

25

13

11

12

136

94

50

36

172

259

1500

21

6

13

13

114

69

36

24

188

188

3000

0

2

4

0

17

0

3

0

37

0

5000

0

0

1

0

7

0

0

0

6

0

4-NOPD 10

 

 

 

 

 

 

 

528

 

 

2-AA 2.5

140

 

105

 

941

 

592

 

 

Sodium azide 10

 

1402

 

 

 

1222

 

 

 

 

MMS 1μL

 

 

 

 

 

 

 

 

 

1849

4-NOPD 40

 

 

 

130

 

 

 

 

 

 

2-AA 10

 

 

 

 

 

 

 

 

810

 

4 -NOPD: 4-nitro-o-phenylene-diamine

2 -AA: 2-aminoanthracene

MMS: methylmethanesulphonate

Conclusions:
N,N', N''-Tributyl-1-methylsilanetriamine has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471, compliant with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or TA 102 in the initial plate incorporation experiment or the repeat assay using the preincubation method up to limit concentrations. Appropriate positive, solvent (ethanol) and negative (untreated) controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

N,N’,N’’-Tricyclohexyl-1-methylsilanetriamine has been tested in a valid bacterial reverse mutation assay, according to the OECD TG 471 (1989), and in compliance with GLP, using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or TA 3538 (Hazleton, 1989). No increase in the number of revertants was observed in any test strain, with or without metabolic activation. Appropriate positive and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test. The study was considered reliability 2, because the range of strains tested did not include a strain to detect cross-linking mutagens.

In order to provide data for the ability of the registered (target) substance to act as a cross-linking mutagen data on two source substances are provided.

N,N', N''-Tributyl-1-methylsilanetriamine has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471, compliant with GLP (Bioservice, 2004a). No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537  or TA 102 in the initial plate incorporation experiment or the repeat assay using the preincubation method up to limit concentrations. Appropriate positive, solvent (ethanol) and negative (untreated) controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test. The study was considered reliability 1.

1,1-Dimethyl-N,N'-bis(1-methylpropyl)silanediamine has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471, in compliance with GLP (Bioservice, 2004b). No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537  or TA 102 in the initial or the repeat experiments using the plate incorporation method up to limit concentrations. Appropriate positive, solvent and negative controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test. The study was considered reliability 1.

N,N’,N’’-Tricyclohexyl-1-methylsilanetriamine has been tested for ability to cause chromosome aberrations in Chinese hamster ovary cells according to OECD TG 473 and in compliance with GLP (Eurofins, 2015). No increase in the number of cells with aberrations was observed either with or without metabolic activation in Chinese hamster V79 cells. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of this study.

N,N’,N’’-Tricyclohexyl-1-methylsilanetriamine has been tested for mutagenicity in mouse lymphoma L5178Y cells according to OECD TG 490, and in compliance with GLP (Eurofins, 2016). No test-substance induced increase in the number of mutations was observed. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study. The study was considered reliability 1.

Read across justification

No data are available for mutagenicity to a bacterial strain capable of detecting cross-linking or oxidising mutagens, therefore data are read-across from the related substances 1,1-dimethyl-N,N'-bis(1-methylpropyl)silanediamine (CAS 93777-98-1) and N,N',N''-tributyl-1-methylsilanetriamine (CAS 16411-33-9). Non-testing methods including read-across from surrogate substances are able to provide information on genetic toxicity (REACH Guidance part 07a, R.7.7.3). In the case of genetic toxicity the presence or absence of functional groups that are known to be related to genetic toxicity is considered important, as the presence or absence of reactive groups and molecular substructures is associated with mutagenic and carcinogenic properties of chemicals (Benigni and Bossa, 2006). Consideration is therefore given to the structural similarity, particularly presence or absence of structural alerts for genetic toxicity, when selecting surrogate substances for genetic toxicity endpoints.

Read-across hypothesis

The hypothesis is that the source (read-across) substances and target (registration) substance have similar systemic toxicological properties because they hydrolyse to similar silanol hydrolysis products (methylsilanetriol and dimethylsilanediol) and alkylamines (cyclohexylamine for the target substance, and n-butylamine and sec-butylamine for the source substances). None of the substances or hydrolysis products have structural alerts for genetic toxicity.

Read-across justification

(a) Structural similarity of parent substances and silicon-containing hydrolysis products

The registration and read-across substances are structurally similar in that they are all silylamines with one or two methyl groups and two or three alkylamine groups bound to a central silicon atom. The target substance has one methyl group and three cyclohexylamine groups bound to silicon. The first source substance (CAS 16411-33-9) has one methyl group and three n-butylamine groups bound to silicon. The difference between this read-across substance and the target substance is that the three cyclohexyl groups in the target substance are replaced by three n-butyl groups in the source substance. The second source substance (CAS93777-98-1) has two methyl groups and two sec-butylamine groups bound to silicon. The differences between this read-across substance the target substance are that (i) there are two methyl groups and two amine groups in the source substance and one methyl group and three amine groups in the target substance, and (ii) the amine groups are sec-butylamine in the source substance and cyclohexylamine in the target substance.

All three substances hydrolyse very rapidly to methylsilanetriol or dimethylsilanediol and an alkylamine.

The registered substance, N,N',N''-tricyclohexyl-1-methylsilanetriamine CAS 15901-40-3 hydrolyses rapidly (hydrolysis half-lives at 25°C, < 3 min at pH 4; < 2 min at pH7 and 5.1 min at pH9). The products of hydrolysis are the amine-functional leaving group, cyclohexylamine (CAS 108-91-8) and the silanol, methylsilanetriol (CAS 2445-53-6).

N,N',N''-Tributyl-1-methylsilanetriamine CAS 16411-33-9 hydrolyses rapidly (hydrolysis half-lives at 25°C, < 2 min at pH 4, 7 and 9). The products of hydrolysis are the amine-functional leaving group, n-butylamine (CAS 109-73-9) and the silanol, methylsilanetriol (CAS 2445-53-6).

1,1-Dimethyl-N,N'-bis(1-methylpropyl)silanediamine CAS 93777-98-1 hydrolyses rapidly (hydrolysis half-lives at 25°C, < 2 min at pH 4, 7 and 9). The products of hydrolysis are the amine-functional leaving group, sec-butylamine (CAS 13952-84-6) and the silanol, dimethylsilanediol (CAS 1066-42-8).  

The substances and their hydrolysis products are shown in the table below (see attachment for structures).

 

Target

Source1

Source2

Parent

N,N',N''-tricyclohexyl-1- methylsilanetriamine

CAS 15901-40-3

 

N,N',N''-Tributyl-1- methylsilanetriamine

CAS 16411 -33 -9

1,1-Dimethyl-N,N'-bis(1-methylpropyl)silanediamine

CAS93777-98-1

Silanol hydrolysis product

Methylsilanetriol

CAS2445-53-6

Methylsilanetriol

CAS2445-53-6

Dimethylsilanediol

CAS 1066-42-8

Amine hydrolysis product

Cyclohexylamine

CAS108-91-8

n-butylamine

CAS109-73-9

sec-butylamine

CAS13952-84-6

(b) Lack of structural alerts

None of the substances or hydrolysis products has structural alerts for genotoxicity (Benigni et al., 2008).

(c) Lack of genetic toxicity of the silanol hydrolysis product

Dimethylsilanediol is not mutagenic in a study which included an appropriate 5th strain (unpublished data). No data are available for methylsilanetriol, but other substances which also hydrolyse to methylsilanetriol give negative results in appropriate strains.

(d) Lack of genetic toxicity of the non-silicon hydrolysis product

The non-silicon hydrolysis product of the registered product, cyclohexylamine does not have data in the disseminated dossier from a bacterial mutagenicity study which tested an appropriate strain. However, a wide range of in vitro and in vivo data are presented which conclude that the substance is not a genetic toxin. In addition, the registered substance has been tested for mutagenicity and cytogenicity to mammalian cells, and no evidence for genetic toxicity has been observed. The data available for the rapidly hydrolysing read-across substances indicates that n-butylamine and sec-butylamine are not of concern for genetic toxicity

Benigni and Bossa (2006). Current Computer-Aided Drug Design 2, (2), 169-176. Benigni et al (2008). The Benigni/Bossa rule base for mutagenicity and carcinogenicity JR Scientific report EUR 23241 EN.

Justification for classification or non-classification

N,N’,N’’-Tricyclohexyl-1-methylsilanetriamine contains no structural elements which may be of concern for potential mutagenic activity.

In vitro tests are negative, including bacterial mutagenicity, mammalian cytogenicity and mammalian mutagenicity studies.