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Diss Factsheets

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 S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 (OECD Test Guideline 471) (Laboratory of Pharmacology and Toxicology KG 2002).
Cytogenicity in mammalian cells: negative with and without metabolic activation in cultured human lymphocytes (OECD Test Guideline 473) (Harlan Laboratories Ltd 2010a).
Mutagenicity in mammalian cells: negative with and without metabolic activation in L5178Y mouse lymphoma cells (OECD Test Guideline 476) (Harlan Laboratories 2010b).

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:
2002-04-03 to 2002-05-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
100-5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: none given in report
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100 and TA 1535 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 without metabolic 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 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 102 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthracene amide
Remarks:
TA 98, TA 102, TA 1537 with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
TA 100, TA 1535 with metabloic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation;
DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

NUMBER OF REPLICATIONS: 3 plates per concentration and 2 independent experiments

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: other: condition of bacterial lawn
Evaluation criteria:
A response is considered positive if there is a statistically significant dose-related increase in the number of revertants relative to the solvent control, by at least 2-fold (TA 98, 100 and 102) or 3-fold (TA 1535 or 1537), and that this is reproducible and revertants are confirmed by streaking to histidine-free agar plates.
Statistics:
Mann and Whitney U-test and Spearman's rank correlation coefficient.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Preincubation method: 316 µg/plate -S9; 3160 µg/plate +S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Preincubation method: 5000 µg/plate -S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Preincubation method: 1000 µg/plate - S9 and +S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Plate incorporation method: 5000 µg/plate -S9; 316 µg/plate + S9 Preincubation method: 316 µg/plate -S9; 3160 µg/plate +S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Pre-incubation method: 316 µg/plate -S9; 1000 µg/plate +S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1 Plate incorporation test average number of revertants per plate (mean of 3 plates)

 

Concentration (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

5000

35.3

34.3

179.7

157.3

150.7

151.7

0.0*

0.0*

4.0*

3.7*

3160

31.7

26.3

134.0

137.3

172.3

120

39.7*

0.0*

2.7*

5.7*

1000

40.3

19.0

122.7

128.0

161.7

155.7

22.7

0.0*

5.0

5.3*

316

32.0

27.3

121.3

166.0

172.3

202

28.0

1.3

2.7

5.3

100

46.7

28.3

109.3

159.7

197.3

240.7

22.7

37.7

4.7

4.3

Negative control 100 µL/plate

32.0

36

119.0

153.0

220.7

292.0

16.3

27.7

5.3

3.7

Positive control

817.0

333

447.3

491.7

1231.0

905.7

957.3

841.3

290.3

290.7

 * = scarce background lawn

Table 2 Preincubation test average number of revertants per plate (mean of 3 plates)

Concentration (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

5000

0.0*

0.0*

31.0*

72.7*

1.3*

0.0*

0.0*

0.0*

0.0*

0.0*

3160

10.3*

0.0*

65.3*

80.3*

0.0*

0.0*

0.0*

7.0*

0.0*

2.3*

1000

10.7*

50.0*

93.0*

116.3*

74.3*

0.0*

0.0*

18.7*

0.0*

0.0*

316

12.3

25.7

119.7

135

280

340.7

3.3

20.7

3

13.3

100

19.7

29.7

102.3

132.3

275.3

370.7

11

17.3

3.7

12.3

Negative control 100 µl/plate

32.3

35

119.7

126

246.7

364.7

12

18.7

6.3

14.7

Positive control

303.3

313

464

477

1131

1153.7

891

984.3

286.3

288.7

* = scarce background lawn

Conclusions:
(3-chloropropyl)triethoxysilane has been tested in a valid and reliable study according to OECD Test Guideline 471 and in compliance with GLP. No increase in the number of revertants was observed in any strain with or without activation in either the initial plate incorporation assay or the subsequent preincubation assay. The solvent and positive controls gave the expected results. It is concluded that the 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:
2009-09-21 to 2009-11-30
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:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
primary culture, other: peripheral human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone and beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
18.75 - 250 µg/ml (4 h exposure, +/-S9; 20 h exposure -S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: recommended by sponsor and formed clear solution suitable fo rdosing
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
ACTIVATION: S9 mix contained NADP as cofactor and S9 was present at a final concentration of 2%

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 4 hours (with and without activation) 24 hours (without activation)
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 µg/ml) two hours before harvest
STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: Duplicate cultures.

NUMBER OF CELLS EVALUATED: 2000 (mitotic index); 100/culture (chromosome damage)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A result was considered positive if the percentage of cells with aberrations, excluding gaps markedly exceeded that seen in the concurrent control,
either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
Only applied in event of a positive result.
Species / strain:
primary culture, other: peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
150 µg/ml (4 h exp -S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: controls were within the range of historical data

Table 1Results of Chromosome Aberration Test 4 hours incubation, without metabolic activation (S9)

 

Concentration µg/ml

No. of cells examined

Mitotic index % mean

Cells with aberrations % mean including gaps

Cells with aberrations % mean excluding gaps

Vehicle control

200

100

0.5

0.5

18.75

200

82

0.5

0

37.5

200

111

1

0.5

75

200

107

0

0

150

200

33

2

1.5

MMC 0.4

100a

55

59

56

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed

 

Table 2 Results of Chromosome Aberration Test 4 hours incubation, with metabolic activation (S9)

Concentration µg/ml

No. of cells examined

Mitotic index % mean

Cells with aberrations % mean including gaps

Cells with aberrations % mean excluding gaps

Vehicle control

200

100

0.5

0

75

200

95

0

0

150

200

90

0

0

200

200

45

0

0

CP 5

150

31

26.7

26.7

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed

Table 3 Results of Chromosome Aberration Test 24 hours incubation, without metabolic activation (S9)

Concentration µg/ml

No. of cells examined

Mitotic index % mean *

Cells with aberrations % mean including gaps

Cells with aberrations % mean excluding gaps

Vehicle control

200

100

0.5

0.5

37.5

200

124

1

1

75

200

132

1.5

1

150

200

86

0.5

0

200

200

25

b

b

MMC 0.2

100a

41

33

33

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed

b = Too toxic for metaphase analysis

Conclusions:
(3-chloropropyl)triethoxysilane has been tested in a valid and reliable study according to OECD Test Guideline 473 and in compliance with GLP. No increase in the number of cells with aberrations was observed with or without activation. Vehicle and positive controls gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations in peripheral human lymphocytes under the conditions of the test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-12-15 to 2010-01-18
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:
1997 (prior to guideline 490)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone, beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
4.69 to 150 µg/ml (4 and 24 h exposure without activation); 9.38 to 300 µg/ml (4 hour exposure with activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle:
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
ACTIVATION: S9 mix contained NADP as cofactor and S9 was present at a final concentration of 2%
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 4 hour (with and without activation); 24 hour (without activation)
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

SELECTION AGENT (mutation assays): 4 µg/ml 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate treatments

NUMBER OF CELLS EVALUATED: 2000 cells plated in selective medium for mutant frequency; 2 cells per well to evaluate viability

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

OTHER EXAMINATIONS:
- Other: Large and small colony evaluation

OTHER:
Evaluation criteria:
To be considered positive, a substance must induce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. The mutation frequency must also exceed the vehicle value by the Global Evaluation Factor (GEF) of 126 x 10-06 (International Workshop on Genotoxicity Test Procedures, Moore et al 2003, 2006)
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
150 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid

Table 1 Summary of Results, 4 hour treatment

Treatment (µg/ml)

 

 

4-hours -MA

Treatment (µg/ml)

4-hours +MA

%RSG

RTG

MF

 

%RSG

RTG

MF

0

100

1

122.75

0

100

1

153.76

4.69

99

NE

NE

9.38

90

1.23

125.84

9.38

103

NE

NE

18.75

90

1.16

151.25

18.75

100

0.92

129.75

37.5

92

1.17

135.38

37.5

111

0.94

131.85

75

85

1.05

169.31

75

96

0.91

148.54

150

60

0.68

148.29

100

76

0.7

140.85

200

3

0.02

333.47

125

35

0.33

118.37

250

1

NE

NE

150

5

0.04

178.71

300

1

NE

NE

EMS 400

79

0.7

650.26

CP 2

57

0.2

1243.63

NE not evaluated

RSG Relative suspension growth

RTG Relative total growth

MF mutant frequency

Table 2 Summary of Results, 24 hour treatment

Treatment (µg/ml)

24-hours -MA

%RSG

RTG

MF

0

100

1

102.37

4.69

96

NE

NE

9.38

92

0.94

97.25

18.75

94

0.98

108.58

37.5

89

0.99

93.55

75

72

0.88

73.5

100

49

0.57

129.81

125

30

0.41

81.87

150

3

NE

NE

EMS 150

61

0.49

911.5

NE not evaluated

RSG Relative suspension growth

RTG Relative total growth

MF mutant frequency

Conclusions:
(3-chloropropyl)triethoxysilane has been tested in a valid and reliable study according to OECD Test Guideline 476 and in compliance with GLP. No increase in the number of revertants was observed with or without metabolic activation. The vehicle and positive controls gave the expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Information is available from reliable studies for all the required in vitro endpoints. The results of most of the studies were in agreement. A positive result was obtained in one study in a single strain of bacteria in the presence of activation, but only when ethanol was used as the solvent. This result was not confirmed in a more recent study also using ethanol as solvent, nor in the other available bacterial mutagenicity assays, so it is concluded that the positive response was not biologically relevant, and the most recent study was chosen as the key study.

No test substance related increase in the number of revertants relative to the solvent control was observed when (3-chloropropyl)triethoxysilane (CAS 5089-70-3; EC 225-805-6) was tested in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 in accordance with OECD Test Guideline 471 and in compliance with GLP. The results obtained with and without metabolic activation in either the initial plate incorporation assay or the subsequent pre-incubation assay were in agreement. The solvent (ethanol) and positive controls were included and gave the expected results. The test substance was therefore concluded to be negative for mutagenicity to bacteria under the conditions of the test (Laboratory of Pharmacology and Toxicology KG, 2002).

(3-Chloropropyl)triethoxysilane has been tested for mutagenicity in bacteria according to a protocol that is similar to OECD Test Guideline 471 and  in compliance with GLP using the pre-incubation method and Salmonella strains (TA98, TA100, TA1535, TA1537) and E.coli strains (WP2 uvrA and WP2), in the presence and absence of metabolic activation.

No increase in the number of revertants was detected in any strain with or without metabolic activation when tested using DMSO as the vehicle. When the study was repeated using ethanol as solvent, there were difficulties with dosing levels, so parts of the experiment had to be repeated. An increase in the number of revertants (5.6-fold) was observed in strain TA 1535 in the presence of activation only. The experiment was not repeated to confirm if the positive result was reproducible. It is concluded that the test substance is positive for mutagenicity to bacteria in the presence of metabolic activation when ethanol is used as solvent (Dow Corning Corporation, 1995).

(3-Chloropropyl)triethoxysilane has been tested for mutagenicity in bacteria according to a protocol that is similar to OECD Test Guideline 471 and in compliance with GLP using the pre-incubation method and Salmonella strains (TA98, TA100, TA1535, TA1537) and E.coli strains (WP2 uvrA and WP2), in the presence and absence of metabolic activation.

No increase in the number of revertants was observed in any strain at any test concentration in the presence or the absence of metabolic activation. Vehicle and positive controls gave expected results. It is concluded that the test substance is negative for mutagenicity under the conditions of the test (Dow Corning Corporation, 1994).

(3-Chloropropyl)triethoxysilane has been tested for ability to cause chromosome aberrations in human lymphocytes cells according to OECD Test Guideline 473 and in compliance with GLP. Three treatment conditions were used for the study; 4-hour exposure in the absence of metabolic activation with a 20-hour expression period; 4-hour exposure in the presence of metabolic activation at a 2% final concentration with cell harvest after a 20-hour expression period and a 24-hour continuous exposure in the absence of metabolic activation. No statistically significant increase in the number of cells with aberrations was observed with or without activation. Vehicle and positive controls gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations in peripheral human lymphocytes under the conditions of the test (Harlan Laboratories Ltd, 2010a).

(3-Chloropropyl)triethoxysilane has been tested for mutagenicity to mammalian cells in mouse lymphoma L5178Y cells according to OECD Test Guideline 476 and in compliance with GLP. The cells were treated with the test material at eight dose levels, in duplicate, together with vehicle (Acetone) and positive controls for 4 hours both with and without metabolic activation, and for 24 hours without metabolic activation. No increase in the number of revertants was observed with or without metabolic activation. The vehicle and positive controls gave the expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test (Harlan Laboratories, 2010b).

Justification for classification or non-classification

Based on the in vitro available data, (3-chloropropyl)triethoxysilane (CAS 5089-70-3) does not require classification for germ cell mutagenicity in accordance with Regulation (EC) No 1272/2008.