Registration Dossier

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 (OECD TG 471) (BioServices, 1997).

Cytogenicity in mammalian cells: negative with and without metabolic activation in peripheral human lymphocytes (OECD TG 473) (CRL, 2018).

Mutagenicity in mammalian cells: read across from analogous substance 3-aminopropyltriethoxysilane CAS 919-30-2: negative in CHO K-1 cells (OECD TG 476) (Hüls AG, 1998b).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-08-07 to 1997-09-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Properties of test substance not determined by testing laboratory
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
75-5000 µg/plate (Test 1); 33-5000 µg/plate (Test 2)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All Strains (with 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 activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100 TA 1535 (without activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 (without activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA (without activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 60 minutes

- Expression time (cells in growth medium): 48 - 72 hours

- Fixation time (start of exposure up to fixation or harvest of cells): 12 hours

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation and the deletion of the uvrB gene.

Cultures of tester strains TA 98 and TA 100 must demonstrate the presence of the pKM101 plasmid R-factor.

All WP2uvrA cultures must demonstrate the deletion in the uvrA gene.

Results must be within range of historical data: TA 98 10-50, TA 100 80-240, TA 1535 5-45, TA 1537 3-21, WP2uvrA 10-60.

A result is positive if the number of revertants is significantly increased compared with the solvent control to at least 2-fold of the solvent control for TA 98, TA 100 and WP2uvrA and 3-fold of the solvent control for TA 1535 and TA 1537 in both experiments.

Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking on histidine-free agar
plates.

Cytotoxicity is defined as a reduction in the mean number of revertants per plate by >50% compared with the mean vehicle control. The reduction
must be accompanied by an abrupt dose-dependant drop in the revertant count, and/or a reduction in background lawn.

Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
600 - 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate, without activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: Results were within range of historical control data

Table 2: Dose range-finding study, Number of revertants per plate (2 plates per strain)

TA 100

WP2 uvrA

Concentration (µg/Plate)

Plate 1

+ MA

Plate 2

- MA

Cytotoxic (Yes/No)

Plate 1

+ MA

Plate 2

- MA

Cytotoxic (Yes/No)

0

130

95

No

17

9

No

6.7

137

102

No

8

15

No

10

120

105

No

15

14

No

33

135

100

No

16

7

No

67

102

91

No

15

15

No

100

114

105

No

31

12

No

333

138

103

No

13

19

No

667

135

89

No

17

18

No

1000

95

98

No

15

16

No

3333

60

89

No

11

15

No

5000

59

66

No

11

9

Yes

*solvent control with DMSO

Table 3: Experiment 1 Mutagenicity Assay, Number of revertants per plate (mean of 3 plates)

 

TA98

TA100

TA1535

Conc.
µg/plate

— MA

+

MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

0*

14

16

No

112

125

No

10

12

No

75

16

18

No

129

119

No

8

8

No

200

16

18

No

100

140

No

9

7

No

600

13

17

No

109

118

No

6

9

No

1800

7

15

No

8

70

Yes

3

10

Yes

5000

0

17

Yes

0

61

Yes

0

10

Yes

Positive control

553

1377

No

528

1103

No

397

100

No

*solvent control with DMSO

Table 4: Experiment 1 Mutagenicity Assay, Number of revertants per plate (mean of 3 plates)

 

TA1537

WP2uvrA

Conc.
µg/plate

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

0*

5

7

No

13

15

No

75

3

5

No

15

14

No

200

4

4

No

15

15

No

600

1

4

Yes

14

17

No

1800

1

1

Yes

11

14

No

5000

0

2

Yes

1

15

Yes

Positive control

860

141

No

389

407

No

*solvent control with DMSO

Table 5: Experiment 2 Mutagenicity Assay, Number of revertants per plate (mean of 3 plates)

 

TA98

TA100

TA1535

Conc.µg/plate

— MA

+

MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

0*

12

18

No

131

140

No

9

11

No

33

13

17

No

112

134

No

9

11

No

100

13

13

No

98

124

No

7

9

No

333

12

13

No

123

117

No

8

9

No

1000

12

17

No

104

105

No

6

7

No

5000

10

7

Yes

30

67

Yes

1

4

Yes

Positive control

749

1149

No

645

986

No

482

94

No

*solvent control with DMSO

Table 6: Experiment 2 Mutagenicity Assay, Number of revertants per plate (mean of 3 plates)

 

TA1537

WP2 uvrA

Conc.
µg/plate

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

0*

5

8

No

13

14

No

33

5

4

No

13

11

No

100

5

4

No

13

11

No

333

5

5

No

11

10

No

1000

3

3

Yes

13

9

No

5000

1

1

Yes

5

12

Yes

Positive control

638

110

No

402

248

No

*solvent control with DMSO

Conclusions:
Interpretation of results: negative with and without metabolic activation

3-(trimethoxysilyl)propylamine has been tested in a valid and reliable study according to OECD 471 and under GLP. No mutagenic effect was observed for the test substance tested up to limit concentration in any of the test strains with and without metabolic activation in Salmonella typhimurium strains TA 98, 100, 1535, 1537 and E.coli WP2 uvrA. The result of the first experiment was confirmed in an independent repeat experiment. 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
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)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age).
- Suitability of cells: recommended in OECD TG 473
- Cell cycle length, doubling time or proliferation index: Average generation time (AGT):
Dose-range finding study: age 26, AGT = 13.5 h and age 27, AGT = 15.2 h
First cytogenetic assay: age 28, AGT = 13.4 h (absence of S9-mix)
age 26, AGT = 14.4 h (presence of S9-mix)
Second cytogenetic assay: age 32, AGT = 14.6 h

- Sex, age and number of blood donors if applicable: see above
- Whether whole blood or separated lymphocytes were used if applicable: lymphocyte cultures: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel, Europe Ltd., Dartford, United Kingdom) was added

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) (Life technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 40 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.6 - 37.1°C)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg) activated rat liver S9.
Test concentrations with justification for top dose:
500, 1000 and 2000 µg/ml. No cytotoxicity was observed at the limit dose, which was the lowest dose at which precipitation was observed.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: none given in study report
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
ACTIVATION: Phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg) activated rat liver S9.
S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP ; 4 µmol HEPES. S9-mix contained 50% (v/v) S9-fraction.
0.2 mL S9-mix was added to 5.3 mL cell culture. The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 +/- 2 hours
- Exposure duration: Experiment 1: 3 hours with and without metabolic activation; Experiment 2: 24 and 48 hours without metabolic activation
- Fixation time (start of exposure up to fixation or harvest of cells): Experiment 1: 24 hours Experiment 2: 24 and 48 hours

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/mL medium)

STAIN (for cytogenetic assays): 5% (v/v) Giemsa (Merck)

NUMBER OF REPLICATIONS: Duplicate cultures

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/mL medium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper

NUMBER OF CELLS EVALUATED: at least 1000 cells

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Not applicable

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;
- Any supplementary information relevant to cytotoxicity: The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%).

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not applicable

Rationale for test conditions:
According to protocol
Evaluation criteria:
A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:
Fisher’s exact test, one-sided
Key result
Species / strain:
lymphocytes: peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
. The test item precipitated in the culture medium at the limit dose.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: The pH and osmolarity of a concentration of 2000 µg/mL were 9.3 and 0.433 mOsm/kg, respectively, (compared to 8.2 and 0.433 mOsm/kg in the solvent control) in the solubility test. Concentrations of 1000 µg/ml and 500 µg/ml had a pH of 8.9 and 8.7, respectively.
- Precipitation: precipitation was observed only at the highest (limit) dose.
- Definition of acceptable cells for analysis: Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed..
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: the mitotic index of the cultures tested in the dose range-finding study was as follows (% of control):
Without metabolic activation (-S9-mix)
3 h exposure time, 24 h fixation time
Control 100
500 97
1000 96
2000 72
MMC-C; 0.5 µg/mL 87
MMC-C; 0.75 µg/mL 53

With metabolic activation (+S9-mix)
3 h exposure time, 24 h fixation time
Control 100
500 89
1000 90
2000 86
CP; 10 µg/mL 72


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
- Negative (solvent/vehicle) historical control data: The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database . The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database .

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic index

Table 1 Chromosome Aberrations in Human Lymphocyte Cultures Treated with
3-(trimethoxysilyl)propylaminein the Absence of S9-Mix in the First Cytogenetic Assay (3 H Exposure Time, 24 H Fixation Time
)

Conc

DMSO

(1.0% v/v)

500 µg/mL

1000 µg/mL

2000 µg/mL

MMC-C

0.5 µg/mL

MMC-C

0.75 µg/mL

Culture

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

 

 

B

Mitotic

Index (%)

 

100

 

 

97

 

 

96

 

 

72

 

 

87

 

 

53

 

No. of

Cells scored

150

150

300

150

150

300

150

150

300

150

150

300

150

150

300

150

143

293

No. of

Cells with

aberrations

(+ gaps) a)

3

1

4

0

1

1

0

0

0

1

1

2

16

8

24***)

17

18

35***)

No. of

Cells with

aberrations

(- gaps)

2

0

2

0

1

1

0

0

0

1

1

2

16

8

24***)

17

17

34***)

g’

1

1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

 

g”

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

b’

1

 

 

 

 

 

 

 

 

 

 

 

8

7

 

10

7

 

b”

1

 

 

 

 

 

 

 

 

 

 

 

3

2

 

4

3

 

m’

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m”

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

exch.

 

 

 

 

1

 

 

 

 

1

1

 

6

1

 

6

8

 

dic

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

d’

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

misc.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

total aberr

(+ gaps)

3

1

 

0

1

 

0

0

 

1

1

 

17

9

 

20

19

 

total aberr

(- gaps)

2

0

 

0

1

 

0

0

 

1

1

 

17

9

 

20

18

 

a)    Abbreviations used for various types of aberrations are listed below the tables
misc.
 = (miscellaneous) aberrations not belonging to the ones mentioned above.

*)   Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or
*** P < 0.001.

Table 2 Chromosome Aberrations in Human Lymphocyte Cultures Treated with the test item in the Presence of S9-Mix in the First Cytogenetic Assay (3 H Exposure Time, 24 H Fixation Time)

Conc

DMSO

(1.0% v/v)

500 µg/mL

1000 µg/mL

2000 µg/mL

CP

10 µg/mL

Culture

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

Mitotic

Index (%)

 

100

 

 

86

 

 

84

 

 

81

 

 

58

 

No. of

Cells scored

150

150

300

150

150

300

150

150

300

150

150

300

150

150

300

No. of

Cells with

aberrations

(+ gaps) a)

0

1

1

1

0

1

0

1

1

1

0

1

23

21

44***)

No. of

Cells with

aberrations

(- gaps)

0

1

1

1

0

1

0

1

1

1

0

1

23

18

41***)

g’

 

 

 

 

 

 

 

 

 

 

 

 

 

2

 

g”

 

 

 

 

 

 

 

 

 

 

 

 

 

1

 

b’

 

 

 

 

 

 

 

 

 

1

 

 

14

10

 

b”

 

 

 

 

 

 

 

 

 

 

 

 

5

5

 

m’

 

1

 

1

 

 

 

1

 

 

 

 

2

2

 

m”

 

 

 

 

 

 

 

 

 

 

 

 

2

2

 

exch.

 

 

 

 

 

 

 

 

 

 

 

 

3

1

 

dic

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

d’

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Misc.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

total aberr

(+ gaps)

0

1

 

1

0

 

0

1

 

1

0

 

26

23

 

total aberr

(- gaps)

0

1

 

1

0

 

0

1

 

1

0

 

26

20

 

a)    Abbreviations used for various types of aberrations are listed in below the tables.
misc.
 = (miscellaneous) aberrations not belonging to the ones mentioned above.

*)   Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or
*** P < 0.001.

Table 3 Chromosome Aberrations in Human Lymphocyte Cultures Treated with the test item in the Absence of S9-Mix in the Second Cytogenetic Assay (24 H Exposure Time, 24 H Fixation Time)

Conc

DMSO

(1.0% v/v)

500 µg/mL

1000 µg/mL

2000 µg/mL

MMC-C

0.2 µg/mL

Culture

 A   B    A+B

 A   B    A+B

 A   B    A+B

 A   B    A+B

 A   B    A+B

Mitotic

Index (%)

100

99

101

99

47

No. of

Cells scored

150  150300

150  150300

150  150300

150  150300

150  150300

Culture

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

Mitotic

Index (%)

 

100

 

 

99

 

 

1201

 

 

99

 

 

47

 

No. of

Cells scored

150

150

300

150

150

300

150

150

300

150

150

300

150

150

300

No. of

Cells with

aberrations

(+ gaps) a)

0

0

0

0

0

0

1

0

1

0

0

0

37

41

78***)

No. of

Cells with

aberrations

(- gaps)

0

0

0

0

0

0

1

0

1

0

0

0

34

37

71***)

g’

 

 

 

 

 

 

 

 

 

 

 

 

4

5

 

g”

 

 

 

 

 

 

 

 

 

 

 

 

1

 

 

b’

 

 

 

 

 

 

 

 

 

 

 

 

7

21

 

b”

 

 

 

 

 

 

1

 

 

 

 

 

7

7

 

m’

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m”

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

exch.

 

 

 

 

 

 

 

 

 

 

 

 

22

18

 

dic

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

d’

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

misc.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

total aberr

(+ gaps)

0

0

 

0

0

 

1

0

 

0

0

 

41

51

 

total aberr

(- gaps)

0

0

 

0

0

 

1

0

 

0

0

 

36

46

 

a)    Abbreviations used for various types of aberrations are listed below the tables.
misc.
 = (miscellaneous) aberrations not belonging to the ones mentioned above.

*)   Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or
*** P < 0.001.

Table 4 Chromosome Aberrations in Human Lymphocyte Cultures Treated with the test item in the Absence of S9-Mix in the Second Cytogenetic Assay (48 H Exposure Time, 48 H Fixation Time)

Conc

DMSO

(1.0% v/v)

500 µg/mL

1000 µg/mL

2000 µg/mL

MMC-C

0.1 µg/mL

Culture

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

A

B

A+B

Mitotic

Index (%)

 

100

 

 

98

 

 

90

 

 

95

 

 

86

 

No. of

Cells scored

150

150

300

150

150

300

150

150

300

150

150

300

150

150

300

No. of

Cells with

aberrations

(+ gaps) a)

0

0

0

2

0

2

0

1

1

1

0

1

38

29

67***)

No. of

Cells with

aberrations

(- gaps)

0

0

0

1

0

1

0

0

0

1

0

1

33

27

60***)

g’

 

 

 

1

 

 

 

1

 

 

 

 

4

3

 

g”

 

 

 

 

 

 

 

 

 

 

 

 

1

 

 

b’

 

 

 

 

 

 

 

 

 

1

 

 

7

13

 

b”

 

 

 

 

 

 

 

 

 

 

 

 

11

10

 

m’

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m”

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

exch.

 

 

 

1

 

 

 

 

 

 

 

 

18

11

 

dic

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

d’

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

misc.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

total aberr

(+ gaps)

0

0

 

2

0

 

0

1

 

1

0

 

41

37

 

total aberr

(- gaps)

0

0

 

1

0

 

0

0

 

1

0

 

36

34

 

a)    Abbreviations used for various types of aberrations are listed below the tables.
misc.
 = (miscellaneous) aberrations not belonging to the ones mentioned above.

*)   Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or
*** P < 0.001.

Aberration                              Abbreviation   Description

Chromatid gap                       g'                      An achromatic lesion which appears as an unstained region in the chromatid arm, the size of which is equal to or smaller than the width of the chromatid and the apparently "broken" segments of the chromatid arm are in alignment.

Chromosome gap                   g"                     An achromatic lesion which appears as an unstained region in both chromatids at the same position, the size of which is equal to or smaller than the width of the chromatid and the apparently "broken" segments of the chromatids are in alignment.

Chromatid break                   b'                      An achromatic lesion in a chromatid arm, the size of which is larger than the width of the chromatid. The broken segments of the chromatid arm are aligned or unaligned.

Chromosome break                b"                     An achromatic lesion in both chromatids at the same position, the size of which is larger than the width of the chromatid. The broken segments of the chromatids are aligned or unaligned.

Chromatid deletion                d'                     Deleted material at the end of a chromatid arm.

Minute                                    m'                     A single, usually circular, part of a chromatid lacking a centromere.

Double minutes                      m"                    Two, usually circular, parts of a chromatid lacking a centromere.

Dicentric chromosome           dic                   A chromosome containing two centromeres.

Tricentric                                tric                   A chromosome containing three

chromosome                                                    centromeres.

Ring chromosome                  r                       A ring structure with a distinct lumen.

Exchange figure                     exch.                An exchange(s) between two or more chromosomes resulting in the formation of a tri- or more-armed configuration.

Chromosome                          intra                 A chromosome intrachange is scored

intrachange                                                      after rejoining of a lesion within one

                                                                        chromosome.

Pulverized                              p                      A fragmented or pulverized chromosome

chromosomes

Multiple                                  ma                    A metaphase spread containing ten or

aberrations                                                       more of the above mentioned aberrations (chromatid and chromosome gaps not included). ma is counted as 10 aberrations.

Polyploidy                              poly                 A chromosome number that is a multiple of the normal diploid number.

Endoreduplication     endo   A form of polyploidy in which each centromere connects two or four pairs of chromatids instead of the normal one pair.

Conclusions:
3-(Trimethoxysilyl)propylamine has been tested in a reliable in vitro cytogenetic assay according to OECD TG 473 and under GLP (CRL, 2018). The test substance did not induce statistically and biologically significant increases in the chromosomal aberration frequency of cultivated peripheral human lymphocytes in either the initial 3-hour exposure assay, with and without metabolic activation, or in the repeat experiment in which cells were exposed for 24 and 48 hours without metabolic activation. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations (not clastogenic) in vitro under the conditions of the test.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-10-20 to 1998-12-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.
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:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and Beta-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
200, 600, 1800, 3000, 5000 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle: homogeneous (as determined by visual inspection)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ham's F12 medium with 2 mM Glutamine, 100 IU/ml Penicillin, and 100 µg/ml Streptomycin
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without activation: 300 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ham's F12 medium with 2 mM Glutamine, 100 IU/ml Penicillin, and 100 µg/ml Streptomycin
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with activation: 10 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 4 hours (with and without activation)
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): 9 days
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays): H 10 medium

NUMBER OF REPLICATIONS: triplicate plates, experiment repeated

NUMBER OF CELLS EVALUATED: approx 5x10 E05

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test compound is considered positive in this assay if it causes a statistically significant, dose related increase in mutant frequency at concentrations of test substance resulting in >20% survival, at a level which is significantly above the maximum spontaneous mutant frequency.
Statistics:
t-test
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
>5000 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Cytotoxicity test: Concentrations in the range of 50-5000 µg/ml were tested for induction of cytotoxicity. There was no effect of the test substance on cloning efficiency of the CHO cells in  the presence or absence of metabolic activation at any concentration  tested.

The frequency of mutations was low in all groups. In the absence of metabolic activation mutant frequencies of test substance treated cells were within the range of historical negative controls (i.e., 0-21 mutants per 10 E06 viable cells). The test substance did not produce a significant mutant frequency at any concentration tested in the absence of metabolic activation. In the presence of metabolic activation, the test substance did induce significant increases of the mutant frequencies at the concentration of 600 µg/ml.  The observed mutant frequencies were within the range of the historical negative control (0-23 mutants per 10(6) viable cells) and did not show a dose response relationship, the statistical significance is the result of normal assay variation rather than indicative of a mutagenic effect of the test substance.

Table 1 Mutant frequency (x10E-06) (mean of 5 flasks)

Concentration µg/ml

Experiment 1

Experiment 2

- MA

+ MA

cytotoxicity

- MA

+ MA

cytotoxicity

0 *

3

1

no

4

3

no

200

2

1

no

5

3

no

600

2

5***

no

0

8****

no

1800

0

0

no

0

5

no

3000

1

1

no

5

3

no

5000

0

3

no

1

2

no

Positive control

265**

290**

no

342**

230**

no

* Solvent control with culture medium

** Highly significant, no statistical evaluation

*** Statistically significant, P<0.001 but within the range of the historical negative control

**** Statistically significant 0.1<P<0.05

but within the range of the historical negative control


Conclusions:
Interpretation of results:
negative with and without activation

3-aminopropyltriethoxysilane has been tested in a reliable assay according to OECD TG 476 and under GLP. The results from the repeat experiment were consistent with those from the initial test. Expected results were obtained from medium and positive controls. No increase in the mutant frequency was observed in the absence of activation. In the presence of activation, a statistically significant increase was observed at a single concentration. As this result was within the range of the mutant frequencies of the historical negative controls, and no dose response relationship was observed, the increases are a result of normal assay variation rather than a mutagenic effect of the test substance. It is the opinion of the reviewer that although the test substance hydrolyses in water, and so the use of aqueous cell culture medium may have led to exposure of the test organisms to the products of hydrolysis rather than the test substance itself, this does not invalidate the test, as hydrolysis would occur in exposed organisms. It is concluded that the test substance in not mutagenic to mammalian cells under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Information is available for 3-(trimethoxysilyl)propylamine from reliable studies for in vitro mutagenicity to bacteria and in vitro chromosome aberration in mammalian cells. No further data are available for the registered substance, however, studies are available for the structural analogue substance 3-aminopropyltriethoxysilane (CAS 919-30-2) including an in vitro mutagenicity study in mammalian cells.

Read-across justification

Non-testing methods including read-across from surrogate substances are able to provide information on mutagenic 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

3-aminopropyltriethoxysilane is a close structural analogue of 3-(trimethoxysilyl)propylamine; both substances are

trialkoxysilanes with an aminopropyl group, and both hydrolyse to aminopropylsilanetriol. Hydrolysis is likely to occur

under the conditions of the studies and also in vivo. The non-silanol products of hydrolysis are ethanol and methanol, which

are not expected to contribute to genetic toxicity, as explained below.

Ethanol is negative in Salmonella typhimurium bacterial mutagenicity assays, up to limit concentrations, including an

appropriate 5th strain (TA 102). No evidence for cytogenicity was found in chromosome aberration studies using cultured human lymphocytes or Chinese hamster ovary cells. A mammalian mutagenicity assay using L5178Y cells gave negative results with and without metabolic activation. Ethanol did not induce micronuclei in bone marrow of rats, or chromosome aberrations in rats or hamsters. Ethanol was negative in the most reliable rodent dominant lethal assay (OECD, 2004b)

Methanol has been tested in vitro in bacterial and mammalian mutagenicity assays and in micronucleus and chromosome

aberration assays. The majority of the results were negative OECD, 2004a). In the ECHA disseminated dossier for methanol, the conclusions of all the key in vitro studies and the weight of evidence of the in vivo assays are negative.

The rates of hydrolysis that are relevant to in vitro conditions of pH and temperature have been calculated.

3-(Trimethoxysilyl)propylamine hydrolyses rapidly, with an estimated hydrolysis half-life of 1 h at pH7 and 37.5°C.

3-Aminopropyltriethoxysilane hydrolyses rapidly, with a hydrolysis half-life, calculated from measured values at 20°C, of 3 h at pH7 and 37.5°C.

Analogue approach justification

(a) Structural similarity: 3-Aminopropyltriethoxysilane is a structural analogue of 3-(trimethoxysilyl)propylamine with a similar functional group, with three methoxy or three ethoxy groups and an aminopropyl group bound to silicon. Both substances hydrolyse in contact with water generating 3-aminopropylsilanetriol.

(b) Structural alerts for genotoxicity: 3-Aminopropyltriethoxysilane and 3-(trimethoxysilyl)propylamine do not include structural alerts for genotoxicity.

(c) Lack of genetic toxicity of non-silanol products of hydrolysis.

3-Aminopropyltriethoxysilane was chosen as read-across substance as it forms the same hydrolysis product as that formed by the registered substance, and neither of these substances has any functional groups that are associated with genetic toxicity. The genetic toxicity data available for these and other structural analogue substances are summarised in the table below. Additional information is given in a supporting report (PFA, 2013aa) attached in Section 13 of the IUCLID dossier.

Table 5.7.3. Genetic toxicity data for 3-(trimethoxysilyl)propylamine and related substances

CAS

Substance name

Bacterial mutagenicity

In vitro cytogenicity

Mammalian mutagenicity

In vivo

3179-76-8

3-(diethoxymethylsilyl)propylamine

Negative (Hita Research Laboratory, 2003a)

Positive (- MA) (Hita Research Laboratory, 2003b)

-

-

919-30-2

3-aminopropyltriethoxysilane

Negative (Hüls AG, 1998a)

Negative (Degussa-Hüls AG, 1999)

Negative (Hüls AG, 1998b)

Negative in micronucleus (Bushy Run Research Center, 1988b)

13822-56-5

3-(trimethoxysilyl)propylamine

Negative (BioServices, 1997)

Negative

(Charles River Laboratories, 2018)

-

-

82985-35-1

Bis(trimethoxysilylpropyl)amine

Negative (BSL Bioservice, 2005)

Negative (Bushy Run Research Center, 1990)

-

Equivocal in micro-nucleus1

(Bushy Run Research Center, 1988c)

13497-18-2

Bis(triethoxysilylpropyl)amine

Negative (Biotoxtech, 2010)

Negative (LPT, 2009)

-

-

1 possibly an artefact due to haemorrhage

3-(Trimethoxysilyl)propylamine has been tested in a valid and reliable study according to OECD 471 and under GLP (BioServices, 1997). No mutagenic effect was observed for the test substance tested up to limit concentration with and without metabolic activation in Salmonella typhimurium strains TA 98, 100, 1535, 1537 and E.coli WP2 uvrA. The result of the first experiment was confirmed in an independent repeat experiment. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

3-(Trimethoxysilyl)propylamine has been tested in a reliable in vitro cytogenetic assay according to OECD TG 473 and under GLP (Charles River Laboratories, 2018). The test substance did not induce statistically and biologically significant increases in the chromosomal aberration frequency of cultivated peripheral human lymphocytes in either the initial 3-hour exposure assay, with and without metabolic activation, or in the repeat experiment in which cells were exposed for 24 and 48 hours without metabolic activation. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations (not clastogenic) in vitro under the conditions of the test.

Evidence for lack of mutagenicity to mammalian cells comes from the surrogate substance 3-aminopropyltriethoxysilane which has been tested in a reliable assay conducted according to OECD TG 476 and in compliance with GLP (Hüls AG, 1998b). No increase in the mutant frequency was observed in the absence of activation in Chinese hamster ovary cells. In the presence of activation, a statistically significant increase was observed at a single concentration. As this result was within the range of the mutant frequencies of the historical negative controls, and no dose response relationship was observed, the increases are a result of normal assay variation rather than a mutagenic effect of the test substance. The results from the repeat experiment were consistent with those from the initial test. Expected results were obtained from medium and positive controls. It is the opinion of the reviewer that although the test substance hydrolyses in water, and so the use of aqueous cell culture medium may have led to exposure of the test organisms to the products of hydrolysis rather than the test substance itself, this does not invalidate the test, as hydrolysis would occur in organisms exposed to the surrogate substance or to the target substance; the estimated hydrolysis half-life of 3-(trimethoxysilyl)propylamine is 1 h at pH7 and 37.5°C. At 37.5°C and pH 2 (relevant for conditions in the stomach following oral exposure), the calculated half-life of 3-(trimethoxysilyl)propylamine is approximately 5 seconds. It is concluded that the test substance in not mutagenic to mammalian cells under the conditions of the test.

REFERENCES

Biotoxtech, (2010). Bacterial Reverse Mutation Test of Dynasylan 1122. Testing Laboratory: Biotoxtech Co. Ltd., Chungcheongbuk-do, 363-883, Korea. Study Number: J10318. Owner company: Evonik Degussa. Report date: 2010-11-02

BSL Bioservice (2005). Reverse Mutation Assay using Bacteria (Salmonella typhimurium and Escherichia coli) with Silquest A-

1170 silane. Testing laboratory: BSL Bioservice, Planegg, Germany. Report no.: 52186. Owner company: GE Advanced Materials -

Silicones, Zwijndrecht, Belgium. Report date: 2005-09-12.

Bushy Run Research Center, (1990a). Organofunctional Silane Y-9492 In vitro Chromosomal Aberration Assay in Chinese Hamster

Ovary Cells. Testing laboratory: Bushy Run Research Center, R. D. 4, Melion Road, Export, Pennsylvania 15632. Report no.: 53

-73. Owner company: Union Carbide Chemicals and Plastics Company Inc., Speciality Chemicals Division, Danbury, CT 06817.

Study number: 89-15-18237. Report date: 1990-08-12.

Bushy Run Research Center 1988: Organofunctional Silane A-1100 In Vivo Mouse Micronucleus Study (study report), Testing laboratory: Bushy Run Research Center Mellon Road Export, Pennsylvania 15632 USA, Report no: 51-33, 88-0345-FKM. Owner company; Evonik Degussa, Report date: Apr 12, 1988

Bushy Run Research Center (1988c). Organofunctional Silane Y-9492 In Vivo Mouse Micronucleus Study. Testing laboratory: Bushy

Run Research Center, R. D. 4, Melion Road, Export, Pennsylvania 15632. Report no.: 51-77. Owner company: Union Carbide

Chemicals and Plastics Company Inc., Speciality Chemicals Division, Danbury, CT 06817. Study number: 88-15-18170. Report

date: 1988-10-17.

Degussa-Hüls AG 1999b: Dynasylan AMEO In vitro chromosomal aberration assay (study report), Testing laboratory: Hüls Institutes of analytics, biology and toxicology PO box 1320 D-45764 Marl Germany, Report no: 99-0033-DGM. Owner company; Evonik Degussa, Study number: CA-98 0250, Report date: Feb 2, 1999.

Hita Research Laboratory (2003a). Final report Bacterial Reverse Mutation Test of KBE-902. Testing laboratory: Hita Research

Laboratories, Chemicals Evaluation and Research Institute, Japan 822, Ishii-machi, Hita, Oita 877-0061, Japan. Report no.:

2575. Owner company: Shin Etsu Chemical Co. Ltd. Study number: K01-2930. Report date: 2003-06-25.

Hita Research Laboratory, (2003b) Chromosome aberration test of KBE-902 in mammalian cultured cell. Testing laboratory:

Chemicals Evaluation and Research Institute, Japan, Hita Laboratory. Report no.: K06-1015. Owner company: Shin-Etsu Chemical

Co. Ltd. Report date: 2003-10-17.

Hüls AG (1998). Unpublished Report: Salmonella typhimurium reverse mutation assay (Ames-test) (1998a). Testing laboratory:

Hüls Institutes for analytics, biology and toxicology. Report no.: 98-0111-DGM. Study number: AM-98 10. Report date: 1998-12-17.

LPT (2009). In vitro assessment of the clastogenic activity of Dynasylan® 1122 in cultured human peripheral lymphocytes. Testing laboratory: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Redderweg 8, 21147 Hamburg, Germany. Report no. 23461. Owner company: Evonik Degussa. Report date: 2009-02-26

OECD (2004a): SIDS Initial Assessment Report for SIAM 19, Berlin, Germany, 18-20 October 2004, Methanol, CAS 67-56-1.

OECD (2004b): SIDS Initial Assessment Report for SIAM 19, Berlin, Germany, 19-22 October 2004, Ethanol, CAS 64-17-5.



Justification for classification or non-classification

Based on the available in vitro genotoxicity data, 3-(trimethoxysilyl)propylamine is not classified for mutagenicity according to Regulation 1272/2008/EC.