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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): positive without activation in Salmonella typhimurium strain TA 98, negative with activation in strain TA 98; negative with and without activation in strains TA 100, TA 1535, TA 1537, TA 1538 (similar to OECD Test Guideline 471 and in compliance with GLP) (Bushy Run Research Center, 1994).

Cytogenicity in mammalian cells: data for the the hydrolysis product, 3-(trimethoxysilyl)propylamine (CAS 13822-56-5); negative in peripheral human lymphocytes with and without metabolic activation (according to OECD Test Guideline 443 and in compliance with GLP) (Charles River Laboratories, 2018).

Mutagenicity in mammalian cells: positive with and without metabolic activation in mouse lymphoma L5178Y cells (OECD Test Guideline 476 and in compliance with GLP) (BSL Bioservice, 2012).

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:
1994-07-06 - 1994-10-08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
range of strains does not comply with current guideline
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Test 1: 0.003 - 0.30 µg/plate. Test 2: 0.03 - 3.0 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-Phenylenediamine 0.01 µg/plate
Remarks:
TA98, TA1538 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100, TA1535 without metabolic activation; 0.01 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537 without metabolic activation 0.06 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene 2.5 µg/plate
Remarks:
TA 98, TA100, TA1535, TA1537 and TA1538 with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation

DURATION
- Preincubation period: ?
- Exposure duration: 48 - 72 hours


SELECTION AGENT (mutation assays):

NUMBER OF REPLICATIONS:
3 plates per dose, experiment repeated


DETERMINATION OF CYTOTOXICITY
- Method: Revertant colonies were counted


OTHER:
Evaluation criteria:
A chemical is considered positive if it shows a statistically significant dose dependent and reproducible increase in the number of revertants relative to the solvent control.
Statistics:
None used
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Produced a weak but consistent mutagenic effect
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1a Experiment 1 Number of revertants per plate (mean of 3 plates)

Concentration

µg/plate

TA98

TA100

TA1535

-MA

+MA

-MA

+MA

-MA

+MA

Solvent control

7

18

90

88

25

8

Positive control

294

1500

645

1310

596

90

3

10

-

1

-

3*

-

10

10

14

64

80

134

5

30

12

15

69

72

3*

6

100

17

14

60

71

9*

6

300

12

9

69

50

4*

2*

3000

-

3*

-

8*

-

0*

* Toxic, absence of background lawn, or mean number of colonies < ½ solvent control values.

 

Table 1b Experiment 1 Number of revertants per plate (mean of 3 plates)

Concentration

TA1537

TA1538

µg/plate

-MA

+MA

-MA

+MA

Solvent control

4

3

9

14

Positive control

76

150

451

1285

3

4

-

5

3

10

4

4

11

11

30

4

5

7

9

100

3

5

12

12

300

2

3

2

6*

3000

-

*

 2*

0*

* Toxic: absence of background lawn, or mean number of colonies < ½ solvent control values.

 

Table 2a Experiment 2 Number of revertants per plate (mean of 3 plates)

Concentration

TA98

TA100

TA1535

µg/plate

-MA

+MA

-MA

+MA

-MA

+MA

Solvent control

11

17

59

83

3

5

Positive control

341

1508

688

1572

664

101

3

12

-

59

-

4

-

10

13

20

53

65

4

7

30

15

17

66

74

5

4

100

22

18

65

76

2

1*

300

6

15

57

40*

1*

4

3000

-

0*

-

14*

-

3*

* Toxic, absence of background lawn, or mean number of colonies < ½ solvent control values.

 

Table 2b Experiment 2 Number of revertants per plate (mean of 3 plates)

Concentration

TA1537

TA1538

µg/plate

-MA

+MA

-MA

+MA

Solvent control

3

6

10

9

Positive control

100

175

338

1340

3

2

-

9

-

10

4

5

9

9

30

5

3

11

11

100

5

5

10

12

300

2

3

4*

6

3000

-

2*

-

2*

* Toxic, absence of background lawn, or mean number of colonies < ½ solvent control values.

Conclusions:
3-(trimethoxysilyl)propyl isocyanate has been tested according to a protocol that is similar to OECD 471, and under GLP in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. A slight increase in the number of revertants was observed in strain TA 98 in the absence of metabolic activation, in the initial experiment at a single dose. This result was reproducible in the second experiment, in which the response appeared to be weakly dose-dependent. This was considered to be evidence of a weak mutagenic potential. It is concluded that the test substance is positive for mutagenicity to Salmonella typhimurium TA 98 in the absence of metabolic activation under the conditions of the test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-11-03 to 2012-02-20
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
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: IWGT Recommendations
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment
with and without metabolic activation:
0.2, 0.5, 2.5, 5.0, 7.5 and 10.0 mM

Main Experiment:
with metabolic activation:
0.01, 0.02, 0.05, 0.10, 0.20, 0.50, 1.00 and 2.00 mM

and without metabolic activation:
0.05, 0.10, 0.20, 0.50, 1.00, 1.50, 2.00 and 2.50 mM





Vehicle / solvent:
The test item was dissolved in DMSO (final concentration 1% DMSO v/v).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation 2.5 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation 300 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation M 10 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: dissolved in DMSO
DURATION: 4 h (short-term exposure)
Expression time (cells in growth medium): 48 h
Selection time (if incubation with selection agent): about 14 days

SELECTION AGENT ( mutation assay) 5 µg/mL trifluorothymidine
NUMBER OF REPLICATIONS: one experiment with single exposure; cells were seeded in 4 plates and evaluated
NUMBER OF CELLS SEEDED: 2000 cells per well
DETERMINATION OF CYTOTOXICITY: relative total growth (RTG)

METABOLIC ACTIVATION: S9 mix contained glucose-- phosphate and NADP as co-factors. Protein content of S9 mix was adjusted to give a final protein concentration of 0.75 mg/ml in the cultures.
Evaluation criteria:
The test item is considered mutagenic if following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 per 10^6 cells
- A dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (=40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.

Statistics:
The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
However, due to precipitation of the test item at all concentrations showing an increase in induced mutant frequency above the GEF, results should be evaluated with care.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RTG 12.3% (2 mM, +MA), 6.8% (2.5 mM, -MA)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Pre-experiment for toxicity with and without metabolic activation

Concentration (mM)

Number of cells 4 h after treatment

Number of cells 24 h after treatment

Number of cells 48 h after treatment

SGa

RSGb

 (%)

 

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

Negative control

31900

277000

1080000

771000

1590000

1490000

17.2

11.5

118.1

115.0

Negative control

351000

324000

1140000

889000

1540000

1470000

17.6

13.1

120.8

130.8

Solvent control

331000

258000

920000

644000

1610000

1480000

14.8

9.5

100.0

100.0

Solvent control

331000

293000

926000

721000

1540000

1450000

14.3

10.5

0.2

338000

281000

916000

677000

1530000

1410000

14.0

9.5

96.4

95.5

0.5

343000

295000

941000

652000

1530000

1450000

14.4

9.5

99.0

94.6

2.5

323000

184000

779000

119000

1550000

1360000

12.1

4.1

83.1

40.8

5.0 (P)

276000

73000

325000

51700

862000

45000

2.8

0.1

19.3

1.4

7.5 (P)

175000

92300

158000

79400

152000

69900

0.5

0.2

3.1

2.1

10.0(P)

172000

93700

128000

77500

104000

61000

0.3

0.2

2.1

1.8

P = precipitation RSG = relative suspension growth SG = suspension growth

Summary tables with and without metabolic activation

Treatment (mM)

RTG (%)

MF (mutants/106cells

IMF (mutants/106cells)

Precipitate

 

-S9

-S9

-S9

S9

Negative control

101.2

84.9

/

-

Negative control

97.9

/

-

Solvent control

100.0

73.7

/

-

Solvent control

/

-

0.05

101.9

67.1

-6.7

-

0.10

91.6

75.5

1.7

-

0.20

93.2

99.0

25.3

-

0.50

91.8

69.5

-4.2

-

1.00

86.1

75.6

1.8

-

1.50

43.6

158.1

84.3

-

2.00

21.7

208.3

134.5

+

2.50

6.8

299.2

225.4

+

Positive control 1

80.7

455.2

381.5

-

Positive control 2

63.7

381.5

307.7

-

Treatment (mM)

RTG (%)

MF (mutants/106cells

IMF (mutants/106cells)

Precipitate

 

+S9

+S9

+S9

+S9

Negative control

90.8

87.0

/

-

Negative control

102.2

/

-

Solvent control

100.0

68.9

/

-

Solvent control

/

-

0.01

88.2

41.9

-27.0

-

0.02

92.5

98.8

30.0

-

0.05

90.4

72.3

3.4

-

0.10

103.2

57.6

-11.3

-

0.20

102.4

55.1

-13.7

-

0.50

82.2

153.9

85.0

-

1.00

78.8

80.3

11.4

-

2.00

12.3

218.1

149.3

+

Positive control

53.9

518.2

449.3

-

None of the test substance exceed GEF (global evaluation factor).

Positive control results exceed GEF and were statistically significant: test substance results did not exceed GEF and ere not statistically significant.

Conclusions:
Information on mutagenicity to mammalian cells is available for 3-(trimethoxysilyl)propyl isocyanate from a reliable study conducted according to OECD 476 and in compliance with GLP, using mouse lymphoma L5178Y cells. A statistically significant, biologically-relevant increase in mutant frequency was observed with and without metabolic activation. The Global Evaluation Factor (GEF) was exceeded at two highest evaluated doses without metabolic activation (2.0 and 2.5 mM, relative total growth (RTG) 21.7 and 6.8% respectively), and at the highest dose with metabolic activation (2.0 mM, RTG 12.3%). A dose-response relationship was observed in the main experiment without metabolic activation. In addition, slight evidence for clastogenicity was observed. Appropriate solvent, negative and positive controls were included and gave expected results. It is concluded that the test substance is positive for mutagenicity to mammalian cells, with some evidence for a clastogenic effect. Precipitation of the test item occurred at all concentrations showing an increase in induced mutant frequency above the GEF, so these results should be evaluated with care.
Executive summary:

The test item 3-(trimethoxysilyl)propyl isocyanate was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations used in the main experiment was based on data from the pre-experiment. In the main experiment with metabolic activation 2.00 mM and without metabolic activation2.50 mM were selected as the highest concentrations. The experiment with and without metabolic activation was performed as a 4 h short-term exposure assay.

The test item was dissolved in DMSO.

The test item was investigated at the following concentrations:

with metabolic activation:

0.01, 0.02, 0.05, 0.10, 0.20, 0.50, 1.00 and 2.00 mM

and without metabolic activation:

0.05, 0.10, 0.20, 0.50, 1.00, 1.50, 2.00 and 2.50 mM

Precipitation of the test item was noted in the pre-experimentwith and without metabolic activationand in the main experiment with metabolic activation.

Growth inhibition was observed in the main experiment with and without metabolic activation.

In the main experiment with metabolic activation the relative total growth (RTG) was 12.3% for the highest concentration (2.00 mM) evaluated. The highest concentration evaluated without metabolic activation was 2.50 mM with a RTG of 6.8%. Due to high cytotoxicity the highest concentration was not considered for evaluation of mutagenicity.

In the main experiment a biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation).The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was exceeded by the induced mutant frequency in the higher dose ranges with and without metabolic activation.

In addition, in the main experiment without metabolic activation a dose-response relationship was observed.

Additionally, in the main experiment with and without metabolic activation colony sizing showed clastogenic effects at the highest evaluated concentrations induced by the test item under the experimental conditions .

EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

This study is classified as acceptable. This study satisfies the requirements for Test Guidelines OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward mutation) data.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Micronucleus assay in mouse (oral administration): negative at doses up to the maximum tolerated dose (MTD) of 1000 mg/kg bw (OECD Test Guideline 474 and in compliance with GLP) (Harlan Laboratories, 2011).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-02-16 - 2011-03-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was compliant with the 1997 guideline, which was current at the time of the study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Albino HSD
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd
- Age at study initiation: 6 - 10 weeks old
- Weight at study initiation: 20 - 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: no information
- Housing: Groups of up to 7 in solid floor polypropylene cages with wood flake bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25°C
- Humidity (%): 30 - 70%
- Air changes (per hr): Approx 15 per hour
- Photoperiod (hrs dark / hrs light): 12 dark /12 light

IN-LIFE DATES: From: 2011-02-22 To: 2011-03-10 .
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: None given
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Lot/batch no. (if required): V-4855
.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Arachis oil was used as vehicle. Dosing volume was 10 ml/kg.

All animals were dosed once by gavage using a metal cannula attached to a graduated syringe.
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
Once
Post exposure period:
24 and 48 hours
Remarks:
Doses / Concentrations:
250 -1000 mg/kg bw
Basis:
other: gavage
No. of animals per sex per dose:
7 male mice
Control animals:
yes
Positive control(s):
- cyclophosphamide;
- Justification for choice of positive control(s): Cyclophosphamide is known to produce micronuclei under the conditions of the test
- Route of administration: oral - gavage
- Doses / concentrations: 50 mg/kg - 5 mg/ml
Tissues and cell types examined:
Polychromatic erythrocytes (PCE) were scored for micronuclei and normochromatic erythrocytes (NCE) were counted from bone marrow smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Animals were dosed once and sampled at 24 and 48 hours.

DETAILS OF SLIDE PREPARATION: Immediately following termination both femurs were dissected from each animal, prepared and stained in May-Grunwald/Giemsa, air dried and mounted with a cover slip.

METHOD OF ANALYSIS: Slides were coded and examined under a microscope x1000 magnification.

Evaluation criteria:
A positive mutagenic response would be demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to the vehicle control group. If these criteria were not fulfilled, then the test item would be considered non-genotoxic under the conditions of the test.
Statistics:
The Student's t-test (two tailed) was used and any significant results were confirmed using the one way analysis of variance.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
reduction in PCE/NCE ratio was observed in the 1000 mg/kg test group relative to the vehicle group.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 - 2000 mg/kg bw
- Solubility: The test item was formulated 2 hours before application and was assumed stable for this duration.
- Clinical signs of toxicity in test animals: Animals above 1000 mg/kg bw showed clinical signs of excessive toxicity which included hunched posture, pilo-erection, splayed gait, ataxia, ptosis, dehydration, lethargy, decreased respiratory rate and laboured respiration.
- Evidence of cytotoxicity in tissue analyzed: Not examined
- Rationale for exposure: "It was considered unnecessary to investigate the intraperitoneal route of administration as there was evidence of marked toxicity via the oral route. No sex-related differences were observed, so only male mice were treated in the micronucleus study."
- Harvest times: 24 and 48 hours
- High dose with and without activation: 1000 mg/kg bw, no metabolic activation was used


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): negative
- Ratio of PCE/NCE (for Micronucleus assay): Modest decreases in PCE/NCE ratio were observed in both 24 and 48-hour 1000mg/kg dose groups when compared to the vehicle control group.
- Appropriateness of dose levels and route: Dose levels and route were appropriate.
- Statistical evaluation: no statistically significant effects were observed.
- Other: Two premature deaths were observed in the 48-hour 1000 mg/kg dose group and one premature death was observed in the 24-hour 500 mg/kg dose group. The deaths were considered to be due to variable sensitivity of the mice to the test item.

Table 3 Results of micronucleus assay

Treatment group mg/kg bw and sample time

Number of PCE with micronuclei per 2000 PCE

       PCE/NCE Ratio

Group Mean

      SD

Group Mean

      SD

Control 24 hour

       2.4

      2.5

      0.87

     0.33

1000 48 hour

       1.6

      2.3

      0.67

     0.16

1000 24 hour

       2.7

      3.0

      0.57

     0.17

 500 24 hour

       1.5

      1.0

      0.78

     0.13

 250 24 hour

       0.4

      0.8

      0.80

     0.15

Positive control 24 hour

     31.2**

    14.4

      0.71

     0.09

 

** = P<0.001

Conclusions:
3-(Trimethoxysilyl)propyl isocyanate has been tested in a reliable micronucleus assay according to OECD TG 474 and in compliance with GLP. No statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was observed in peripheral erythrocytes of mice treated with the test substance by oral gavage. Appropriate vehicle and positive controls were included and gave expected results. It is concluded that 3-(trimethoxysilyl)propyl isocyanate is negative for the induction of micronuclei under the conditions of this test.
Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Data for the registered substance 3-(trimethoxysilyl)propyl isocyanate (CAS 15396-00-6) are available from reliable in vitro studies for mutagenicity to bacterial and mammalian cells, and an in vivo micronucleus assay. In vitro cytogenicity data are available for the hydrolysis product, 3-(trimethoxysilyl)propylamine, (CAS 13822-56-5).

3-(Trimethoxysilyl)propyl isocyanate has been tested according to a protocol similar to OECD Test Guideline 471 and in compliance with GLP in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. A slight increase in the number of revertants was observed in strain TA 98 in the absence of metabolic activation, in the initial experiment at a single dose. This result was reproducible in the second experiment, in which the response appeared to be weakly dose-dependent. This was considered to be evidence of a weak mutagenic potential. It is concluded that the test substance is positive for mutagenicity to Salmonella typhimurium TA 98 in the absence of metabolic activation under the conditions of the test (Bushy Run Research Center, 1994).

Bacterial mutagenicity data for the hydrolysis product, 3-(trimethoxysilyl)propylamine (CAS 13822-56-5), have been added to the dataset as suppoting information for completeness.

The hydrolysis product, 3-(trimethoxysilyl)propylamine has been tested in a reliable in vitro cytogenetic assay according to OECD Test Guideline 473 and in compliance with 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.

Information on mutagenicity to mammalian cells is available for 3-(trimethoxysilyl)propyl isocyanate from a reliable study conducted according to OECD Test Guideline 476 (1997) and in compliance with GLP, using mouse lymphoma L5178Y cells (BSL Bioservice, 2012). A statistically significant, biologically-relevant increase in mutant frequency was observed with and without metabolic activation. The Global Evaluation Factor (GEF) was exceeded at two highest evaluated doses without metabolic activation (2.0 and 2.5 mM, relative total growth (RTG) 21.7 and 6.8% respectively), and at the highest dose with metabolic activation (2.0 mM, RTG 12.3%). A dose-response relationship was observed in the main experiment without metabolic activation. In addition, slight evidence for clastogenicity was observed. Appropriate solvent, negative and positive controls were included and gave expected results. It is concluded that the test substance is positive for mutagenicity to mammalian cells, with some evidence for a clastogenic effect. Precipitation of the test item occurred at all concentrations showing an increase in induced mutant frequency above the GEF, so these results should be evaluated with care.

3-(Trimethoxysilyl)propyl isocyanate has been tested in a reliable in vivo micronucleus assay according to OECD Test Guideline 474 (2008) and in compliance with GLP (Harlan Laboratories, 2011). No statistically significance increase in the frequency of micronucleated polychromatic erythrocytes was observed in peripheral erythrocytes of mice treated with the test substance by oral gavage. Appropriate vehicle and positive controls were included and gave expected results. It is concluded that 3-(trimethoxysilyl)propyl isocyanate is negative for the induction of micronuclei under the conditions of this test.

In vitro results for 3-(trimethoxysilyl)propyl isocyanate indicate that the substance has potential for mutagenicity. The in vivo micronucleus assay indicates that the substance is not clastogenic. Further information is required on the potential for mutagenicity, so an in vivo Comet assay is proposed to investigate the genetic toxicity potential further.

Justification for use of data for the hydrolysis product, 3-(trimethoxysilyl)propylamine, (CAS 13822-56-5)

The registered substance, 3-(trimethoxysilyl)propyl isocyanate (CAS 15396-00-6) is a trimethoxysilane with a propyl side-chain that has an isocyanate functional group at the opposite end from the trimethoxysilane group.

The isocyanate group in 3-(trimethoxysilyl)propyl isocyanate (CAS 15396-00-6) hydrolyses very rapidly under physiological conditions (half-life 1 minute at 37°C and pH 7), forming an amine. Therefore, the substance hydrolyses very rapidly to give the hydrolysis product, 3-(trimethoxysilyl)propylamine (CAS 13822-56-5). 3-(Trimethoxysilyl)propylamine (CAS 13822-56-5) has been predicted to hydrolyse rapidly under conditions relevant for oral exposure. The estimated hydrolysis half-life at pH 2 (relevant for oral exposure) and 37.5°C is approximately 5 seconds. Therefore, the test organism is expected to be mainly exposed to the hydrolysis products, 3-aminopropylsilanetriol and methanol.





Justification for classification or non-classification

Insufficient information is available to conclude on the classification for mutagenicity of 3-(trimethoxysilyl)propyl isocyanate according to Regulation (EC) No.1272/2008.