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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assay / Ames test, conducted according to OECD Test Guideline 471 and in compliance with GLP (Infracor, 2000, reliability score 1), triethoxy(3-thiocyanatopropyl)silane (CAS No. 34708-08-2, EC No. 252-161-3) was found to be negative in both the presence and absence of metabolic activation. The tested strains included Salmonella typhimurium: TA98, TA100, TA1535 and TA1537 as well as Escherichia coli WP2.

 

In an in vitro cytogenicity study in mammalian cells (chromosome aberration), conducted according to OECD Test Guideline 473 and in compliance with GLP (Bioservice Scientific Laboratories, 2012, reliability score 1), the potential of triethoxy(3-thiocyanatopropyl)silane to induce structural chromosome aberrations in Chinese hamster V79 cells was evaluated. A dose-related increase in the number of aberrations was observed at all concentrations tested with and without metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance was clastogenic to mammalian cells (caused structural aberrations in chromosomes) under the conditions of the test. No evidence of polyploidy was observed.

 

In an in vitro mutagenicity study, conducted according to OECD Test Guideline 476 and in compliance with GLP (Bioservice Scientific Laboratories, 2011, reliability score 1), a dose-related increase in the mutant frequency was observed in mouse lymphoma L5178Y cells. Therefore, it is concluded that the test substance was positive for mutagenicity in mammalian cells with metabolic activation. No clastogenic effects were observed.          

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:
2000-05-09 to 2000-05-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Male Wistar rats
- source of S9 : RCC (Cytotest Cell Research GmbH), Roßdorf, Germany.
- method of preparation of S9 mix: Wistar male rats received 80 mg/kg bw phenobarbital (i.p) and 80 mg/kg bw beta-naphthoflavone (oral) during 3 consecutive days and were sacrificed 24 h after the last administration. The livers were removed from the animals and homogenised. The S9 mix was prepared freshly before use: one part S9 fraction mixed with 9 parts of a cofactor solution.
- concentration or volume of S9 mix and S9 in the final culture medium: 10%
- quality controls of S9: enzymatic activity, sterility and protein content
Test concentrations with justification for top dose:
50-5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Not reported
Untreated negative controls:
yes
Remarks:
water
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:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100 (without activation)
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
TA 102 (without activation)
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535 (without activation)
Untreated negative controls:
yes
Remarks:
water
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:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA (without activation)
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoflouorene
Remarks:
All strains (with activation)
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
All strains (with activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

ACTIVATION:

DURATION

- Preincubation period: 30 minutes

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

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; background lawn assessment
Evaluation criteria:
For a test compound or a test substance to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. The increase must be accompanied by a dose response towards increasing concentrations of the test article. Single increases in revertant frequencies which are not dose-related and not reproducible in two independent tests are considered non-relevant. However, when increases occur in both tests, this will be taken as an indication of a mutagenic effect.

A reduction in the number of spontaneous revertants, and / or a thinning of the background lawn is indicative of toxicity.
Key result
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: precipitation noted at 1600 and 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA: Results were within range of historical control data

Table 2: Experiment 1 Plate incorporation Number of revertants per plate

 

TA 98

TA 100

TA 102

Conc.
(
µl/ml)

MA

+

MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

Negative Control

13

13

No

129

237

No

380

434

No

0*

15

9

No

104

2219

No

329

351

No

50

18

2

No

103

237

No

398

312

No

160

16

7

No

111

233

No

388

308

No

500

16

5

No

130

225

No

350

347

No

1600

22

7

No

135

223

No

345

298

No

5000

13

7

No

138

199

No

349

268

No

Positive control

100

2216

No

587

1637

No

1205

837

No

Positive control 2

-

1606

No

-

1524

No

-

522

No

*solvent control with DMSO

Table 2: Experiment 1 Plate incorporation Number of revertants per plate

 

TA 1535

TA 1537

WP2 uvrA

Conc.
(
µl/ml)

MA

+

MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

Negative Control

12

15

No

13

16

No

12

12

No

0*

14

8

No

12

15

No

9

11

No

50

13

7

No

14

17

No

11

15

No

160

11

7

No

9

12

No

9

14

No

500

13

9

No

11

16

No

8

12

No

1600

13

10

No

17

11

No

11

14

No

5000

10

10

No

11

9

No

9

12

No

Positive control

576

31

No

43

69

No

185

40

No

Positive control 2

-

142

No

-

116

No

-

20

No

*solvent control with DMSO

Table 3: Experiment 2 Pre incubation Number of revertants per plate

 

TA 98

TA 100

TA 102

Conc.
(
µl/ml)

MA

+

MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

Negative Control

26

30

No

196

197

No

406

365

No

0*

23

33

No

181

182

No

350

361

No

50

31

34

No

182

185

No

391

360

No

160

30

31

No

195

179

No

361

336

No

500

37

31

No

196

174

No

374

359

No

1600

27

35

No

200

175

No

399

334

No

5000

27

28

No

191

200

No

385

277

No

Positive control

137

1966

No

680

1331

No

1390

1173

No

Positive control 2

-

1113

No

-

1064

No

-

471

No

*solvent control with DMSO

Table 3: Experiment 2 Pre incubation Number of revertants per plate

 

TA 1535

TA 1537

WP2 uvrA

Conc.
(
µl/ml)

MA

+

MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

Negative Control

15

11

No

6

10

No

13

11

No

0*

16

9

No

9

9

No

10

13

No

50

13

10

No

8

7

No

13

17

No

160

16

11

No

9

15

No

12

18

No

500

11

10

No

6

10

No

14

16

No

1600

12

11

No

11

13

No

8

16

No

5000

12

12

No

9

14

No

12

12

No

Positive control

555

24

No

39

78

No

222

52

No

Positive control 2

-

128

No

-

71

No

-

22

No

*solvent control with DMSO

Conclusions:
Triethoxy(3-thiocyanatopropyl)silane has been tested for bacterial mutagenicity in an in vitro study conducted in accordance with OECD Test Guideline 471 (1997) and in compliance with GLP using Salmonella typhimurium strains TA 98, 100, 1535, 1537 and 1538, and E.coli WP2uvrA (reliability score 1). No evidence of test substance induced increase in the number of revertants was observed up to limit concentrations in any of the bacteria tested without and with metabolic activation in either the initial plate incorporation assay or the repeat experiment using the preincubation method. Appropriate untreated, solvent and positive controls were included and gave expected results. In conclusion, the test substance is non-mutagenic to bacteria under the conditions of the test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-10-19 to 2011-12-29
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and ß-naphthoflavone-induced rat liver S9 mix containing 8 mM MgCl2; 22 mM KCl 5 mM Glucose-6-phosphate and 4 mM NADP
Test concentrations with justification for top dose:
Pre-experiment:
with and without metabolic activation:
0.020, 0.039, 0.078, 0.16, 0.31, 0.63, 1.25, 2.50, 5.0 and 10.0 mM

Main Experiment :
without metabolic activation:
0.40, 0.60 and 0.80 mM

with metabolic activation:
0.15, 0.30 and 0.50 mM
Vehicle / solvent:
- Vehicle (s)/solvent(s) used: DMSO (final concentration of 1% solvent v/v)
- Justification for choice of solvent/vehicle: The test item could be dissolved in DMSO and was compatible with the survival of the cells.
Untreated negative controls:
yes
Remarks:
900 µg/mL
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
0.83 µg/mL
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
TREATMENT TIME:
4 hours (Main Experiment with and without metabolic activation)

FIXATION INTERVAL: 20 hours (Main Experiment with and without metabolic activation)
NUMBER OF REPLICATIONS: 1 experiment
NUMBER OF CELLS SEEDED: 1 x 10^4 - 5 x 10^4 cells
NUMBER OF CULTURES: two cultures per concentration
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture)
DETERMINATION OF CYTOTOXICITY: Mitotic index, cell density
Evaluation criteria:
There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells (with and without metabolic activation)).


Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
0.6 mM (without metabolic activation)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Results of chromosome analysis                         without metabolic activation                          
     Cytotoxicity Chromatid aberrations         Isochromatid aberrations       rel. Mitotic index (%) rel. Cell density (%)  Poly-ploidy mean % aberrant cells
Scored cells  gaps breaks  inter-changes  other  gaps breaks  inter-changes  other incl. Gaps excl. Gaps
Main Experiment 
negative control 200 - 2 3 0 0 0 0 0 0 87 100 1 2.5 1.5
solvent control 200 - 2 0 0 0 0 0 0 0 100 100 1 1.0 0.0
0.02 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 101 n.d. n.d. n.d. n.d.
0.03 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 95 n.d. n.d. n.d. n.d.
0.06 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 97 n.d. n.d. n.d. n.d.
0.08 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 85 n.d. n.d. n.d. n.d.
0.10 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 91 n.d. n.d. n.d. n.d.
0.20 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 80 n.d. n.d. n.d. n.d.
0.40 mM 200 no 5 12 4 2 1 0 0 0 73 89 2 10.0 7.5
0.60 mM 200 yes 7 22 1 1 1 0 0 1 48 77 1 11.5 8.5
0.80 mM 200 yes 6 28 4 1 0 1 0 0 32 48 0 13.5 12.0
EMS 900 µg/mL 200 - 7 6 8 3 1 0 6 0 105 84 1 12.5 9.5
Results of chromosome analysis
with metabolic activation
     Cytotoxicity Chromatid aberrations         Isochromatid aberrations       rel. Mitotic index (%) rel. Cell density (%)  Poly-ploidy mean % aberrant cells
Scored cells  gaps breaks  inter-changes  other  gaps breaks  inter-changes  other incl. Gaps excl. Gaps
Main Experiment 
negative control 200 - 3 3 2 0 0 0 1 0 101 101 1 4.5 3.0
solvent control 200 - 6 4 0 0 1 0 0 0 100 100 1 5.0 2.0
0.02 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 98 n.d. n.d. n.d. n.d.
0.05 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 96 n.d. n.d. n.d. n.d.
0.08 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 89 n.d. n.d. n.d. n.d.
0.15 mM 200 no 8 10 0 0 0 0 0 2 86 95 0 8.5 6.0
0.30 mM 200 no 3 15 3 1 0 1 0 2 87 94 2 10.0 9.0
0.50 mM 200 no 4 31 6 1 0 0 0 1 83 82 0 15.0 14.5
0.75 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 63 n.d. n.d. n.d. n.d.
1.00 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 45 n.d. n.d. n.d. n.d.
1.25 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 12 n.d. n.d. n.d. n.d.
1.50 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 28 n.d. n.d. n.d. n.d.
CPA 0.83 µg/mL 200 - 7 13 6 0 0 1 0 1 106 99 2 11.0 9.5

n.d. not determined

Conclusions:
Triethoxy(3-thiocyanatopropyl)silane has been tested in an in vitro chromosomal aberration test conducted according to OECD 473 and in compliance with GLP (reliability score 1). A dose-related increase in the number of aberrations was observed at all concentrations tested with and without metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is clastogenic to mammalian cells (causes structural aberrations in chromosomes) under the conditions of the test. No evidence of polyploidy was observed.
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:
2011-09-26 to 2011-11-15
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 containing 8 mM MgCl2; 33 mM KCl; 5 mM Glucose-6-phosphate and 4 mM NADP.
Test concentrations with justification for top dose:
Pre-experiment with [0.2, 0.5 and 2.5 mM] and without metabolic activation [0.2, 0.5, 1 and 2 mM]

Main Experiment
with metabolic activation: 0.05, 0.1, 0.2, 0.5, 0.7, 0.9, 1.1 and 1.3 mM
without metabolic activation: 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1 mM





Vehicle / solvent:
Based on the solubility test DMSO was used as solvent (final concentration 1% DMSO v/v).
Untreated negative controls:
yes
Remarks:
2.5 µg/ml
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Untreated negative controls:
other: 300 µg/ml
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
other: 10 µg/ml
Positive control substance:
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: suspended in medium
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)
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 106 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.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

Summary of mutagenicity data with and without metabolic activation

1 Without metabolic activation

Concentration (mM)

RCE %

RTG %

MF (mutants /10x8 cells)

IMF (mutants /10x8 cells)

GEF exceeded

Statistical significance

Negative control 1

103.2

122.9

69.4

/

/

/

Negative control 2

100.4

121.0

/

/

/

Solvent control 1

100.0

100.0

66.4

/

/

/

Solvent control 2

/

/

/

0.005

101.1

110.1

67.1

0.8

-

-

0.01

99.6

104.1

69.1

2.7

-

-

0.02

109.5

119.3

50.8

-15.5

-

-

0.05

103.2

108.4

85.9

19.5

-

-

0.1

112.3

61.3

74.7

8.3

-

-

0.2

92.6

35.8

93.0

26.7

-

-

0.5

96.8

26.1

113.1

46.7

-

+

1

96.8

13.1

143.3

76.9

-

+

Positive control 1

94.0

99.6

622.7

556.3

+

+

Positive control 2

92.6

88.1

456.8

390.5

+

+

 

2 With metabolic activation

Concentration (mM)

RCE %

RTG %

MF (mutants /10x8 cells)

IMF (mutants /10x8 cells)

GEF exceeded

Statistical significance

Negative control 1

100.0

93.4

88.0

/

/

/

Negative control 2

100.0

106.2

/

/

/

Solvent control 1

100.0

100.0

84.6

/

/

/

Solvent control 2

/

/

/

0.05

103.5

97.3

72.7

-11.9

-

-

0.1

90.2

91.3

107.2

22.6

-

-

0.2

100.7

65.2

127.5

42.9

-

+

0.5

93.7

34.8

161.0

76.4

-

+

0.7

101.4

35.8

130.0

45.5

-

+

0.9

95.8

30.1

186.3

101.8

-

+

1.1

95.8

22.5

226.7

142.1

+

+

1.3

86.0

9.4

236.2

151.6

+

+

Positive control 1

82.5

44.2

817.8

733.3

+

+

 

RCE = Relative cloning efficiency    GEF = Global evaluation factor

RTG = Relative total growth

MF = Mutant frequency                     IMF = Induced mutant frequency

Conclusions:
Triethoxy(3-thiocyanatopropyl)silane has been tested for mutagenicity to mammalian cells in an in vitro study conducted according to OECD Test Guideline 476 and in compliance with GLP using L5178Y cells (reliability score 1). A dose-related increase in mutant frequency above the global evaluation factor was observed at concentrations of 1mM and above in the main experiment with metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is therefore concluded that the test substance is mutagenic to mammalian cells (thymidine kinase locus). No clastogenic effects (increase in number of small colonies) were observed in this study.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In a mammalian comet assay, conducted according to OECD Test Guideline 489 and in compliance with GLP (Eurofins BioPharma Product Testing, 2021, reliability score 1), triethoxy(3- thiocyanatopropyl)silane did not induce biologically relevant DNA-strand breaks in the liver, glandular stomach and duodenum of male Wistar Han rats. Rats were administered triethoxy(3-thiocyanatopropyl)silane undiluted at 25, 50, and 75 mg/kg bw/day via oral gavage. None of the test substance-treated animal slides had significant increases in the % tail DNA compared to the respective negative control (0.9% NaCl). All criteria for a valid assay (0.9% NaCl negative control, ethyl methanesulfonate positive control) were met for the liver, glandular stomach and duodenum. Under the experimental conditions, triethoxy(3-thiocyanatopropyl)silane was not DNA-damaging.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020/11/23 - 2021/03/23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: Approximately 7-9 weeks
- Weight at study initiation: 164-199 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: In groups of 2-3 animals / sex / group / cage, in IVC cages (type III H, polysulphone cages)
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: Tap water, sulphur acidified to a pH of approximately 2.8, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
None, test item administered undiluted
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Triethoxy(3-thiocyanatopropyl)silane was administered undiluted at 25, 50, 75 mg/kg bw/day. The administered volume of the test item was adjusted to the animal’s body weight by dose group:
- 0 mg/kg bw/day: 2 mL/kg bw
- 25 mg/kg bw/day: 0.0315 mL/kg bw
- 50 mg/kg bw/day: 0.0630 mL/kg bw
- 75 mg/kg bw/day: 0.0950 mL/kg bw
- ethyl methanesulfonate (positive control): 10 mL/kg bw
Duration of treatment / exposure:
Over 2 days
Frequency of treatment:
Once a day for two consecutive days
Post exposure period:
None, animals were sacrificed on day 2 (4 hr after dosing)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
animals received 0.9% NaCl as negative control
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
maximum tolerated dose in range-finding study
No. of animals per sex per dose:
5F
Control animals:
yes
Positive control(s):
yes, ethyl methanesulfonate at 230 mg/kg bw, administered 4 hr before sacrifice
Tissues and cell types examined:
The liver, glandular stomach and duodenum were examined. The organs were selected to cover two first-contact organs upon peroral exposure and liver as the metabolising organ.
Details of tissue and slide preparation:
Four hours after the last treatment animals were deeply anaesthetized using ketamine/xylazine. The abdominal aorta was cut and the blood was released. The liver, glandular stomach and duodenum were removed and a part of each was kept in ice-cold mincing buffer for the comet assay. The other part of the isolated organs was preserved in 10% neutral-buffered formalin for histopathological evaluation, if considered necessary by the sponsor.

Organs assigned to the comet assay were rinsed with cold mincing buffer to remove residual blood and stored in mincing buffer on ice until further processing. The times between animal death and removal of the tissues as well as the times to process the tissues and slides were tightly controlled and recorded in the raw data.

The 25 samples of liver, 25 samples of glandular stomach and 25 samples of duodenum were analysed for DNA strand breaks. The samples were kept in ice-cold mincing buffer. The fresh samples were directly prepared according to the following procedure using a small part of the available tissues. The remaining part of all tissues was fixed for enabling a possible histopathological analysis.

Isolation of primary hepatocytes:
A portion of the liver was minced with a pair of scissors to liberate the cells. The cell suspension was left for not more than 15 seconds until bigger fragments of the liver settled on the bottom of the tube. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL low-melting agarose (LMA) solution.

Isolation of duodenum cells:
The duodenum was flushed with a syringe filled with cold mincing buffer to wash out the food. Afterwards a portion of the duodenum was minced with a pair of scissors. The cell suspension was left for not more than 15 seconds until bigger fragments settled on the bottom of the tube. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.

Isolation of glandular stomach cells:
The glandular stomach was cut open and washed free of food using cold water. A portion of the glandular stomach was minced with a pair of scissors. The pieces were further crushed with a pestle to release single cells. The suspension was left for less than 15 seconds to allow large clumps to settle. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.

In accordance with OECD Test Guideline 489, 150 cells/animal/tissue were analysed, if available.

Methodology
Once single cells were obtained, the basic steps of the assay included:

Slide preparation:
The slides used were pre-coated with normal-melting agarose (NMA) and coded with a random number. A volume of 75 µL of cell suspension embedded in low-melting temperature agarose was placed on slides, which were covered with a cover slip and cooled for 10 min on ice (3 slides per animal and tissue).

Lysis:
Cover slips were carefully removed and the slides incubated overnight in chilled lysing solution at 2 - 8 °C in the fridge to lyse cellular and nuclear membranes and allow the release of coiled DNA loops during electrophoresis. After completion of lysis, the slides were rinsed in purified water to remove residual detergent and salts.

Unwinding of DNA and electrophoresis:
Prior to electrophoresis, the slides are incubated in alkaline (pH > 13) electrophoresis solution for 20 min. An incubation period of 20 min is generally considered appropriate for alkali unwinding.

After alkali unwinding, the unwound DNA is electrophoresed under alkaline conditions to allow for the formation of DNA tails. The electrophoretic conditions are 0.7 V/cm and approximately 300 mA, with the DNA being electrophoresed for 30 min. The slides are placed in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with electrophoresis buffer. Slides will be placed in the electrophoresis chamber in a random order.

Neutralization and dehydration of slides:
After electrophoresis, the slides were neutralized by rinsing with neutralization buffer three times for 5 min each. The slides were incubated for approximately 10 – 20 min in ice-cold ethanol and air-dried afterwards.

DNA staining:
Following dehydration, the cells were stained by applying 75 µL gel red staining solution on top of the slides and covering with a cover slip.

Analysis of DNA-strand breaks:
Comet slides are analysed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and by using the Comet Software “Comet Assay IV” (Perceptive Instruments, Software version 2.1.2). The slides will be coded so that the evaluator is not aware of which dose group is evaluated. Each slide is screened for cells in a meandering pattern in the unfrosted area of the slide by an evaluator. The calculation of the different parameters will be done automatically by the Comet Software, but the set front, middle and back lines of the comet may be adjusted manually if they are not correctly set automatically. All cells of the visual field will be scored, except for e.g. overlapping cells, cells with an atypical nucleus, cells with a strong background or “hedgehogs” (cells that exhibit a microscopic image consisting of a small or non-existent head and a large diffuse tail, are considered to be heavily damaged cells). Therefore, cells will be classified into three categories: scorable, non-scorable and “hedgehog”. To avoid artefacts only scorable cells (defined round to oval nucleus) and at least 150 cells per sample on two slides (75 cells per slide) should be scored. The third back-up slide may be scored in case of discordant results. The %-tail intensity will be the parameter for evaluation and interpretation of DNA damage, and is determined by the DNA staining intensity present in the tail region expressed as a percentage of the cell's total staining intensity including the nucleus.
Evaluation criteria:
Increases in DNA damage in the presence of a clear evidence for cytotoxicity during e.g. clinical observations should be interpreted with caution. A positive response should minimally yield a statistically significant increase in the %-tail DNA in at least one dose group at a single sampling time in comparison with the negative control value.

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

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if:
- none of the test concentrations exhibits a statistically significant increase in tail intensity compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data,
- direct or indirect evidence supports exposure of, or toxicity to, the target tissue(s).

To assess the biological relevance of a positive or equivocal result, information on cytotoxicity of the target tissue can be required. Where positive or equivocal findings are observed solely in the presence of a clear evidence for cytotoxicity, the study should be concluded as equivocal for genotoxicity unless there is enough information supporting a more definitive conclusion.
Statistics:
All slides, including those of positive and negative controls were independently coded and blinded before microscopic analysis. The median %-tail DNA for each slide was determined and the mean of the median values was calculated for each of the tissue types from each animal.

For each tissue type, the mean of the individual animal means was then determined to give a group mean % of tail DNA. Normality was tested according to Kolmogorov-Smirnov-test. For the determination of statistical significances, the mean values of each animal per dose group were evaluated with one-way ANOVA (Dunnett’s test) at the 5 % level (p<0.05). The p value was used as a limit in judging for significance levels in comparison with the corresponding negative control.
Key result
Sex:
female
Genotoxicity:
negative
Remarks:
No biologically relevant increase of tail intensity was found after treatment with triethoxy(3-thiocyanatopropyl)silane in any of the dose groups and organs evaluated compared to the negative (0.9% NaCl) controls.
Toxicity:
yes
Remarks:
At 75 mg/kg bw/day. No signs systemic toxicity at 25 or 50 mg/kg bw/day.
Vehicle controls validity:
valid
Remarks:
Tail intensities of the negative (0.9% NaCl) control group were within the historical control limits and, therefore, accepted for addition to the laboratory control data set.
Negative controls validity:
valid
Remarks:
See "vehicle controls validity"
Positive controls validity:
valid
Remarks:
The positive control ethyl methanesulfonate induced a statistically significant increase in DNA damage for all evaluated organs.
Remarks on result:
other: See Table 1 below
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:
- Solubility:
- Clinical signs of toxicity in test animals:
- Evidence of cytotoxicity in tissue analyzed:
- Rationale for exposure:
- Harvest times:
- High dose with and without activation:
- Other:

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route:
- Other: At 75 mg/kg bw/day, animals showed mild to moderate toxic effects such as moving bedding, salivation, reduced spontaneous activity, half eyelid closure, piloerection and abnormal breathing. No signs systemic toxicity at 25 or 50 mg/kg bw. The body weight variation in the main experiment (±9.7%) was below 20%.

Table 1. Summary of mean tail intensities [%] in liver, glandular stomach and duodenum

Dose

Group

Mean tail intensity [%]

Clinical signs

Liver

Glandular stomach

Duodenum

25 mg/kg bw/d

1.17

2.28

1.72

None

50 mg/kg bw/d

0.91

2.23

1.66

None

75 mg/kg bw/d

1.18

3.73

3.56

e.g. Moving bedding, salivation,reduced spontaneous activity, prone position

Statistically significant trend

No

No

No

/

Study positive control (a)

9.56*

16.21*

13.74*

None

Study negative (b) Control

0.74

1.61

1.65

None

Historic control range

Negative Control: 0.07 % – 3.82 %

Positive Control:

2.81 % – 28.08 %

Negative Control:

1.30 % – 5.46%

Positive Control: 5.99% - 34.62%

Negative Control:

0.89 % – 4.06%

Positive Control: 1.77% - 30.75%

/

* = Significantly increased (One-way ANOVA with Dunnett’s test after normality test by Kolmogorov-Smirnov)

(a) ethylmethanesulfonate, 200 mg/kg bw

(b) 0.9% NaCl

Conclusions:
In a study conducted according to OECD Test Guideline 489 and in compliance with GLP (reliability score 1), triethoxy(3-thiocyanatopropyl)silane did not induce biologically relevant DNA-strand breaks in the liver, glandular stomach and duodenum of female Wistar Han rats. Rats were administered undiluted triethoxy(3-thiocyanatopropyl)silane at 25, 50, and 75 mg/kg bw/day via oral gavage. None of the test substance-treated animal slides had significant increases in the % tail DNA compared to the respective negative control (0.9% NaCl). All criteria for a valid assay (0.9% NaCl negative control, ethyl methanesulfonate positive control) were met for the liver, glandular stomach and duodenum. Under the experimental conditions, triethoxy(3-thiocyanatopropyl)silane was not DNA-damaging.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Evidence of clastogenicity and mutagenicity to mammalian cells was obtained from the in vitro cytogenicity and mutagenicity studies. In the in vivo mammalian comet assay, triethoxy(3-thiocyanatopropyl)silane was not DNA-damaging.

 

Triethoxy(3-thiocyanatopropyl)silane has been tested in a study conducted in accordance with OECD Test Guideline 471 (1997) and in compliance with GLP using Salmonella typhimurium strains TA 98, 100, 1535, 1537 and 1538, and E. coli WP2uvrA (Infracor, 2000, reliability score 1). No evidence of test substance induced increase in the number of revertants was observed up to limit concentrations in any of the bacteria tested with and without metabolic activation in either the initial plate incorporation assay or the repeat experiment using the preincubation method. Appropriate untreated, solvent and positive controls were included and gave expected results. It is concluded that the test substance was non-mutagenic to bacteria under the conditions of the test.

 

Information on the potential for triethoxy(3-thiocyanatopropyl)silane to cause chromosome aberrations in mammalian cells is available from a study conducted according to OECD Test Guideline 473 and in compliance with GLP (Bioservice Scientific Laboratories, 2012, reliability score 1). A dose-related increase in the number of aberrations was observed at all concentrations tested with and without metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance was clastogenic to mammalian cells (caused structural aberrations in chromosomes) under the conditions of the test. No evidence of polyploidy was observed.

 

Triethoxy(3-thiocyanatopropyl)silane has been tested for mutagenicity to mammalian cells in a study conducted according to OECD Test Guideline 476 and in compliance with GLP using mouse L5178Y cells (Bioservice Scientific Laboratories, 2011, reliability score 1). A dose-related increase in mutant frequency above the global evaluation factor was observed at concentrations of 1mM and above in the main experiment with metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is therefore concluded that the test substance was mutagenic mammalian cells (thymidine kinase locus). No clastogenic effects (increase in number of small colonies) were observed in this study.

 

Triethoxy(3-thiocyanatopropyl)silane has been tested in an in vivo mammalian alkaline comet assay conducted according to OECD Test Guideline 489 and in compliance with GLP (Eurofins BioPharma Product Testing, 2021, reliability score 1). Female Wister Han rats were administered triethoxy(3-thiocyanatopropyl)silane undiluted at 25, 50, and 75 mg/kg bw/day via oral gavage on two consecutive days. Female rats were used in the main experiment as the dose range finding study showed them to be the more sensitive gender. Triethoxy(3-thiocyanatopropyl)silane did not induce biologically relevant DNA-strand breaks in the rat liver, glandular stomach or duodenum. None of the test substance-treated animal slides had significant increases in the % tail DNA compared to the respective negative control (0.9% NaCl). The negative control % tail DNA was within the laboratory’s historical range, and the positive control (ethyl methanesulfonate at 230 mg/kg bw) had a statistically significant increase in % tail DNA compared to the negative control. Thus, all criteria for a valid assay were met for the liver, glandular stomach and duodenum. Under the experimental conditions, triethoxy(3-thiocyanatopropyl)silane was not DNA-damaging.

 

The potential for mutagenicity and clastogenicity observed in the in vitro studies in mammalian cells was not confirmed in the in vivo mammalian alkaline comet assay. Thus, it is concluded that triethoxy(3-thiocyanatopropyl)silane is neither clastogenic nor mutagenic.

Justification for classification or non-classification

Based on the available in vivo data, triethoxy(3-thiocyanatopropyl)silane does not require classification for germ cell mutagenicity according to Regulation (EC) No 1272/2008.