Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro:

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 Escherichia coli WP2 uvrA (OECD TG 471) (BioReliance 2001).

Cytogenicity in mammalian cells: negative with and without metabolic activation in Chinese hamster V79 cells (OECD 473) (BSL Bioservice, (2012a).

Mutagenicity in mammalian cells: positive without metabolic activation in L5178Y mouse lymphoma cells (OECD TG 476) (BSL Bioservice (2012b).

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:
2000-10-06 - 2001-01-03
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
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Without metabolic activation: 50, 150, 500, 1500 and 5000 µg/plate; with metabolic activation - 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: Based on the sponsor's request due to compatibility with the target cells and solubility of the test article
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 75 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene 1.0 µg/plate and 10 µg/plate
Remarks:
TA98, TA100, TA1535, TA1537 - 1.0 µg/plate and WP2 uvrA - 10 µg/plate all with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA without metabolic activation 1000 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA98 without metabolic activation 1.0 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100 and TA1535 without metabolic activation 1.0 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

ACTIVATION: S9 mix contained 10% S9, glucose-6-phosphate and NADP as co-factors. 0.5 ml S9 mix, 100 µl of tester strain and 25 µl of vehicle or test article were added to 2ml top agar, giving a final concentration of approximately 2% S9.

DURATION
- Preincubation period: 60+/-2 minutes at 37ºC
- Exposure duration: 48 - 72 hours at 37ºC+/-2ºC

SELECTION AGENT (mutation assays): histidine deficient agar

NUMBER OF REPLICATIONS: triplicate plates, experiment repeated

DETERMINATION OF CYTOTOXICITY
- Method: other: condition of background lawn

Evaluation criteria:
A result is positive if the increase in mean revertants at the peak of the dose response in strains TA1535 and TA1537 is equal to or greater than three times the mean negative control value. In strains TA 98, TA 100 and E. coli WP2 uvrA the result is considered positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean negative control value.
Statistics:
None stated in report
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:
cytotoxicity
Remarks:
toxicity observed at 667 µg/plate with metabolic activation, none was observed without metabolic activation thus 5000 µg/plate was highest dose without metabolic activation. No precipitate was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
cytotoxicity
Remarks:
toxicity observed at 667 µg/plate with metabolic activation, none was observed without metabolic activation thus 5000 µg/plate was highest dose without metabolic activation. No precipitate was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Summary of results – Experiment B1

Dose µg/ml

+/- metabolic activation

Average revertants per plate (mean of 3 plates)

TA98

TA100

TA1535

TA1537

WP2 uvrA

Solvent control

-

14

90

13

5

13

50

-

15

83

11

6

14

150

-

14

74

11

5

11

500

-

13

53

12

5

11

1500

-

12

36

7

4

10

5000

-

9

14

8

3

6

Positive control

-

282

557

595

1490

90

Solvent control

+

23

94

15

7

13

5.0

+

16

94

13

8

n.t.

15

+

15

101

16

7

 n.t.

50

+

16

84

12

7

13

150

+

14

93

8

5

14

500

+

11

11

8

4

11

1500

+

2

10

6

2

14

5000

+

n.t

n.t.

n.t.

n.t.

10

Positive control

+

773

941

171

161

66

Repeated assay due to excessive toxicity in Experiment B1

 

Dose µg/ml

+/-

metabolic activation

Average revertants per plate

TA100

TA1537

Solvent control

-

108

6

          15       

-

 

8

50

-

84

11

150

­-

85

6

500

­-

93

6

1500

-

83

7

5000

-

83

6

Positive control

-

407

763

Independent repeat assay

Dose µg/ml

+/- metabolic activation

Average revertants per plate

TA98

TA100

TA1535

TA1537

WP2 uvrA

Solvent control

-

12

86

9

5

12

5.0

-

 n.t

n.t 

 n.t

 n.t

14

15

-

10

95

13

6

11

50

-

13

98

11

5

12

150

-

13

99

11

5

11

500

-

13

113

12

5

12

1500

-

11

70

11

7

15

5000

-

8

63

15

7

n.t 

Positive control

-

92

359

274

752

209

Solvent control

+

26

120

13

7

12

5.0

+

19

116

12

4

 n.t.

15

+

22

105

13

7

10

50

+

26

80

16

5

13

150

+

26

109

11

6

11

500

+

18

97

13

3

14

1500

+

5

66

11

5

13

5000

+

 n.t

 n.t.

 n.t.

 n.t.

12

Positive control

+

509

1129

100

118

290

Repeat assay due to unacceptable vehicle control in independent repeat assay

 

Dose µg/ml

+/-

metabolic activation

Average revertants per plate

WP2 uvrA

Solvent control

-

18

50

-

17

150

­-

16

500

­-

16

1500

-

20

5000

-

19

Positive control

-

597

Conclusions:
3-(triethoxysilyl)propanethiol has been tested for mutagenicity to bacteria in a study which was conducted according to OECD Test Guideline 471 and in compliance with GLP. No evidence of a test substance related increase in the number of revertants was observed with or without activation in the initial or the repeat experiments. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-10-19 to 2012-02-19
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:
Liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix
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, 2.5, 5 and 10 mM

Experiment I:
without metabolic activation: 0.031, 0.063 and 0.125 mM
with metabolic activation: 0.2, 0.3 and 0.4 mM

Experiment II:
without metabolic activation: 0.125, 0.15 and 0.20 mM
with metabolic activation: 0.25, 0.35 and 0.45 mM

Vehicle / solvent:
-Vehicle (s)/solvent(s) used: Dimethylsulfoxide (DMSO)
-Justification for choice of solvent/vehicle: The test item 3-(triethoxysilyl)propanethiol could be dissolved in DMSO. The solvent was compatible with the survival of the cells and the S9 activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation 400 and 600 µg/mL
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation 0.83 µg/mL
Details on test system and experimental conditions:
TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 hours (Experiment II without metabolic activation)

FIXATION INTERVAL: 20 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independent experiments
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)
(except concentration 0.031 mM (exp. I without metabolic activation): 300 cells
and 0.3 mM (exp. I with metabolic activation): 400 cells)
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:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 0.125 mM (-S9)
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
Experiment I                              
negative control 200 - 2 2 0 1 0 0 0 0 100 100 1 2.5 1.5
solvent control 200 - 6 3 1 1 0 0 0 0 92 101 5 5.0 2.5
0.004 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.
0.008 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 99 n.d. n.d. n.d. n.d.
0.016 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.
0.031 mM 300 no 3 4 1 3 0 0 1 0 96 100 2 4.0 3.0
0.063 mM 200 no 4 2 1 0 0 0 1 0 99 96 4 3.5 1.5
0.125 mM 200 yes 1 1 0 1 1 0 2 0 43 47 0 3.0 2.0
0.25 mM - n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
0.5 mM - n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
1.0 mM - n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
2.0 mM - n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
3.0 mM - n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
4.0 mM - n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
EMS (600 µg/mL) 200 - 3 14 2 0 0 1 2 0 87 101 1 10.0 9.0
Experiment II                                  
negative control 200 - 1 1 0 0 0 0 1 0 97 94 1 1.5 1.0
solvent control 200 - 3 2 0 0 0 0 2 0 100 100 1 3.5 2.0
0.0063 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.
0.0125 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.
0.025 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 108 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. 93 n.d. n.d. n.d. n.d.
0.075 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 111 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. 96 n.d. n.d. n.d. n.d.
0.125 mM 200 no 1 1 0 0 0 0 0 0 73 97 1 1.0 0.5
0.15 mM 200 yes 2 2 0 0 0 0 0 0 59 88 2 2.0 1.0
0.20 mM 200 yes 1 1 0 0 0 0 0 0 45 84 0 1.0 0.5
EMS (400 µg/mL) 200 - 7 12 3 1 1 0 2 0 74 91 0 13.0 9.0
                               
                             
                               
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
Experiment I                              
negative control 200 - 1 5 0 0 0 0 0 0 100 100 1 3.0 2.5
solvent control 200 - 6 4 0 0 1 0 0 0 99 99 1 5.0 2.0
0.0125 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 90 n.d. n.d. n.d. n.d.
0.025 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 105 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. 94 n.d. n.d. n.d. n.d.
0.1 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 90 n.d. n.d. n.d. n.d.
0.2 mM 200 no 4 5 2 0 0 0 0 0 85 99 2 5.0 3.5
0.3 mM 400 yes 4 10 0 2 0 0 2 1 50 80 1 4.5 3.5
0.4 mM 200 yes 7 1 1 1 0 0 0 1 42 64 2 5.0 2.0
0.5 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0 n.d. n.d. n.d. n.d.
0.75 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
1.0 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
CPA (0.83 µg/mL) 200 - 9 14 6 0 0 1 1 3 105 98 2 12.0 10.5
Experiment II                                  
negative control 200 - 4 4 1 0 0 0 0 0 89 105 0 4.5 2.5
solvent control 200 - 6 4 0 1 0 0 0 0 100 100 0 5.0 2.5
0.04 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 102 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. 91 n.d. n.d. n.d. n.d.
0.15 mM - no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 99 n.d. n.d. n.d. n.d.
0.25 mM 200 no 2 1 1 0 0 0 0 0 75 81 0 2.0 1.0
0.35 mM 200 yes 1 2 0 1 0 0 0 0 64 66 0 2.0 1.5
0.45 mM 200 yes 3 3 0 0 0 0 0 0 43 58 0 3.0 1.5
0.55 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 5 n.d. n.d. n.d. n.d.
0.65 mM - yes n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
CPA (0.83 µg/mL) 200 - 10 15 8 0 0 0 0 1 79 106 1 12.0 10.0
n.d.: not determined
Conclusions:
3-(triethoxysilyl)propanethiol has been tested in a reliable in vitro chromosomal aberration test conducted according to OECD Test guideline 473 and in compliance with GLP. No evidence for the induction of structural chromosomal aberrations was observed in the V79 Chinese hamster cell line with and without metabolic activation. It is concluded that the test substance is negative for clastogenicity (induction of chromosome aberrations) under the conditions of the test.

Executive summary:

To investigate the potential of 3-(triethoxysilyl)propanethiol to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay was carried out.

The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations (except concentration 0.031 mM (exp. I without metabolic activation): 300 cells

and 0.3 mM (exp. I with metabolic activation): 400 cells)

The test item 3-(triethoxysilyl)propanethiol could be dissolved in DMSO

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 0.031, 0.063 and 0.125 mM

with metabolic activation: 0.2, 0.3, 0.4 mM

Experiment II:

without metabolic activation: 0.125, 0.15 and 0.20 mM

with metabolic activation: 0.25, 0.35, 0.45 mM

No precipitation of the test item was noted with and without metabolic activation at all concentrations evaluated in both experiments.

Toxic effects of the test item were noted with and without metabolic activation in experiment I and II.

In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.

EMS (400 and 600 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

The positive controls induced the appropriate responses.

There was no evidence of 3 -(triethoxysilyl)propanethiol induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5375; OECD 473 for in vitro cytogenetic mutagenicity data. 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-26 to 2011-12-13
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)
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:
Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment I with and without metabolic activation: 0.2, 0.5 and 2.5 mM
Pre-experiment II without metabolic activation (24 h long-term exposure): 0.01, 0.08, 0.10, 0.30, 0.50 and 0.70 mM

Experiment I
with metabolic activation:
0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70 mM
and without metabolic activation:
0.01, 0.02, 0.05, 0.10, 0.20, 0.30, 0.40, 0.48, 0.56 mM
Experiment II
with metabolic activation:
0.12, 0.25, 0.35, 0.52, 0.58, 0.64, 0.68, 0.72 mM
and without metabolic activation:
0.04, 0.08, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35 mM




Vehicle / solvent:
Based on the results of the solubility test DMSO was used as solvent (final concentration 1% DMSO v/v).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
2.5 and 3.5 µg/ml
Positive control substance:
ethylmethanesulphonate
Remarks:
200 µg/ml and 300 µg/ml
Positive control substance:
methylmethanesulfonate
Remarks:
10 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: dissolved in DMSO
DURATION: 4 h (short-term exposure), 24 h (long-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: two separate experiments (I+II) 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 10E+06 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 and without
Genotoxicity:
positive
Remarks:
without metabolic activation (additionally a clastogenic effect was noted)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid

Summary of mutagenicity data with and without metabolic activation

Concentration mM

RCE %

Mean number cultures/well

MF (mutants/ 10x6cells

IMF (mutants/10x8 cells)

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

Negative control 1

101.2

107.1

9.3

15.5

74.9

103.4

-

-

Negative control 2

106.0

99.6

12.0

11.8

90.8

88.9

-

-

Solvent control 1

100.0

100.0

11.0

14.3

86.3

106.2

-

-

Solvent control 2

11.8

17.5

106.7

138.9

-

-

0.04

-

107.1

-

17.5

-

118.2

-

-4.4

0.08

-

108.6

-

19.3

-

127.6

-

5.0

0.10

-

95.9

-

17.5

-

146.8

-

24.2

0.12

114.7

-

10.0

-

63.8

-

-32.7

-

0.15

-

104.1

-

18.0

-

129.9

-

7.3

0.20

-

100.4

-

35.8

-

312.4

-

189.8

0.25

102.0

97.4

9.8

33.3

78.0

303.1

-18.5

180.6

0.30

-

98.1

-

23.0

-

192.0

-

69.4

0.35

103.6

74.2

9.3

12.5

72.1

156.1

-24.4

33.5

0.52

113.1

-

10.0

-

65.4

-

-31.1

-

0.58

102.8

   -

13.0

-

104.7

-

8.2

-

0.64

90.8

-

8.0

-

77.5

-

-19.0

-

0.68

102.8

-

8.8

-

69.9

-

-26.6

-

0.72

90.0

-

6.8

-

65.7

-

-30.8

-

Positive control 1

93.2

63.7

37.8

77.5

426.2

2262.2

329.7

2139.6

Positive control 2

-

50.9

-

50.0

-

1353.4

-

1230.8

RCE= Relative cloning efficiency

MF = Mutant frequency

IMF = Induced mutant frequency

Conclusions:
3-(triethoxysilyl)propanethiol has been tested for mutagenicity to mammalian cells (thymidine kinase locus) in a reliable study conducted according to OECD Test Guideline 476 and in compliance with GLP. An increase in the mutant frequency was observed in mouse lymphoma L5178Y cells in the absence of metabolic activation. In addition, an increase in the number of small colonies was observed without metabolic activation, suggesting a clastogenic effect. The test substance is considered to be mutagenic without metabolic activation under the conditions of the study.
Executive summary:

The test item 3-(triethoxysilyl)propanethiol 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 experiments was based on data from the pre-experiments. In Experiment I with metabolic activation 0.70 mM and without metabolic activation 0.56 mM were selected as the highest concentrations. In Experiment II with metabolic activation 0.72 mM and without metabolic activation 0.35 mM were selected as the highest concentrations. Experiment I with and without metabolic activation and Experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

The test item was dissolved in DMSO (final concentration 1% DMSO v/v).

The test item was investigated at the following concentrations:

Experiment I

with metabolic activation:

0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70 mM

and without metabolic activation:

0.01, 0.02, 0.05, 0.10, 0.20, 0.30, 0.40, 0.48, 0.56 mM

Experiment II

with metabolic activation:

0.12, 0.25, 0.35, 0.52, 0.58, 0.64, 0.68, 0.72 mM

and without metabolic activation:

0.04, 0.08, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35 mM

Precipitation of the test item (oily droplets) was noted in the pre-Experiment with metabolic activation at concentrations of 2.5 mM and higher and without metabolic activation at concentrations of 0.2 mM and higher. In Experiment I without metabolic activation precipitation was noted at concentrations of 0.48 mM and higher.

Growth inhibition was observed in Experiment I and II with and without metabolic activation.

In Experiment I with metabolic activation the relative total growth (RTG) was 21.7% for the highest concentration (0.70 mM) evaluated. The highest concentration evaluated without metabolic activation was 0.56 mM with a RTG of 3.2%. In Experiment II with metabolic activation the relative total growth (RTG) was 11.2% for the highest concentration (0.72 mM) evaluated. The highest concentration evaluated without metabolic activation was 0.35 mM with a RTG of 4.2%. Concentrations showing extremely reduced relative total RTG values (below 10%) were not considered for evaluation of mutagenicity.

A slight dose-response relationship was observed in Experiment I without metabolic activation.

In Experiment II 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 two concentrations (0.20 mM and 0.25 mM).

Additionally, in Experiment II without metabolic activation an increase in small colonies was noted combined with mutagenicity. 

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 Guideline OPPTS 870.5300, OECD476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo mammalian cell study: DNA damage and/or repair (mammalian alkaline comet assay, oral administration): negative in cells of glandular stomach, duodenum and liver (OECD 489) (Eurofins with BSL Bioservice 2020).

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:
22 June 2020 to 03 November 2020
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 (incl. QA statement)
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Remarks:
Crl: W I(Han)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Sex: male animals were used in the main study as no differences were observed between the male and female animals used in the preliminary dose range-finding study.
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approximately 7-9 weeks old
- Weight at dosing in main experiment: 253-304 g
- Assigned to test groups randomly: yes. Prior to randomization detailed clinical observations were made to assure a good health condition. Animals showing pathological signs before administration were excluded from the study. Supplementary animals from the same delivery were provided in exchange.
- Fasting period before study: no
- Housing: The animals were kept in groups of 2-3 animals / sex / group / cage in IVC cages (type III H, polysulphone cages) on Altromin saw fibre bedding.
- Diet: Altromin 1324 maintenance diet for rats and mice ad libitum. (Altromin Spezialfutter GmbH & Co. KG Im Seelenkamp 20, D-32791 Lage)
- Water tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals, ad libitum.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: Not given in study report
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: none
- Justification for choice of solvent/vehicle: the substance hydrolyses rapidly in contact with water, so no vehicle was used
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was administered undiluted.
Duration of treatment / exposure:
2 days
Frequency of treatment:
Daily
Post exposure period:
Animals were sacrificed 4 hours after second administration of test substance
Dose / conc.:
250 mg/kg bw (total dose)
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
No. of animals per sex per dose:
Five
Control animals:
yes
Positive control(s):
- Positive control substance: ethylmethanesulfonate;
- Justification for choice of positive control(s): none given in report. This is a standard positive control substance for the in vivo mammalian alkaline comet assay
- Route of administration: oral
- Doses / concentrations: 200 mg/kg bw, single dose
Tissues and cell types examined:
Cells of the liver, glandular stomach and duodenum were examined
Details of tissue and slide preparation:
In vivo phase (BSL Bioservice)
CRITERIA FOR DOSE SELECTION: The highest dose was based on the maximum tolerated dose, defined as the highest dose that will be tolerated without evidence of study-limiting toxicity, determined in a dose range-finding study. In this preliminary study, 2 male and 2 female animals were dosed with the limit dose of 2000 mg/kg bw daily for 2 consecutive days. Clinical signs were observed in all animals, and the male and female rats were euthanised 4 hours after the second treatment due to severity of symptoms and out of consideration of animal welfare. Based on these effects, a dose of 500 mg/kg bw was administered orally on 2 consecutive days with a 24-hour interval to one male and one female animal. After the second application adequate toxicity was not observed at this dose, and a further two animals (one male, one female) were dosed orally with 1000 mg/kg bw on two consecutive days. Adequate toxicity was observed at this dose level, and no gender specific differences were determined. Based on these results, the main study was conducted with male rats and 1000 mg/kg bw, which is the limit dose according to the OECD test guideline 489, was selected as the highest dose in the main experiment.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Treatment occurred during two consecutive days. Animals were sacrificed 4 hours after the second dose. Sampling occurred after termination of the animals.

Tissue Sampling
Four hours after last treatment 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 isolated organs was preserved in 10% neutral-buffered formalin for histopathological evaluation, if considered necessary by the sponsor.
Organs assigned to 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.

Ex vivo phase
Tissue preparation:
Isolation of primary hepatocytes:
A portion of the liver was minced with a pair of scissors to isolate the cells. The cell suspension was kept 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 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 kept 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 kept 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.

DETAILS OF 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 were incubated in alkaline (pH > 13) electrophoresis solution for 20 min. After alkali unwinding, the single-stranded DNA was electrophoresed under alkaline conditions to enable the formation of DNA tails. The electrophoretic conditions were 0.7 V/cm and approximately 300 mA, with the DNA being electrophoresed for 30 min. The slides were placed in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with electrophoresis solution. Slides were placed in the electrophoresis chamber in a random order.
Neutralisation 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.

METHOD OF ANALYSIS:
Comet slides were analysed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and the Comet Software ‘Comet Assay IV’ (Perceptive Instrument, software version 2.1.2). The slides were coded so that the evaluator was not aware of which dose group was evaluated.
The calculation of the different parameters was done automatically by the Comet Software, but the set front, middle and back lines of the comet may be adjusted manually if they were not set correctly automatically. All cells of the visual field were 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 were classified into three potential categories scorable, non-scorable and “hedgehog” (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).
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) were scored. The third back-up slide was scored in case of discordant results. The %-tail intensity is the parameter for evaluation and interpretation of DNA damage, and was 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:
The assay is considered acceptable if:
- the concurrent negative control data are considered acceptable for addition to the laboratory historical control database,
- the concurrent positive controls should induce responses that are compatible to those previously generated and included in the historical positive control database and produce a statistically significant increase compared with the concurrent negative control,
- three doses and, if available, 150 cells per organ of each animal have been analysed.

A test item is considered to be clearly positive if:
- acceptability criteria are fulfilled
- 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
A test item is considered clearly negative if:
- acceptability criteria are fulfilled
- 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).
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 vehicle control.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
some signs of systemic toxicity were observed in the high dose animals
Vehicle controls validity:
not applicable
Remarks:
No vehicle was used; the negative control, 0.9% NaCl, was valid.
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000, 1000 and 500 mg/kg bw
- Solubility: not relevant
- Clinical signs of toxicity in test animals:
2000 mg/kg bw: Clinical signs were observed in all animals, and the male and femal rats were euthanised 4 hours after the second treatment due to severity of symptoms and out of consideration of animal welfare.
500 mg/kg bw: after the second application the only clinical sign left was moving bedding in both genders, which had resolved after 30 minutes.
1000 mg/kg bw: after the second application reduced spontaneous activity, piloerection, moving bedding and salivation, prone position, ataxia and chromodacryorrhea were present. 4 hours later most of the animals recovered from the symptoms. No gender specific differences were determined. Based on these results, the main study was conducted with male rats and 1000 mg/kg bw, which is the limit dose according to the OECD test guideline 489, was selected as the highest dose in the main experiment.
- Evidence of cytotoxicity in tissue analysed: not applicable
- Rationale for exposure: initial limit dose showed excessive toxicity, the second dose was too low to elicit adequate toxicity, so a third dose was administered


RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: both route of exposure and dose were appropriate
- Results and statistical evaluation: no statistically significant increase in the mean tail intensity relative to the negative control was observed in liver, duodenum and glandular stomach cells of male animals treated with the test substance.

Summary of Mean Tail intensities [%] in liver, glandular stomach and duodenum

Dose

 

Mean Tail Intensity

[%]

 

Group

Liver

Glandular stomach

Duodenum

Clinical Signs

PC

11.83*

16.86*

11.36*

None

NC

1.62

1.88

1.79

None

LD

1.69

2.68

4.16

Moving bedding, salivation

MD

2.01

2.94

3.73

Moving bedding, salivation

HD

0.68

4.44

2.35

e.g. reduced spontaneous activity, prone position, half eyelid closure, piloerection

Statistically significant trend

Yes**

No

No

/

Historic Control Range

Negative Control: 0.00° % – 6.11 %

Positive Control: 3.14 % – 31.43 %

Negative Control:

0.89 % – 8.73%

Positive Control: 4.66% - 31.23%

Negative Control:

0.00° % – 7.47%

Positive Control: 3.89% - 33.14%

/

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

**     statistically significant concentration-dependency was noted but not considered biologically relevant

°      A negative value was obtained due to the calculation of the LCL. However, as no negative tail intensities can be measured, the LCL was set to 0.

 

NC: Negative Control (0.9% NaCl)

LD: Low Dose

PC: Positive Control (Ethyl methanesulfonate: 200 mg/kg bw)

MD: Mid dose

HD: High Dose

n.a.: Not available

Conclusions:
In an in vivo mammalian alkaline comet assay conducted in accordance with OECD test guideline 489 and in compliance with GLP, 3-(triethoxysilyl)propanethiol was administered by oral gavage to groups of five male Wistar rats at doses of 250, 500 and 1000 mg/kg bw daily for two consecutive days. The test substance did not induce biologically relevant DNA-strand breaks in liver, glandular stomach and duodenum. The results of the negative control were within laboratory historical control values, and the positive control substance induced a statistically significant increase in tail intensity; the validity criteria were therefore met. It is concluded that 3-(triethoxysilyl)propanethiol is negative for the induction of DNA damage in the in vivo mammalian alkaline comet assay, under the conditions of the experiment.
Executive summary:

3-(Triethoxysilyl)propanethiol has been tested in a reliable in vivo mammalian alkaline comet assay conducted according to OECD Test Guideline 489 and in compliance with GLP. In an initial dose range-finding study toxicity was observed but there were no relevant differences between male and female animals. Based on the results of the preliminary study, the test substance was administered to male Wistar rats by oral gavage at doses of 250, 500 and 1000 mg/kg bw. The animals were dosed twice over two days. 0.9% sodium chloride was used as a negative control; the positive control was ethyl methanesulfonate, administered once by oral gavage at a dose of 200 mg/kg bw.

No signs of systemic toxicity were observed in the low and mid dose groups, except moving bedding and slight salivation in two animals per group. The animals treated with the 1000 mg/kg bw showed moving bedding, salivation, reduced spontaneous activity, prone position, piloerection, hunched posture and half eyelid closure.

The animals were sacrificed four hours after the last treatment, and cells from the liver, the glandular stomach and the duodenum were isolated, embedded in agarose, lysed and DNA allowed migrating under electrophoresis conditions. 150 cells per animal tissue were evaluated. DNA migration during electrophoresis was determined and expressed as tail intensity.

The tail intensities of the negative control group were within the historical control limits, and the mean values for the test groups were within the range of the concurrent negative control. The positive control induced a statistically significant increase in DNA damage for all evaluated organs.

In conclusion, under the experimental conditions reported in this study, the test item 3‑(triethoxysilyl)propanethiol did not induce biologically relevant DNA-strand breaks in liver, glandular stomach and duodenum after oral administration to rats.

Therefore, 3-(triethoxysilyl)propanethiol is considered to be negative for the induction of DNA damage under these experimental conditions in the in vivo mammalian alkaline comet assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro

3-(Triethoxysilyl)propanethiol has been tested for mutagenicity to bacteria in a study which was conducted according to OECD Test Guideline 471 and in compliance with GLP (BioReliance 2001). No evidence of a test substance related increase in the number of revertants was observed with or without activation in the initial or the repeat experiments. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

The potential for induction of damage to chromosomes in mammalian cells was investigated by testing 3-(triethoxysilyl)propanethiol in a study conducted according to OECD Test Guideline 473 and in compliance with GLP (BSL Bioservice 2012a). No evidence for the induction of structural or numerical chromosome aberrations was observed when the substance was tested up to cytotoxic concentrations with and without metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the substance is not clastogenic or aneugenic (does not induce structural or numerical chromosome aberrations) under the conditions of the test.

3-(Triethoxysilyl)propanethiol has been tested for mutagenicity to mammalian cells (thymidine kinase locus) in a reliable study conducted according to OECD Test Guideline 476 and in compliance with GLP (BSL Bioservice 2012b). An increase in the mutant frequency was observed in mouse lymphoma L5178Y cells in the absence of metabolic activation. In addition, an increase in the number of small colonies was observed without metabolic activation, suggesting a clastogenic effect. The test substance is considered to be mutagenic without metabolic activation under the conditions of the study.

In vivo

3-(Triethoxysilyl)propanethiol has been tested in a reliable in vivo mammalian alkaline comet assay conducted according to OECD Test Guideline 489 and in compliance with GLP (Eurofins with BSL Bioservice 2020).

In an initial dose range-finding study toxicity was observed but there were no relevant differences between male and female animals. Based on the results of the preliminary study, the test substance was administered to male Wistar rats by oral gavage at doses of 250, 500 and 1000 mg/kg bw. The animals were dosed twice over two days. 0.9% sodium chloride was used as a negative control; the positive control was ethyl methanesulfonate, administered once by oral gavage at a dose of 200 mg/kg bw.

No signs of systemic toxicity were observed in the low and mid dose groups, except moving bedding and slight salivation in two animals per group. The animals treated with the 1000 mg/kg bw showed moving bedding, salivation, reduced spontaneous activity, prone position, piloerection, hunched posture and half eyelid closure.

The animals were sacrificed four hours after the last treatment, and cells from the liver, the glandular stomach and the duodenum were isolated, embedded in agarose, lysed and the DNA was forced to migrate under electrophoresis conditions. 150 cells per animal tissue were evaluated. DNA migration during electrophoresis was determined and expressed as tail intensity.

The tail intensities of the negative control group were within the historical control limits, and the mean values for the test groups were within the range of the concurrent negative control. The positive control induced a statistically significant increase in DNA damage for all evaluated organs.

In conclusion, under the experimental conditions reported in this study, the test item 3-(triethoxysilyl)propanethiol did not induce biologically relevant DNA-strand breaks in liver, glandular stomach and duodenum after oral administration to rats.

Therefore, 3-(triethoxysilyl)propanethiol is considered to be negative for the induction of DNA damage under these experimental conditions in the in vivo mammalian alkaline comet assay.





Justification for classification or non-classification

The potential for mutagenicity indicated in the in vitro mammalian cell gene mutation assay was not confirmed in an in vivo mammalian alkaline comet assay, so no classification is required for germ cell mutagenicity for 3-(triethoxysilyl)propanethiol in accordance with Regulation (EC) No 1272/2008.