3-trimethoxysilylpropane-1-thiol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial mutagenicity: negative, with and without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or E. coli WP2 uvrA (OECD 471) (Bioreliance, 2001).

Cytogenicity in mammalian cells: negative, with and without metabolic activation in V79 cells (OECD 473) (Eurofins, 2017).

Mutagenicity in mammalian cells: negative, with and without metabolic activation in mouse lymphoma L5178Y (OECD 490) (Eurofins, 2018).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-09-01 to 2000-10-09

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

Target gene:

histidine operon (Salmonella strains)
tryptophan operon (E. coli strain)

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:

preliminary assay: 5000 µg/plate
75-5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: compatibility with the target cells

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains, + MA, 1.0 µg/plate (Salmonella) 10 µg/plate (E. coli)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98, - MA, 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:

TA 100 TA 1535, - MA, 1.0 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537, - MA, 75 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA, - MA, 1000 µg/plate

Details on test system and experimental conditions:

ACTIVATION:
S9 mix:
- 10% S9
- 5 mM glucose-6-phosphate
- 4 mM ß-nicotinamide-adenine dinucleotide phosphate
- 8 mM MgCl2
- 33 mM KCl
- 100 mM phosphate buffer (pH 7.4)

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 60±2 min at 37±2°C
- Exposure duration: 48-72 hours at 37±2°C

SELECTION AGENT (mutation assays): minimal agar

NUMBER OF REPLICATIONS: triplicate plates; 2 independent experiments

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

Evaluation criteria:

For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article.
Data sets for TA 1535 and TA 1537 were judged positive if the increase in mean revertants is ≥ 3x the mean vehicle control value.
Data sets for TA 98, TA 100 and WP2 uvrA were judged positive if the increase in mean revertants is ≥ 2x the mean vehicle control value.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: No mutagenic potential

Preliminary toxicity assay: number of revertants per plate

Concentration (µg/plate)	TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA
Vehicle	22	12	128	118	18	14	7	5	15	1
6.7	25	14	125	128	22	17	4	4	11	1
10	19	10	103	112	23	13	5	3	19	1
33	24	16	113	110	17	14	4	3	17	1
67	22	19	93	C	18	11	5	4	14	1
100	23	17	132	111	21	21	4	3	12	1
333	21	11	99	100	18	18	4	4	10	1
667	26	11	111	102	12	16	5	5	16	1
1000	25	16	99	116	19	17	4	4	14	1
3333	21	10*	98	81*	17	10*	6	2*	22	1
5000	18*	7**	64**	0***	14*	0***	2***	0***	13*	14**

* = background lawn slightly reduced

** = background lawn severely reduced

*** = background lawn extremely reduced

C = contaminated

 

Experiment B1: Number of revertants per plate (mean of 3 plates)

Concentration (µg/plate)	TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
Vehicle	20	21	92	116	12	9	5	7	10	12
100	19	26	99	115	-	13	-	7	13	10
105	-	-	-	-	10	-	6	-	-	-
333	22	29	87	119	-	10	-	5	14	12
348	-	-	-	-	12	-	6	-	-	-
1000	25	26	93	95	-	10	-	6	10	11
1045	-	-	-	-	10	-	4	-	-	-
3333	18	25	63**	79*	-	7**	-	4*	11	10
3485	-	-	-	-	9*	-	3**	-	-	-
5000	1***	7**	4***	60**	0***	1***	0****	0***	10*	10
Positive control	567	608	408	488	231	35	580	40	368	162

* = background lawn slightly reduced

** = background lawn severely reduced

*** = background lawn extremely reduced

**** = background lawn absent

Experiment B2: Number of revertants per plate (mean of 3 plates)

Concentration (µg/plate)	TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
Vehicle	13	13	85	97	6	8	4	8	11	10
75	13	15	86	108	9	7	3	4	10	12
200	15	19	89	123	9	7	3	5	11	15
600	12	16	98	85	10	7	4	4	11	10
1800	10*	16	78*	100	3**	9	3*	4	9*	11
5000	0***	0***	0***	0***	0***	8**	0***	1***	7**	10*
Positive control	308	258	311	385	94	39	218	45	266	57

* = background lawn slightly reduced

** = background lawn severely reduced

*** = background lawn extremely reduced

Conclusions:

3-Trimethoxysilylpropane-1-thiol has been tested in a valid bacterial reverse mutation assay, according to the OECD TG 471 (1989), and under GLP, using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or E.coli WP2 uvrA.
No increase in the number of revertants was observed in any test strain, with or without metabolic activation. Appropriate positive and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 February 2017 to 22 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: chromosome aberration in mammalian cells

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature. The sample was recovered with nitrogen after opening and sealed with parafilm.
- Stability under test conditions: Unstable after repeated contact to air. Minimise contact with moisture in air. Close container immediately after use.
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was stable in DMSO. The solvent was compatible with the survival of the cells and the S9 activity.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: The test substance was stable in DMSO.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: DMSO was used as solvent. From the highest test item stock solution (1 M) separate dosing solutions of the test item were prepared for each of the concentrations by serial dilution. From these DMSO dosing solutions 1 % (v/v) was added to the cells in MEM cell culture medium. The pH value was found to be within the physiological range (pH 7.0 ± 0.4).
- Preliminary purification step (if any): None
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Eurofins, Germany
- Suitability of cells: widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens
- Cell cycle length, doubling time or proliferation index: 12-14 hours
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable: The V79 cells were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich, as large stock cultures allowing the repeated use of the same cell culture batch in experiments.
- Modal number of chromosomes: not applicable
- Normal (negative control) cell cycle time: not applicable

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: in 75 cm2 cell culture plastic flasks at 37 °C in a 5% carbon dioxide atmosphere (95% air)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw)

Test concentrations with justification for top dose:

A pre-experiment was conducted under identical conditions as described for the main experiment. The following concentrations were tested without and with S9 mix:
0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.5, 5, 7.5, 10 mM. On the basis of the data and the observations from the pre-experiment and taking into account the recommendations of the guidelines, the following concentrations were selected for the main experiment:
Experiment I: 0.5, 1.0, 1.2, 1.3, 1.4, 1.5, 1.7 mM (+S9 mix) and 0.25, 0.5, 0.7, 0.8, 1.0, 1.2, 1.3, 1.5 mM (-S9 mix); repetition of Experiment I: 0, 0.9, 1.1, 1.3 and 1.5 mM (-S9 mix)
Experiment II: 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.1, 1.25, 1.5 mM (-S9 mix).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 5 x 105 cells per flask were seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and subcultures were made 3-4 days after seeding. About 1 x 104 cells/mL were seeded into cell culture flasks with complete culture medium.

ACTIVATION: An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-phosphate-buffer pH 7.4.


DURATION
- Exposure duration: Experiment I, with and without metabolic activation 4 hours exposure; repeat of Experiment I, without metabolic activation 4 hours exposure; Experiment II, without metabolic activation 21 hours exposure. In Experiment I without metabolic activation an increase in cells with aberrations was observed, which was considered not biologically relevant due to variation between cultures, so this experiment was repeated. A different batch of test material test was used in the repeat experiment I as the first batch had expired.
- Expression time (cells in growth medium): 17 hour exposure duration for Experiment I. Cell were prepared immediately after exposure in Experiment II.
- Selection time (if incubation with a selection agent): 2.5 hours incubation with Colcemid
- Fixation time (start of exposure up to fixation or harvest of cells): at 21 hours after start of treatment.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cell cultures.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 600

DETERMINATION OF CYTOTOXICITY
- Method: relative increase in cell count (RICC)
- Any supplementary information relevant to cytotoxicity: not applicable

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not applicable

Rationale for test conditions:

A pre-experiment was conducted under identical conditions as described for the main experiment. On the basis of the data and the observations from the pre experiment and taking into account the recommendations of the guidelines, the test concentrations were selected.

Evaluation criteria:

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data

Statistics:

Fisher´s exact test was performed to verify the results in the experiment.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1.2 mM and higher (-/+ S9 mix) for Experiment , at 1.1 mM and higher (-S9) for Experiment I repetition, and at 0.6 mM and higher (- S9 mix) for Experiment II

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Evaporation from medium: not reported
- Water solubility: not reported
- Precipitation: No precipitation of the test item was noted without and with metabolic activation after the incubation in experiment I and II.
- Definition of acceptable cells for analysis: not reported
- Other confounding effects: not reported

RANGE-FINDING/SCREENING STUDIES: According to the guidelines the highest recommended concentration was 10 mM. The test item was dissolved in DMSO. No precipitation of the test item was noted. The highest dose group evaluated in the pre-experiment was 1 mM. The relative increase in cell count (RICC) was used as parameter for toxicity. The concentrations tested in the main experiment are based on the results obtained in the pre-experiment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Historical laboratory control data were presented with ranges in the study report. Results for positive control were within ranges of historical positive control data.
- Negative (solvent/vehicle) historical control data: Historical laboratory control data were presented with ranges in the study report. Results for negative control were within ranges of historical positive control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC
- Other observations when applicable: No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

Remarks on result:

other: See "Remarks"

Remarks:

In experiment I without metabolic activation an increase of aberrant cells above the historic control range was noted at concentrations of 1.0 mM and higher. These results varied between the duplicate cultures and the mean increase was not reflected by both cultures. These inductions were considered to be not biologically relevant. To verify the outcome of the first experiment without metabolic activation, a repetition experiment was performed. No biologically relevant increase above the historic control range was determined after incubation with the test item in the same concentration range used in experiment I (without metabolic activation).

Table 1:  Summary: Experiment I, 4 -hour exposure without metabolic activation

Dose Group	Concentration [mM]	RICC [%]	Mean % Aberrant Cells	Mean % Aberrant Cells	Historical Laboratory Negative Control Range (LCL, UCL)	Precipi-tation^a	Statistical Signifi-cance^b
			incl. Gaps	excl. Gaps
C	0	92	2.3	1.0		-	-
S	0	100	4.7	1.7		-	-
1	0.5	103	3.3	2.0	- 0.28 - 3.7% aberrant cells, excl. gaps	-	-
2	1	75	6.5	4.2		-	-
3	1.2	41	8.0	4.5		-	+
EMS	600 µg/mL	78	9.7	6.0		-	+

Table 2:  Summary: Experiment I, 4 -hour exposure with metabolic activation

Dose Group	Concentration [mM]	RICC [%]	Mean % Aberrant Cells	Mean % Aberrant Cells	Historical Laboratory Negative Control Range (LCL, UCL)	Precipi-tation^a	Statistical Signifi-cance^b
			incl. Gaps	excl. Gaps
C	0	94	3.0	1.0		-	-
S	0	100	5.0	2.3		-	-
4	0.8	84	3.3	1.7	- 0.23% -3.95%aberrant cells, excl. gaps	-	-
5	1	76	3.7	2.7		-	-
6	1.2	47	5.7	3.3		-	-
CPA	1.11 µg/mL	67	50.9	50.9		-	+

Table 3: Experiment I (repetition), 4 -hour exposure without metabolic activation

Dose Group	Concentration [mM]	RICC [%]	Mean % Aberrant Cells	Mean % Aberrant Cells	Historical Laboratory Negative Control Range (LCL, UCL)	Precipitation	Statistical Signifi-canceb
			incl. Gaps	excl. Gaps
C	0	111	2.3	1.3		-	-
S	0	100	2.0	0.7		-	-
3	0.9	96	1.0	0.0		-	-
5	1.1	69	2.0	1.3	-0.28 - 3.7% aberrant cells, excl. gaps	-	-
7	1.3	69	1.0	1.0		-	-
9	1.5	35	0.0	0.0		-	-
EMS	900 µg/mL	77	7.3	5.7		-	+

Table 3:  Summary: Experiment II, 21 -hour exposure without metabolic activation

Dose Group	Concentration [mM]	RICC [%]	Mean % Aberrant Cells	Mean % Aberrant Cells	Historical Laboratory Negative Control Range (LCL, UCL)	Precipi-tation^a	Statistical Signifi-cance^b
			incl. Gaps	excl. Gaps
C	0	101	3.0	2.7		-	-
S	0	100	2.0	1.3		-	-
2	0.2	91	1.3	0.3	- 0.20% - 2.71% aberrant cells, excl. gaps	-	-
3	0.4	80	1.3	0.0		-	-
4	0.6	41	2.0	1.0		-	-
EMS	400 µg/mL	62	13.0	11.0		-	+

C:  Negative Control (Culture Medium)

S:   Solvent Control (DMSO)

EMS: Ethylmethanesulfonate

CPA:  Cyclophosphamide

RICC:    Relative Increase in Cell Count, calculated by the increase in cell number of the test groups compared to the solvent control groups.

Conclusions:

3-Trimethoxysilylpropane-1-thiol has been tested for ability to cause chromosome aberrations in Chinese hamster V79 cells according to OECD TG 473 and under GLP (Eurofins, 2018). No increase in the number of cells with aberrations was observed in experiment I with metabolic activation, 4-hour exposure, or in experiment 2 without activation, 21-hour exposure. An increase of aberrant cells above the historical control range was observed in experiment I without metabolic activation, 4-hour exposure, at concentrations of 1.0 mM and higher. These results varied between the duplicate cultures and the mean increase was not reflected by both cultures. These inductions were considered to be not biologically relevant. To verify the outcome of the first experiment without metabolic activation, a repetition experiment was performed. In the repeat of experiment I without metabolic activation, no biologically relevant increase above the historical control range was observed. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of this study.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8th November 2017 to 21 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: gene mutation in mammalian cells

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: unstable after repeated contact to air
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was diluted in DMSO. The vehicle was chosen based on pre-experiment for solubility. The solvent was compatible with the survival of the cells and the S9 activity.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was diluted in DMSO. The vehicle was chosen based on pre-experiment for solubility. The solvent was compatible with the survival of the cells and the S9 activity.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Target gene:

They are heterozygous at the Thymidine Kinase (TK) locus in order to detect mutation events at the TK- locus.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Eurofins Munich stock cultures
- Suitability of cells: recommended in Test Guideline
- Cell cycle length, doubling time or proliferation index: 10-12 h, cloning efficiency, usually more than 50%
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable: Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.
- Modal number of chromosomes: a near diploid karyotype (40 +/- 2 chromosomes)
- Normal (negative control) cell cycle time: not applicable

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: not specified
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) induced rat live S9

Test concentrations with justification for top dose:

without metabolic activation: 0.1, 0.2, 0.6 and 0.7 mM
with metabolic activation: 0.1, 0.2, 0.6, 0.7 and 0.8 mM
The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 10 mM. Six concentrations [0.2, 0.5, 2.5, 5.0, 7.5 and 10 mM] were tested without and with metabolic activation. Dose groups that were not evaluable due to very high toxicity and precipitation of the test item are not reported.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The vehicle was chosen based on pre-experiment for solubility. The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1 x 10⁷ cells

ACTIVATION: S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations below: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP n 100 mM sodium-phosphate-buffer pH 7.4.


DURATION
- Preincubation period:
- Exposure duration: Short-term exposure of 4 hours, with and without metabolic activation
- Expression time (cells in growth medium): 2 days in total at 37 °C in 5% CO2/95% humidified air after 4-hour exposure
- Selection time (if incubation with a selection agent): 2000 cells/well in 200 µL selective medium with TFT incubated for about 12 days at 37 °C in 5% CO2/95% humidified air.
- Fixation time (start of exposure up to fixation or harvest of cells): at the end of expression time

SELECTION AGENT (mutation assays): TFT

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: The cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated for at least 6 days at 37 °C in a humidified atmosphere with 5% CO2.mitotic index.
- Any supplementary information relevant to cytotoxicity: not specified

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not applicable

Rationale for test conditions:

The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 10 mM. Six concentrations [0.2, 0.5, 2.5, 5.0, 7.5 and 10 mM] were tested without and with metabolic activation. Dose groups that were not evaluable due to very high toxicity and precipitation of the test item are not reported.

Evaluation criteria:

The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- a dose-dependent increase in mutant frequency is detected.

Statistics:

Statistical significance

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest concentration tested.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: within the physiological range
- Effects of osmolality: within the physiological range
- Evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: No precipitation of the test item was noted in the pre-experiment / main experiment.
- Definition of acceptable cells for analysis: not specified
- Other confounding effects: not specified

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 10 mM. Six concentrations [0.2, 0.5, 2.5, 5.0, 7.5 and 10 mM] were tested without and with metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: within ranges of historical positive control data
- Negative (solvent/vehicle) historical control data: within ranges of historical negative control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Growth inhibition was observed in the experiment without and with metabolic activation. The relative total growth (RTG) was 13.8% (without metabolic activation) and 12.0% (with metabolic activation) for the highest concentration evaluated.

- Other observations when applicable: Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. An extension of the GEF by the induced mutant frequency in combination with an increased occurrence of small colonies is an indication for potential clastogenic effects and/or chromosomal aberrations. Thus based on the non-mutagenic effects of 3-trimethoxysilylpropane-1-thiol (CAS 4420-74-0), an assessment of clastogenicity was not feasible.

Table 1: Pre-Experiment for Toxicity, without metabolic activation

Test Group	Concen-tration [mM]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]
C1	0	242000	930000	1370000	12.7	106.0
C2	0	334000	1090000	1170000	12.8	106.1
S1	0	295000	924000	1330000	12.3	100.0
S2	0	317000	1050000	1120000	11.8	100.0
1	0.2	278000	894000	1100000	9.8	81.8
2	0.5	298000	828000	1130000	9.4	77.8
3	2.5	82200	23300	11800	0.0	0.3

C:  Negative control

S:  Solvent control(1% DMSO)

a:  Suspension Growth, SG = [((value 24h x 30) / 1x10⁷) x ((value 48 h x 20) / (value 24 h*x20))]; : for value 24 h > 3x10⁵then value 24 h = 3x10⁵

b:  Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

Table 2: Pre-Experiment for Toxicity, with metabolic activation

Test Group	Concen-tration [mM]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]
C1	0	246000	800000	1490000	11.9	86.4
C2	0	278000	893000	1420000	12.7	91.9
S1	0	316000	962000	1470000	14.1	100.0
S2	0	290000	948000	1420000	13.5	100.0
1	0.2	291000	907000	1600000	14.5	105.1
2	0.5	281000	798000	1410000	11.3	81.5
3	2.5	114000	46000	33500	0.1	0.7

C: Negative control

S: Solvent control(1% DMSO)

a: Suspension Growth, SG = [((value 24h x 30) / 1x10⁷) x ((value 48 h x 20) / (value 24 h*x20))];: for value 24 h > 3x10⁵then value 24 h = 3x10⁵

b:  Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

Table 3: Summary: Main Experiment, without and with metabolic activation

Test Group	Conc.  [mM]	RCE^a[%]	RTG^b[%]	MF^c[mutants/ 10^6cells]	IMF^d[mutants/ 10^6cells]	GEF^eexceeded	Statistical Significant Increase^f	Precipitate
Exposure without S9
C1	0	104.0	120.3	59.4	/	/	/	-
C2	0	129.6	142.1	59.4	/	/	/	-
S1	0	100.0	100.0	73.2	/	/	/	-
S2	0	100.0	100.0	73.2	/	/	/	-
1	0.1	117.6	118.5	61.0	-12.2	-	-	-
2	0.2	97.4	99.7	76.2	3.0	-	-	-
3	0.6	97.4	35.6	70.1	-3.1	-	-	-
4	0.7	90.0	13.8	56.6	-16.6	-	-	-
EMS	300 µg/mL	92.8	89.6	939.9	866.7	+	+	-
MMS	10 µg/mL	74.2	74.4	804.3	731.1	+	+	-
Exposure with S9
C1	0	125.2	118.8	65.1	/	/	/	-
C2	0	98.8	105.0	65.1	/	/	/	-
S1	0	100.0	100.0	64.0	/	/	/	-
S2	0	100.0	100.0	64.0	/	/	/	-
1	0.1	102.0	104.7	86.7	22.7	-	+	-
2	0.2	112.6	113.8	89.0	25.0	-	-	-
3	0.6	105.3	47.6	61.7	-2.3	-	-	-
4	0.7	73.5	21.2	61.8	-2.2	-	-	-
5	0.8	76.7	12.0	73.1	9.1	-	-	-
B[a]P	1.5 µg/mL	91.5	66.7	668.9	604.9	+	+	-

C: Negative Controls

S: Solvent Controls (1% DMSO)

a: Relative Cloning Efficiency, RCE = [(CE dose group / CE of corresponding controls) x 100]

Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

b: Relative Total Growth, RTG = (RSG x RCE)/100

c: Mutant Frequency,

MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

d: Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

e: Global Evaluation Factor, GEF (126); +:  GEF exceeded, -: GEF not exceeded

f: statistical significant increase in mutant frequency compared to solvent controls (Mann Whitney test, p<0.05).

+: significant; -not significant

EMS: Ethylmethanesulfonate

MMS: Methylmethanesulfonate

B[a]P: Benzo[a]pyrene

Conclusions:

3-Trimethoxysilylpropane-1-thiol (CAS 4420-74-0) has been tested for mutagenicity to mammalian cells in Mouse lymphoma L5178Y cells according to OECD TG 490, and under GLP (Eurofins, 2018). No test-substance induced increase in the number of mutations was observed when tested up to precipitating concentration. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

3 -Trimethoxysilylpropane-1-thiol has been tested in a valid bacterial reverse mutation assay, according to the OECD TG 471 (1997), and under GLP, using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or E. coli WP2 uvrA (Bioreliance, 2001).

No increase in the number of revertants was observed in any test strain, with or without metabolic activation. Appropriate positive and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

In another bacterial mutagenicity study (Evonik, 1989), which was comparable to guideline, a positive result was reported in S. typhimurium TA 1538 only, with and without metabolic activation. These results, however, are not supported by the key study (BioReliance, 2001) and another two reliable bacterial mutagenicity studies (Wacker, 1990; Evonik, 1988). Therefore, the test substance is considered to be negative for mutagenicity to bacteria.

3-Trimethoxysilylpropane-1-thiol has been tested for ability to cause chromosome aberrations in Chinese hamster V79 cells according to OECD TG 473 and under GLP (Eurofins, 2018). No increase in the number of cells with aberrations was observed in experiment I with metabolic activation, 4-hour exposure, or in experiment 2 without activation, 21-hour exposure. An increase of aberrant cells above the historical control range was observed in experiment I without metabolic activation, 4-hour exposure, at concentrations of 1.0 mM and higher. These results varied between the duplicate cultures and the mean increase was not reflected by both cultures. These inductions were considered to be not biologically relevant. To verify the outcome of the first experiment without metabolic activation, a repetition experiment was performed. In the repeat of experiment I without metabolic activation, no biologically relevant increase above the historical control range was observed. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of this study.

3-Trimethoxysilylpropane-1-thiol (CAS 4420-74-0) has been tested for mutagenicity to mammalian cells in mouse lymphoma L5178Y cells according to OECD TG 490, and under GLP (Eurofins, 2018). No test-substance induced increase in the number of mutations was observed when tested up to precipitating concentration. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study.

Justification for classification or non-classification

Based on the available data, no classification for mutagenicity is required for 3-trimethoxysilylpropane-1-thiol according to Regulation (EC) No 1272/2008.