Trimethoxypropylsilane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (bacterial reverse mutation assay): negative with and without metabolic activation in S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 (according to OECD 471, 1983)

Mammalian cytogenicity (chromosome aberration): negative with and without metabolic activation in cultured peripheral human lymphocytes (according to OECD 473, 2016)

Gene mutation (mammalian cells): negative with and without metabolic activation in L5178Y/TK+/- -3.7.2C mouse lymphoma cells (according to OECD 490, 2016)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Mar - 24 May 2022

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:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

TK locus

Species / strain / cell type:

mammalian cell line, other: L5178Y/TK+/- -3.7.2C mouse lymphoma cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells (American Type Culture Collection, Manassas, USA).
- Suitability of cells: recommended test system in OECD TG 490.

For cell lines:
- Absence of Mycoplasma contamination: yes, the stock cultures were checked for mycoplasma contamination.
- Methods for maintenance in cell culture: stock cultures of the cells were stored in the ultra-low freezer set to maintain -150°C. Cell density was kept below 1E+6 cells/mL.
- Periodically ‘cleansed’ of spontaneous mutants: yes, prior to testing, the mouse lymphoma cells were grown for 1 day in growth medium containing 1E-4 M hypoxanthine, 2E-7 M aminopterine and 1.6E-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on growth medium containing hypoxanthine and thymidine only. After this period cells were returned to growth medium for at least 1 day before starting the experiment.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Basic medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum.
Exposure medium: cells were exposed to the test material in basic medium supplemented with 5% to 10% (v/v) heat-inactivated horse serum.
Selective medium: selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).
Non-selective medium: non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum.

All incubations were carried out in a humid atmosphere (80 - 100%, actual range 40.8 – 103.0%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 33.8 – 37.7°C).

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH, Giessen, Germany (S9 homogenate prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight)).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPES. The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix. The concentration of the S9-fraction in the exposure medium was 4% (v/v).

Test concentrations with justification for top dose:

Experiment 1: 4, 8, 16, 31, 63, 125, 250, 500 and 1000 μg/mL (3 hour exposure with and without S9)
Experiment 2: 4, 8, 16, 31, 63 and 125 µg/mL (24 hour exposure without S9)

Concentrations for the main experiments were selected based on the results of a dose range finding study.

For Experiment 1, initial concentrations of 0.5, 1, 2, 4, 8, 16, 31, 63, 125, 250 and 500 μg/mL were tested, however, since none of the dose levels showed precipitation in the exposure medium an additional experiment was performed with concentrations of 1000 and 1643 μg/mL. Both of the additional concentrations precipitated, therefore, the concentrations selected for determination of mutant frequency were 4, 8, 16, 31, 63, 125, 250, 500 and 1000 μg/mL.

For Experiment 2, concentrations of 4, 8, 16, 31, 63, 125, 250, 500, 1000 and 1643 µg/mL were tested. Precipitation was observed from 63 μg/mL, therefore, the concentrations selected for determination of mutant frequency were 4, 8, 16, 31, 63 and 125 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dried tetrahydrofuran (THF)

- Justification for choice of solvent/vehicle: the test material formed a clear colourless solution in dried THF.

- Justification for percentage of solvent in the final culture medium: the final concentration of the solvent in the exposure medium was 0.25% (v/v), therefore, not exceeding 1% for an organic solvent, as per the OECD guideline requirement.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dried THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single cultures for the test item and positive controls; duplicate cultures for the solvent control
- Number of independent experiments: 3 (Experiment 1 + additional experiment and Experiment 2)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 8E+6 cells (1E+6 cells/mL for 3 hour treatment) or 6E+6 cells (1.25E+5 cells/mL for 24 hour treatment)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Experiment 1 + additional experiment: 3 hours (with and without S9)
Experiment 2: 24 hours (without S9)
- Harvest time after the end of treatment: immediately after treatment

FOR GENE MUTATION:
- Expression time: 2 days
- Selection time: 11 days
- Fixation time: 13 days
- Method used: microwell plates for the mouse lymphoma assay
- If a selective agent is used: 5 µg/mL trifluorothymidine (TFT)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: for determination of the cloning efficiency (CEday2) the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutant frequency (MF) a total number of 9.6E+5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6E+5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 days.
After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
- Criteria for small (slow growing) and large (fast growing) colonies: the small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative suspension growth (RSG); relative total growth (RTG); relative cloning efficiency (RCE)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: mutant frequency (MF)

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutant frequency was evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test material is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF (controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test material is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test material is considered negative (not mutagenic) in the mutation assay if none of the tested concentrations reaches a mutant frequency of MF (controls) + 126.

Species / strain:

mammalian cell line, other: L5178Y/TK+/- -3.7.2C mouse lymphoma cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

In Experiment 1, precipitation was observed at the highest concentration tested (1000 µg/mL). In Experiment 2, precipitation was observed at the two highest concentrations tested (62.5 and 125 µg/mL).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE RANGE-FINDING STUDY:
In the dose-range finding test, L5178Y mouse lymphoma cells were initially treated with a test material concentration range of 125 to 1643 µg/mL in the absence of S9-mix with 3- and 24-hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.

After 3 and 24 hours, the test material precipitated in the exposure medium at all concentrations and therefore the experiment was rejected. The dose range finding test was repeated with a test material concentration range of 3.9 to 125 µg/mL. No precipitate was observed in the exposure medium after 3 and 24 hours up to and including the top dose level of 125 µg/mL.

The pH and osmolarity at a concentration of 125 μg/mL were 7.1 and 0.359 Osm/kg, respectively (compared to 7.1 and 0.358 Osm/kg in the solvent control).

Table 1 (attached background material) shows the cell counts of the cultures from the 3 hours of treatment with various concentrations of the test material after 24 and 48 hours of subculture, the calculated suspension growth and the relative suspension growth. Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test material concentration of 125 μg/mL compared to the solvent control.

Table 2 (attached background material) shows the cell counts of the cultures after 24 hours of treatment with various concentrations of the test material and after 24 hours of subculture and the calculated suspension growth and the relative suspension growth.
No toxicity in the relative suspension growth was observed up to test material concentrations of 125 μg/mL compared to the solvent control.

MUTATION EXPERIMENT
Summary tables presented in the field "any other information on results incl. tables" show the percentages of cell survival and the mutation frequencies for various concentrations of the test material. Individual colony counts of cloning and selective plates and cell counts during sub-culturing are listed in Table 7 to Table 11 (attached background material).

First Mutagenicity Test:
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test:
Without and with S9-mix: 0.5, 1, 2, 4, 8, 16, 31, 63, 125, 250 and 500 μg/mL.

Since none of the dose levels showed precipitate, an additional experiment was performed with the dose levels of 1000 and 1643 μg/mL. Both dose levels precipitated in the culture medium after 3 hours exposure time.

Evaluation of toxicity: no significant toxicity was observed, the dose levels selected to measure mutant frequencies at the TK-locus were:
- Initial experiment: 4, 8, 16, 31, 63, 125, 250 and 500 μg/mL.
- Additional experiment: 1000 μg/mL.

Evaluation of mutagenicity: no biologically relevant increase in the mutant frequency at the TK locus was observed after treatment with the test material either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second Mutagenicity Test:
Based on the results of the dose-range finding test and Experiment 1, the following dose levels were selected for mutagenicity testing: 4, 8, 16, 31, 63, 125, 250, 500, 1000 and 1643 µg/mL.

The test material precipitated from 63 µg/mL in the exposure medium. The test material was tested beyond the limit of the solubility to obtain adequate mutagenicity data.

Evaluation of toxicity: no toxicity was observed. The dose levels selected to measure mutant frequencies at the TK-locus were: 4, 8, 16, 31, 63 and 125 µg/mL.

Evaluation of mutagenicity: no biologically relevant increase in the mutant frequency at the TK locus was observed after treatment with the test material. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Acceptability Criteria:
- The absolute cloning efficiency of the solvent controls (CEday2) was between 65 and 120%.
- The mean mutant frequency found in the solvent control cultures was within the range of the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database (See Table 5, attached background material).
- Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutant frequency. In addition, the mutant frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database (see Table 6, attached background material). It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
- The suspension growth over the two-day expression period for cultures treated with dried THF was between 11 and 17 (3-hour treatment) and 113 and 121 (24-hour treatment) (See Tables 7, 8 and 10, attached background material).

HISTORICAL CONTROL DATA
- see attached background information

Experiment 1 - 3 h Exposure - Without Metabolic Activation

Concentration [µg/mL]	Relative suspension growth [%]	CEday2 [%]	Relative cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Small colony	Large colony
0 (dried THF)	100	88	100	100	130	39	84
0 (dried THF)		89			79	26	50
0 (dried THF)*	100	104	100	100	99	55	39
0 (dried THF)*		79			144	80	56
4	105	105	119	125	87	32	51
8	116	68	77	90	141	49	86
16	103	35	40	41	166	30	133
31	96	81	92	89	79	19	58
63	77	94	107	83	88	45	39
125	89	69	79	70	137	45	86
250	88	105	119	106	88	27	57
500	106	50	57	60	178	77	93
1000*^#	85	88	96	82	124	54	64
MMS, 15	79	29	33	26	673	338	302
MMS, 15*	82	58	63	52	1049	383	512

MMS = methylmethanesulfonate

* = data from the additional experiment

^# = test item precipitation in exposure medium

 

Experiment 1 - 3 h Exposure - With Metabolic Activation

Concentration [µg/mL]	Relative suspension growth [%]	CEday2 [%]	Relative cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Small colony	Large colony
0 (dried THF)	100	116	100	100	60	18	40
0 (dried THF)		94			68	23	44
0 (dried THF)*	100	70	100	100	131	70	55
0 (dried THF)*		88			108	59	45
4	112	105	100	112	77	23	51
8	102	91	87	89	74	28	44
16	109	98	93	102	58	24	33
31	65	102	97	64	73	22	49
63	74	89	84	63	87	30	54
125	80	116	111	89	74	20	51
250	104	107	102	105	66	27	37
500	88	89	84	75	85	34	48
1000*^#	67	81	103	69	131	51	73
CP, 5	70	40	38	27	1311	484	661
CP, 5*	27	62	79	21	1003	297	563

CP = cyclophosphamide

* = data from the additional experiment

^# = test item precipitation in exposure medium

 

Experiment 2 - 24 h Exposure - Without Metabolic Activation

Concentration [µg/mL]	Relative suspension growth [%]	CEday2 [%]	Relative cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Small colony	Large colony
0 (dried THF)	100	118	100	100	45	6	38
0 (dried THF)		94			59	8	50
4	108	78	74	80	59	11	47
8	101	97	91	92	46	14	31
16	102	94	89	91	52	17	34
31	91	108	102	93	62	17	43
63^#	104	97	91	95	59	9	50
125^#	99	88	83	82	74	11	61
MMS, 5	96	95	90	86	373	81	267

MMS = methylmethanesulfonate

^# = test item precipitation in exposure medium

Conclusions:

Trimethoxy(propyl)silane has been tested for gene mutation in L5178Y/TK+/- -3.7.2C mouse lymphoma cells, in a study which was conducted according to OECD 490 and in compliance with GLP. No evidence of a biologically relevant increase in the mutant frequency was observed with or without metabolic activation in short- and long-term treatments. In conclusion, trimethoxy(propyl)silane was not mutagenic in the TK mutation test system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-01-31 to 1996-02-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

yes

Remarks:

deviations to current OECD TG 471: only 2-aminoanthracene used as +S9 positive control and no TA 102 or E.coli strains used.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his Operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: rfa, uvrB (TA 98 and 100: pKM101)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
- source of S9: Cytotest Cell Research GmBH & Co. KG, Roβdorf, Germany
- method of preparation of S9 mix: the S9 mix was freshly prepared before use. One part of S9 fraction was mixed with 9 parts of a cofactor solution resulting in the following mixture: 10% S9 fraction, 33 mM KCl, 8 mM MgCl2, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM Na2HPO4/NaH2PO4 (pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium:
Plate incorporation: 0.5 mL S9 mix was added to 2 mL top agar, 0.1 mL of cell suspension and 0.1 mL test material (or solvent or positive control), giving a final concentration of approximately 2% S9.
Pre-incubation: 0.5 mL S9 mix was mixed with 0.05 mL test material (or solvent or positive control) and 0.1 mL of cell suspension, then incubated for 30 min at 30 °C. At the end of the incubation period 2 mL top agar was added, giving a final concentration of approximately 2% S9.

Test concentrations with justification for top dose:

50 - 5000 µg/plate

The maximum concentration tested (5000 µg/plate) was based on recommendations in OECD TG 471.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: It is assumed by the reviewer that the solvent was chosen for its solubility properties and relative non-toxicity to bacteria.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE: in agar (plate incorporation); preincubation

DURATION
- Pre-incubation period: 30 minutes
- Incubation time: 72 hours

NUMBER OF REPLICATIONS: 3 plates per test concentration in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: background lawn assessment

METHODS FOR MEASUREMENTS OF GENOTOXICITY
- Method: number of revertant colonies vs. solvent control

Evaluation criteria:

For a test substance to be considered positive, it must cause at least a doubling in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test article.
The increase must be accompanied by a dose response towards increasing concentrations of the test article.

Validity criteria:
- in the solvent control, each tester strain culture must exhibit a characteristic mean number of spontaneous revertants.
- to ensure that appropriate numbers of bacteria are plated, overnight culture titers must be in excess of 10E+8 bacteria/mL.
- the mean of each positive control must exhibit a significant increase in the number of revertants over the mean value of the respective vehicle control.
- normally, at least four non-toxic dose levels are required to evaluate the assay data. Exceptions from this requirement, however, may be justified.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg/plate (pre-incubation method) with and without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg/plate (pre-incubation method) with and without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg/plate (pre-incubation method) with and without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg/plate (pre-incubation method) with and without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Titres of the overnight cultures
All bacterial titers of the overnight cultures were in excess of 10E+8 bacteria/mL.

Mutagenicity test
Summary results of the Salmonella reverse mutation assay are presented in the field “Any other information on results incl. tables”, full study results are attached as background material.
In the plate incorporation test with TA1537, all plates treated with either the test compound or controls revealed a total absence of the bacterial background lawn. This test was therefore not accepted by the study director and was repeated.
In the pre-incubation test, a reduced growth or total absence of the bacterial background lawn was observed at the top concentration of the test compound (5000 µg/plate). Bacterial growth at the 4 lower dose levels was, however, not significantly affected by the test compound.
All four bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without metabolic activation by S9 mix.
Negative (solvent) controls were also tested with each strain, and the mean numbers of spontaneous revertants were considered acceptable.
In both experiments, no indication of test compound induced mutagenicity was observed with either one of the four tester strains TA98, TA100, TA1535 and TA1537, with or without metabolic activation. With strain TA1535 in the plate incorporation test without S9 mix, the lowest test compound concentration of 50 µg/plate resulted in a revertant frequency being 2 times above that of the DMSO reference. As this effect was not accompanied by any kind of dose response relationship and was not repeated in the pre-incubation test, it is considered to have occurred by chance, being not indicative of a mutagenic effect.
All criteria for a valid study were met as described.

Table 1: Experiment 1 Plate incorporation assay - Number of revertants per plate (mean of 3 plates)

	TA98			TA100			TA1535
Conc. µg/plate	— MA	+ MA	Cytotoxic (yes/no)	—  MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	21	42	No	91	109	No	8	11	No
50	24	46	No	141	121	No	16	11	No
160	20	45	No	134	131	No	8	10	No
500	25	36	No	113	134	No	7	16	No
1600	25	39	No	130	135	No	9	11	No
5000	30	31	No	137	115	No	9	15	No
Positive Control	142	1324	No	544	1474	No	322	195	No

*solvent control with DMSO

 

Table 1 Cont.: Experiment 1 Plate incorporation assay - Number of revertants per plate (mean of 3 plates)

	TA1537
Conc. µg/plate	— MA	+ MA	Cytotoxic (yes/no)
0*	2	23.4	No
50	6	14	No
160	8	16	No
500	9	9	No
1600	6	13	No
5000	2	16	No
Positive Control	3**	152	No

*solvent control with DMSO

** this control appears to be not valid, because outside range of historical controls, though study report states “All criteria for a valid study were met”

 

Table 2: Experiment 2 Pre incubation assay - Number of revertants per plate (mean of 3 plates)

	TA98			TA100			TA1535
Conc. µg/plate	— MA	+ MA	Cytotoxic (yes/no)	—  MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	23	35	No	147	119	No	9	11	No
50	19	38	No	108	111	No	6	13	No
160	20	29	No	110	137	No	6	13	No
500	18	25	No	104	137	No	6	11	No
1600	21	32	No	123	148	No	6	13	No
5000	6	12	Yes	48	76	Yes	0	1	Yes
Positive Control	110	1264	No	527	1466	No	306	220	No

*solvent control with DMSO

 

Table 2 Cont.: Experiment 2 Pre incubation assay Number of revertants per plate (mean of 3 plates)

	TA1537
Conc. µg/plate	— MA	+ MA	Cytotoxic (yes/no)
0*	5	15	No
50	6	10	No
160	5	11	No
500	5	9	No
1600	3	12	No
5000	0	5	Yes
Positive Control	100	164	No

*solvent control with DMSO

Conclusions:

In a bacterial mutagenicity assay according to OECD 471 and GLP, no mutagenic effect was observed for the test substance tested up to cytotoxic concentration in any of the test strains in two independent experiments without and with metabolic activation. The test substance is non-mutagenic in the test strains used.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Nov 2021 - 07 Jan 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: blood was collected from healthy adult, non-smoking volunteers
- Normal cell cycle time (negative control): average generation time (AGT) 12.7 - 12.9 h

For lymphocytes:
- Sex, age and number of blood donors: sex not specified; dose range finder (31 years), first cytogenetic study (31 years), second cytogenetic study (30 years)
- Whether whole blood or separated lymphocytes were used: Whole blood. Samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Mitogen used for lymphocytes: phytohaemagglutinin, 9 mg/mL

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively) and 30 U/mL heparin. All incubation's were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 35 - 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.3 – 37.3°C).

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: rat S9 homogenate (Trinova Biochem GmbH, Giessen, Germany), prepared from male Sprague Dawley rats that were dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPES.
The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin).
- concentration of S9 in the final culture medium: 1.8% (v/v)

Test concentrations with justification for top dose:

3 h treatment with and without metabolic activation: 500, 1000 and 1642.7 µg/mL

24 h treatment without metabolic activation: 500, 1000 and 1642.7 µg/mL

The doses were based on the results of a dose range finder study. No cytotoxicity or precipitation was observed at a concentration of 1642.7 µg/mL (0.01 M), therefore, this concentration was used as the highest concentration of the test item and is the limit dose recommended by OECD TG 473.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)

- Justification for choice of solvent/vehicle: a solubility test was performed based on visual assessment. The test item formed a clear colorless solution in tetrahydrofuran.

- Justification for percentage of solvent in the final culture medium: the final concentration of the solvent in the culture medium was 0.25% (v/v), therefore, not exceeding 1% for an organic solvent, as per the OECD guideline requirement.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: first experiment 3 h (with and without S9); second experiment 24 h (without S9)
- Harvest time after the end of treatment: first experiment 20 - 22 h (24 h fixation time; with and without S9); second experiment 0 h (24 h fixation time; without S9)
- Spindle inhibitor: colchicine (0.5 µg/mL) was added for the last 2.5 - 3 h of the culture period
- Methods of slide preparation and staining technique used including the stain used: at the end of the culture period, cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol:acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol /diethyl ether and cleaned with a tissue. The slides were marked with the study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 6.7% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter, slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper.
- Number of cells spread and analysed per concentration: 300 (150 per replicate culture)
- Criteria for scoring chromosome aberrations: only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed. The number of cells with aberrations and the number of aberrations was calculated.
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- see Appendix 3 (attached background material) for definitions of chromosome aberrations scored in metaphase portraits

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)
- Any supplementary information relevant to cytotoxicity: the mitotic index of each culture was determined by counting the number of metaphases from 2000 cells (1000 per replicate culture)

Evaluation criteria:

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

Statistics:

Fisher’s exact test, one-sided

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: 7.761 and 298 mOms at 1642.7 µg/mL (solvent control: 7.707 and 308 mOms)

RANGE-FINDING/SCREENING STUDIES:
In the dose-range finding test, test item concentrations were 63, 125, 250, 500, 1000 and 1642.7 µg/mL with and without S9-mix. No precipitation or cytotoxicity was observed at any concentrations, therefore, 1642.7 µg/mL (0.01M) was selected as the top concentration for the main experiment (see Table 1, attached background material for results of the dose range finder).

STUDY RESULTS (see field "any other information on results incl. tables" for summary tables; see "attached background material" for full tabulated data, including historical control data and statistical analysis)
- The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database (see Table 7).
- The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells.
- In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database (see Table 8).
- It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o No test-item related effects on mitotic index were observed under any of the experimental conditions (see Table 2 and 5)
- Genotoxicity results:
o The test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations under any of the test conditions (see Table 3, 4 and 6)
o The test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Summary of Results of Experiment 1

Test item	Concentration	Mitotic Index	Total No. of Aberrant Cells^#
	in µg/mL	in %	with gaps	without gaps
Exposure period 3 h, fixation time 24 h, without S9 mix
THF	0.25% (v/v)	100	0	0
MMC-C	0.5	89	60****	60****
Test substance	500	99	1	1
	1000	97	1	1
	1642.7	100	1	1
Exposure period 3 h, fixation time 24 h, with S9 mix
THF	0.25% (v/v)	100	0	0
CP	10	74	68****	67****
Test substance	500	98	2	2
	1000	100	1	1
	1642.7	95	0	0

MMC-C: Mitomycin C; CP: Cyclophosphamide (positive controls)

^# 300 cells scored (150 per culture)

****P < 0.0001

Summary of Results of Experiment 2

Test item	Concentration	Mitotic Index	Total No. of Aberrant Cells^#
	in µg/mL	in %	with gaps	without gaps
Exposure period 24 h, fixation time 24 h, without S9 mix
THF	0.25% (v/v)	100	1	1
MMC-C	0.2	50	49****	46****
Test substance	500	99	0	0
	1000	99	0	0
	1642.7	100	0	0

MMC-C: Mitomycin C (positive control)

^# 300 cells scored (150 per culture)

****P < 0.0001

Conclusions:

Trimethoxy(propyl)silane has been tested for clastogenicity in cultured human peripheral lymphocytes, in a study which was conducted according to OECD 473 and in compliance with GLP. No evidence of a test substance related increase in the number of cells with aberrations was observed with or without metabolic activation in short- and long-term treatments. No test item-related polyploidy or endoreduplication were noted in the experiments with or without metabolic activation. In conclusion, the test substance did not induce chromosome aberrations in human lymphocytes under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity (mutagenicity) in bacteria

The mutagenicity of trimethoxy(propyl)silane (CAS 1067-25-0) in bacteria was assessed in a GLP experiment according to OECD 471 with Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 (Hüls AG, 1996). Deviations to the latest guideline were the absence of a fifth strain (E. coli WP2 uvrA or S. typhimurium TA102) and use of 2-aminoanthracene as the sole positive control substance in the presence of S9-mix. No further information is given, if the S9-mix has been characterised with a second substance. The tester strains were treated using the plate incorporation and pre-incubation methods, both with and without S9-mix. The concentrations tested were 50 - 5000 µg/plate. No evidence for a test-substance related increase in the number of revertants was observed when tested up to cytotoxic concentration in any of the Salmonella typhimurium strains (TA 1535, TA1537, TA98 and TA100) in two independent experiments without and with metabolic activation. Appropriate positive and solvent controls were included and gave expected results. Under the conditions of this study, the registered substance was concluded to be non-mutagenic in the strains tested.

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

Trimethoxy(propyl)silane (CAS 1067-25-0) has been tested for clastogenicity in cultured human peripheral lymphocytes in a study which was conducted according to OECD 473 and in compliance with GLP (CRL, 2022). Human peripheral lymphocytes were cultured and treated with trimethoxy(propyl)silane (diluted in THF) at concentrations of 500, 1000 and 1642.7 µg/mL (3 h, ±S9 and 24 h, –S9) in the main experiments, the latter concentration equally 10 mM, the highest recommended concentration in OECD 473. The spindle inhibitor colchicine (0.5 µg/ml) was added for the last 2.5 - 3 hours of the culture period. 24 hours after the start of treatment the cells were fixed and then stained with Giemsa and 300 metaphases were scored per concentration. Cytotoxicity was assessed by determination of the mitotic index (2000 cells per concentration). No cytotoxicity or precipitation was observed in any of the treatment conditions. No evidence of a test substance related increase in the number of cells with aberrations was observed with or without metabolic activation in short- and long-term treatments in the main experiments. No test item-related polyploidy or endoreduplication were noted in the experiments with or without metabolic activation. The results of the solvent and positive controls were within the range of the historical control data. In conclusion, the test substance did not induce chromosome aberrations in human peripheral lymphocytes under the conditions of the test.

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

In the in vitro mammalian cell mutagenicity study (CRL, 2022), the test item trimethoxy(propyl)silane (CAS 1067-25-0) was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The study was conducted according to OECD 490 and in compliance with GLP. L5178Y cells were treated with trimethoxy(propyl)silane (diluted in dried THF) at concentrations of 4 – 1000 µg/L for a short-term treatment (3 h, ±S9) and 4 – 125 µg/L for a long-term treatment (24 h, –S9). Cytotoxicity was assessed by determination of relative total growth and genotoxicity was assessed by determination mutant frequency. No significant cytotoxicity was observed, but precipitation was evident at the top concentration(s) in each treatment condition. No biologically relevant increase of mutants was found after treatment with the test item in the short-term treatment (3h, ±S9) and long-term treatment (24 h, –S9). Colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation). Appropriate solvent and positive controls were included in the test and gave the expected results. It was therefore concluded that the test substance is not mutagenic to mammalian cells under the conditions of the test.

Justification for classification or non-classification

The available data on genetic toxicity from the test substance trimethoxy(propyl)silane (CAS 1067-25-0) do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.