Trimethoxypropylsilane

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Trimethoxypropylsilane

EC Number:

213-926-7

EC Name:

Trimethoxypropylsilane

Cas Number:

1067-25-0

Molecular formula:

C6H16O3Si

IUPAC Name:

trimethoxy(propyl)silane

Test material form:

liquid

Method

Target gene:

TK locus

Species / strain

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.

Controls

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.