Dimethoxymethylsilane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay: negative with and without metabolic activation in S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 (OECD 471, RA from CAS 2031-62-1)

Mammalian cytogenicity: negative with and without metabolic activation in cultured peripheral human lymphocytes (OECD 473)

Mammalian gene mutation: negative with and without metabolic activation in L5178Y/TK+/- -3.7.2C mouse lymphoma cells (OECD 490)

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 Feb - 26 Apr 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:

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+06 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-04 M hypoxanthine, 2E-07 M aminopterine and 1.6E-05 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days in 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 37.7 – 103.0%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.2 – 38.9°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 had 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: 7.8, 16, 31, 63, 125, 250, 500 and 1051.9 µg/mL (3 hour exposure with and without S9)
Experiment 2: 16, 31, 63, 125, 250, 500, 750 and 1051.9 µg/mL (24 hour exposure without S9)

Concentrations for the main experiments were selected based on the results of a dose range finding study. The highest tested concentration was 1051.9 µg/mL exposure medium (0.01 M), as recommended in OECD TG 490. At this concentration no significant cytotoxicity was observed, therefore, 1051.9 µg/mL was selected as the highest concentration for the main experiments.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: a solubility test was performed based on visual assessment. The test item formed a clear colorless solution in DMSO.
- Justification for percentage of solvent in the final culture medium: the final concentration of the solvent in the culture medium was 1% (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:

DMSO

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: 2 (excluding dose range finder)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 8E+06 cells (1E+06 cells/mL for 3 hour treatment) or 6E+06 cells (1.25E+05 cells/mL for 24 hour treatment)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Experiment 1: 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 or 12 days
- Fixation time: 13 to 14 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+05 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+05 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 or 12 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:

The test item was tested up to the maximum recommended concentration of 1051.9 µg/mL (0.01 M) in Experiments 1 and 2. In Experiment 1, the test item precipitated at the 1051.9 µg/mL with and without S9-mix. In Experiment 2, no precipitation was observed.

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 treated with a test material concentration range of 63 to 1051.9 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.
The highest concentration which did not precipitate directly in the exposure medium was 1051.9 μg/mL. The pH and osmolarity at a concentration of 1051.9 μg/mL were 7.2 and 0.480 Osm/kg, respectively (compared to 7.2 and 0.474 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 1051.9 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 1051.9 μg/mL compared to the solvent control.

STUDY RESULTS
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).
After 3 hours, the test material precipitated in the exposure medium at a concentration of 1051.9 µg/mL with and without metabolic activation. After 24 hours, no test item precipitation was observed.

Evaluation of toxicity:
No significant toxicity was observed in Experiments 1 and 2, therefore, the highest 8 dose levels were evaluated for mutant frequency.

Evaluation of the mutagenicity:
No biologically relevant increase in the mutant frequency at the TK locus was observed in either experiment (Experiment 1, 3 hour treatment with and without S9; Experiment 2, 24 hour treatment without S9). For both experiments, 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 mutant frequency found in the solvent control cultures was within 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 DMSO was between 9 and 20 (3 hour treatment) and 85 and 91 (24 hour treatment)

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 (DMSO)	100	116	100	100	65	31	32
0 (DMSO)		95			73	27	43
7.8	79	110	104	83	63	30	30
16	103	108	102	105	76	39	34
31	112	97	91	102	69	22	45
63	100	101	95	95	66	31	33
125	108	83	78	85	62	24	36
250	122	98	93	113	62	26	34
500	112	105	99	111	52	18	33
1051.9^#	103	93	88	90	66	18	46
MMS, 15	79	62	59	46	749	249	418

MMS = methylmethanesulfonate

^# = 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 (DMSO)	100	104	100	100	95	34	56
0 (DMSO)		105			102	26	71
7.8	114	101	97	110	70	34	33
16	103	110	105	108	92	39	49
31	92	94	90	83	109	18	88
63	131	91	87	114	135	38	90
125	57	111	107	61	94	22	69
250	143	83	79	113	97	31	62
500	96	99	95	91	90	30	56
1051.9^#	127	101	97	123	97	42	50
CP, 5	126	81	78	98	670	329	252

CP = cyclophosphamide

^# = 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 (DMSO)	100	102	100	100	89	46	39
0 (DMSO)		97			59	28	30
16	97	110	110	108	52	22	28
31	93	95	96	89	74	18	54
63	87	110	110	96	53	20	31
125	97	102	103	100	58	25	32
250	96	110	110	106	51	16	33
500	71	129	129	92	52	19	31
750	96	91	92	88	58	20	37
1051.9	81	115	115	93	72	35	34
MMS, 5	75	85	85	64	678	395	195

MMS = methylmethanesulfonate

Conclusions:

Dimethoxy(methyl)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, dimethoxy(methyl)silane was not mutagenic in the TK mutation test system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Oct 2021 to 14 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:

adopted 29 July 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) 12.6 - 12.9 h

For lymphocytes:
- Sex, age and number of blood donors: sex not specified; dose range finder (31 years), first cytogenetic study (25 years), second cytogenetic study (31 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 29 - 94%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.7 – 37.5°C).

Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

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: 250, 500, and 1051.9 µg/mL

24 h treatment without metabolic activation: 250, 500, and 1051.9 µg/mL

The doses were based on the results of a dose range finder study. No cytotoxicity was observed in the dose range finder. A concentration of 1051.9 µg/mL (0.01 M) showed precipitation in the culture medium directly after addition of the test item, which disappeared after 5 minutes. 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: dried DMSO

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

- Justification for percentage of solvent in the final culture medium: the final concentration of the solvent in the culture medium was 1% (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 DMSO

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, but tested up to precipitating concentrations

Remarks:

and up to the limit dose of 0.01 M

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: 7.8 and 492 mOms at 500 µg/mL (blank 7.8 and 438 mOms)

RANGE-FINDING/SCREENING STUDIES:
A concentration of 1051.9 µg/mL (0.01 M) showed precipitation in the culture medium directly after addition of the test item, which disappeared after 5 minutes. Therefore, this concentration was used as the highest concentration in the range finder and is the limit dose recommended by OECD TG 473. In the dose range finding test, blood cultures were treated with 1051.9, 500, 250, 125, 63 and 31 µg/mL with and without S9-mix. No cytotoxicity was observed (see Table 1, attached background material).

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, in the attached background material).
- 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, in the attached background material).
- 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, in the attached background material).
- 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, in the attached background material).
o The test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Summary 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
Dried DMSO	1.0% (v/v)	100	1	1
MMC-C	0.5	93	57****	54****
Test substance	250	98	1	1
	500	92	0	0
	1051.9	95	0	0
Exposure period 3 h, fixation time 24 h, with S9 mix
Dried DMSO	1.0% (v/v)	100	3	0
CP	10	78	53****	51****
Test substance	250	98	1	1
	500	98	0	0
	1051.9	98	0	0

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

^# 300 cells scored (150 per culture)

****P < 0.0001

 

Summary 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
Dried DMSO	1.0% (v/v)	100	3	3
MMC-C	0.2	63	79****	79****
Test substance	250	96	0	0
	500	98	0	0
	1051.9	92	2	2

MMC-C: Mitomycin C (positive control)

^# 300 cells scored (150 per culture)

****P < 0.0001

 

Conclusions:

Dimethoxy(methyl)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.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to the attached read-across justification.

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The source substances showed no genotoxic potential in bacteria. A similar genotoxicity potential in bacteria is assumed for the target substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reliable studies for in vitro gene mutation in mammalian cells and in vitro cytogenicity in mammalian cells are available for dimethoxy(methyl)silane (CAS 16881-77-9). No in vitro gene mutation in bacteria study is available, however, reliable data is available for the structural analogue substance diethoxy(methyl)silane (CAS 2031-62-1).

 

The key bacterial mutagenicity study on diethoxy(methyl)silane (CAS 2031-62-1) was conducted in compliance with GLP and according to OECD TG 471 (BSL BIOSERVICE, 2007c).

Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 were treated with doses of 31.6 - 5000 µg/plate with and without a metabolic activation system (phenobarbital- and ß-naphthoflavone-induced rat liver S9-mix) in a plate-incorporation and a pre-incubation assay. DMSO was used as the vehicle.

No evidence for a test-substance related increase in the number of revertants was observed in any of the strains. No evidence of cytotoxicity or precipitation was observed at any dose level. Appropriate positive and solvent controls were included and gave the expected results.

Under the conditions of this study, diethoxy(methyl)silane was concluded to be non-mutagenic in the Salmonella typhimurium strains.

 

Dimethoxy(methyl)silane was tested for cytogenicity in cultured human peripheral lymphocytes in a study that was conducted according to OECD TG 473 and in compliance with GLP (CRL, 2022).

Human peripheral lymphocytes were cultured and treated with the test item in the absence and presence of a metabolic activation system (phenobarbital- and ß-naphthoflavone-induced rat liver S9-mix) at concentrations of 250, 500 and 1051.9 µg/mL (3 h, ± S9 and 24 h, - S9). The top concentration of 1051.9 µg/mL was 0.01 M, as required in the test guideline. DMSO was used as the vehicle. Cytotoxicity was assessed by determination of the mitotic index (readout of 1000 cells).

No cytotoxicity was observed at any of the tested concentrations. No evidence of a test item related increase in the number of cells with aberrations was observed with or without metabolic activation in short- and long-term treatments. Both, in the absence and presence of S9-mix, the test item showed no increase in the number of polyploid cells or cells with endoreduplicated chromosomes. The results of the solvent and positive controls were within the range of the historical control data.

In conclusion, dimethoxy(methyl)silane was not clastogenic in human peripheral lymphocytes under the conditions of the test.

 

Dimethoxy(methyl)silane was tested for mutagenicity in L5178Y/TK+/- -3.7.2C mouse lymphoma cells in a study that was conducted according to OECD TG 490 and in compliance with GLP (CRL, 2022, Draft).

L5178Y/TK+/- -3.7.2C mouse lymphoma cells were treated with dimethoxy(methyl)silane in the absence and presence of a metabolic activation system (phenobarbital- and ß-naphthoflavone-induced rat liver S9-mix) at concentrations of 7.8 – 1051.9 µg/mL (3 h, ± S9) and 16 – 1051.9 µg/mL (24 h, - S9). The top concentration of 1051.9 µg/mL was 0.01 M, as required in the test guideline. DMSO was used as the vehicle. Cytotoxicity was assessed by determination of relative total growth.

No significant cytotoxicity was observed. For the 3 h treatment, the test item precipitated in the exposure medium at a concentration of 1051.9 µg/mL. In the 24 h treatment, no precipitation was observed up to the highest concentration of 1051.9 µg/mL. No biologically relevant increase in the mutant frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls. Appropriate solvent and positive controls were included in the test and gave the expected results.

In conclusion, dimethoxy(methyl)silane was not mutagenic in L5178Y/TK+/- -3.7.2C mouse lymphoma cells under the conditions of the test.

Justification for classification or non-classification

The available genetic data on dimethoxy(methyl)silane and the structural analogue substance diethoxy(methyl)silane do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.