Trimethoxy(methyl)silane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without metabolic activation in strains TA98, TA100, TA1535 and TA1537 Escherichia coli WP2 (OECD Test Guideline 471 and in compliance with GLP) (BioReliance, 2004a).

Cytogenicity in mammalian cells: positive with metabolic activation in CHO cells (OECD Test Guideline 473 and in compliance with GLP) (BioReliance, 2004b).

Mutagenicity in mammalian cells: positive with metabolic activation in L5178Y mouse lymphoma cells (OECD Test Guideline 476 and in compliance with GLP) (TNO, 2002a).

The selected key studies were chosen because they are the most reliable and recent available studies for the registered substance. They were conducted according to appropriate OECD guidelines and in compliance with GLP.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-10-09 to 2003-12-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Testing Guidelines of the ICH, S2A and S2B documents for Genotoxicity Testing of Pharmaceuticals, 1996 and 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

bacteria, other: Salmonella typhimurium: TA-1535, TA-1537, TA-98 and TA-100; Escherichia coli: WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

100, 333, 1000, 3333 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: none given in report

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48-72 hours
- Expression time (cells in growth medium): 48-72 hours

NUMBER OF REPLICATIONS: plated in triplicate; initial assay had an unacceptable vehicle control value, so the experiment was repeated with preincubation

DETERMINATION OF CYTOTOXICITY
- Method: other: reduction in mean number of revertants; condition of background lawn

Evaluation criteria:

Responses (number of revertants) to the test article were compared to concurrent negative and positive controls. A dose-related increase in at least one strain over a minimum of two increasing concentrations. Increase must be greater than or equal 3.0 times control for strains TA 1535 an dTA 1537, or 2.0 times for all other strains.

Statistics:

No statistical evaluation required.

Key result

Species / strain:

bacteria, other: Salmonella typhimurium: TA-1535, TA-1537, TA-98 and TA-100; |Escherichia coli: WP2 uvrA

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

Positive controls validity:

valid

Additional information on results:

Neither precipitate nor appreciable toxicity was observed.
All strains were tested in a preliminary toxiicty assay, with and without metabolic activation. No toxicity was observed.

Table 1 Experiment B1/B2: Plate incorporation - number of revertants per plate (mean of three plates)

Concentration  µg/plate	TA98		TA100		TA1535		TA1537		WP2
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
0	11	23	125	210	13	10	8	9	12	10
100	11	23	116	210	14	9	9	11	14	13
333	11	23	138	202	13	9	9	7	11	9
1000	11	21	147	180	13	10	7	7	8	11
3333	12	25	130	213	11	10	7	9	11	12
5000	11	20	134	211	16	10	8	10	10	9
Positive	64	391	454	1108	366	121	407	101	117	477

Table 2 Experiment B4: Plate incorporation, repeat with dried vehicle - number of revertants per plate (mean of three plates)

Concentration  µg/plate	TA98		TA 100
	- MA	+ MA	- MA
0	17	34	184
100	15	28	155
333	16	33	178
1000	18	32	179
3333	19	40	191
5000	15	32	184
Positive	196	1422	568

Table 3 Experiment B3: Preincubation - number of revertants per plate (mean of three plates)

Concentration µg/plate	TA98		TA100		TA1535		TA1537		WP2
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
0	14	22	130	223	16	13	3	9	14	16
100	13	21	146	230	14	11	4	9	11	17
333	12	21	122	198	14	16	6	7	11	15
1000	15	22	129	232	21	12	4	6	12	12
3333	13	26	139	239	16	14	2	7	13	13
5000	18	23	154	246	15	12	4	7	14	13
Positive	441	578	463	702	277	69	262	79	331	116

Conclusions:

Methyltrimethoxysilane has been tested according to OECD 471 and under GLP. No increase in the frequency of revertants relative to control was observed with or without activation at any concentration, in any of the test organisms. The findings of the initial plate incorporation assay were confirmed in the repeat pre-incubation assay. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-10-16 to 2003-11-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster ovary (CHO-K1) cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9, NADP as cofactor

Test concentrations with justification for top dose:

170.3, 340.6, 681.2 and 1362.4 µg/ml. The cells treated with 170.3 µg/ml were not evaluated for chromosome aberrations.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based information provided by sponsor and compatibility with the target cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with activation

Details on test system and experimental conditions:

ACTIVATION: 1 ml S9 mix containing 20 µl Aroclor induced rat liver S9, NADP as cofactor, added to 4 ml of medium
METHOD OF APPLICATION: in medium;
DURATION
- Preincubation period: none
- Exposure duration: 4 or 20 hours (without activation), 4 hours with activation
- Expression time (cells in growth medium): 0 or 16 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid added 2 h prior to harvest
STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: duplicate cultures per concentration

NUMBER OF CELLS EVALUATED: mitotic index in 500 cells, aberrations in 200 cells (100 per duplicate flask)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; other: cell count and percentage viability

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication:yes

Evaluation criteria:

A test substance is considered positive if the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant. (p

Statistics:

Fisher's exact test; in the event of a positive result, Cochran Armitage test used to measure dose-responsiveness

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

with activation

Cytotoxicity / choice of top concentrations:

other: > 1362.4 μg/ml

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: cell growth inhibition was between 7% and 17% in the highest dose tested.

COMPARISON WITH HISTORICAL CONTROL DATA: control values were within historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

The percentage of cells with structural or numerical aberrations in the  non-activated 4 and 20 hour exposure groups was not significantly  increased above that of the solvent control at any dose level. The  percentage of cells with structural aberrations in the S9 activated 4  hour exposure group was significantly increased above that of the solvent  control at 1362.4 ug/mL. The Cochran-Armitage test was also positive for  a dose response. The percentage of cells with numerical aberrations in  the test article-treated group was not significantly increased above that  of the solvent control at any dose level. The results of the assay are  summarized in the following table:

WITHOUT S9:
Treatment time: 4 hr
Recovery time: 16 hr
Harvest time: 20 hr
Toxicity* at highest dose scored: 16%
Mitotic Index Reduction**: None
LED for Structural Aberrations (µg/mL): None
LED for Numerical Aberrations (µg/mL): None

WITHOUT S9:
Treatment time: 20 hr
Recovery time: 0 hr
Harvest time: 20 hr
Toxicity* at highest dose scored: 4%
Mitotic Index Reduction**: 9%
LED for Structural Aberrations (µg/mL): None
LED for Numerical Aberrations (µg/mL): None

WITH S9:
Treatment time: 4 hr
Recovery time: 16 hr
Harvest time: 20 hr
Toxicity* at highest dose scored: 22%
Mitotic Index Reduction**: 16%
LED for Structural Aberrations (µg/mL): 1362.4
LED for Numerical Aberrations (µg/mL): None

Where: 
* Cell growth inhibition
** Relative to solvent control at high dose evaluated for
chromosome aberrations
LED = Lowest Effective Dose

Table 1: 4 h treatment without activation (totals and percentages from 200 cells)

Treatment		Solvent Control	Positive control*	340.6 µg/ml	681.2 µg/ml	1362.4 µg/ml
Cytotoxicity		no	no	no	no	no
Chromatid aberrations	gaps	1	3	0	2	2
	breaks	1	14	1	0	0
	interchanges	0	13	1	1	.
Chromosome aberrations	breaks	0	0	0	0	0
	dicentric	0	0	1	2	3
	ring	0	0	0	0	0
Percent aberrant cells	Numerical	2.5	1.5	2	3	3
	Structural	0.5	29.0	1.5	1.5	2
Mitotic index		7.3	8.1	8.4	7.5	7.6

* 100 cells evaluated

 

Table 2: 4 h treatment with activation (totals and percentages from 200 cells)

Treatment		Solvent Control	Positive control*	340.6 µg/ml	681.2 µg/ml	1362.4 µg/ml
Cytotoxicity		no	yes	no	no	no
Chromatid aberrations	gaps	1	4	0	0	2
	breaks	1	30	0	0	7
	interchanges	0	19	0	0	13
Chromosome aberrations	breaks	0	0	0	0	0
	dicentric	0	3	2	2	2
	ring	0	0	0	0	0
Percent aberrant cells	Numerical	2	1.5	1.5	3.5	4.0
	Structural	0.5	22.5	1.0	3.0	9.5
Mitotic index		9.3	8.6	8.4	8.2	7.8

* 100 cells evaluated

 

Table 3: 20 h treatment without activation (totals and percentages from 200 cells)

Treatment		Solvent Control	Positive control*	340.6 µg/ml	681.2 µg/ml	1362.4 µg/ml
Cytotoxicity		no	no	no	no	no
Chromatid aberrations	gaps	1	6	0	1	1
	breaks	1	28	0	0	0
	interchanges	0	18	0	1	0
Chromosome aberrations	breaks	0	0	0	0	0
	dicentric	0	2	3	4	4
	ring	0	1	0	0	0
Percent aberrant cells	Numerical	2	1.5	2	1.5	1.5
	Structural	0.5	20	1.5	2.5	2.0
Mitotic index		7.9	7.4	6.8	7.0	6.3

* 100 cells evaluated

Conclusions:

Trimethoxy(methyl)silane has been tested in a valid study conducted according to OECD 473 and in compliance with GLP. The test substance induced a statistically significant dose related increase in the number of structural aberrations in Chinese hamster ovary (CHO-K1) cells in the presence of activation. No test-substance related genotoxicity was observed in the absence of metabolic activation. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is positive for the induction of chromosome aberrations in the presence of activation under the conditions of the study.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-03-19 to 2002-04-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

13 to 1360 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: none given in report

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: none
- Exposure duration: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 44-48 hours
- Selection time (if incubation with a selection agent): 10-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 48-68 hours

SELECTION AGENT (mutation assays): TFT

NUMBER OF REPLICATIONS: eight single dose levels

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

OTHER EXAMINATIONS
- Other: small and large colonies

Evaluation criteria:

A response was considered to be positive if the induced mutant frequency was more than 100 mutants per 1 000 000 clonable cells, at less than 90% cytotoxicity. A response was considered equivocal if the induced mutant frequency was less than 100 but more than 50. In addition it should be concentration-related increase was observed.

Statistics:

No statistical analysis was performed

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

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

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

other: equivocal

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

At the highest concentrations evaluated for mutagenicity the relative total growth was 115% and 85% in the absence and presence of S9 mix, respectively.

In the absence of S9 mix, an increase of the mutant frequency was observed at a concentration of 26 µg/ml test substance. This result would not be considered a positive response if assessed using the criteria set out in the draft guideline for in vitro mammalian cell gene mutation assays using the thymidine kinase gene (OECD 2014).

In the presence of S9 a dose related increase of the mutant frequency was observed at concentrations above 560 µg/ml. Only the highest concentration induced an increase in mutant frequency which exceeded the global evaluation factor for the microwell method of 160 E-06. At concentrations causing a positive response in mutant frequency in the presence of S9 mix, relatively more small than large colonies were formed (mean: 73% and 27%).

Table 1: Treatment in the absence of S9-mix: Number of revertants

Treatment	Dose µg/ml	Relative suspension growth %	Relative total growth %	Mutant frequency (*10E06)	Induced mutant frequency	Mutant colonies large %	Mutant colonies small %
MMS	0.1 mM	48	20	2737	2548	36	64
Methyltrimethoxysilane	1362	112	115	163	-26	51	49
	1000	95	104	157	-32	47	53
	750	111	96	204	15	36	64
	560	112	128	140	-49	nd	nd
	420	105	116	149	-40	nd	nd
	210	87	80	201	12	nd	nd
	105	98	96	185	-4	nd	nd
	53	99	90	195	6	nd	nd
	26	103	96	264	75	nd	nd
DMSO	0	102	92	225	36	27	73
	0	98	107	153	-36	46	54

 nd: not determined

Induced mutant frequency determined by reviewer from mutant frequency (treated) – mean mutant frequency (solvent controls)

Table 2: Treatment in the presence of S9-mix: Number of revertants

Treatment	Dose µg/ml	Relative suspension growth %	Relative total growth %	Mutant frequency (*10E06)	Induced mutant frequency	Mutant colonies large %	Mutant colonies small %
MCA	10	76	62	889	761.5	45	55
Methyltrimethoxysilane	1362	91	85	289	161.5	24	76
	1000	97	106	249	121.5	30	70
	750	105	112	188	60.5	nd	nd
	560	106	110	143	15.5	nd	nd
	420	104	100	130	2.5	nd	nd
	210	109	124	124	-3.5	nd	nd
	105	105	98	136	8.5	nd	nd
	53	102	nd	n.d.a	nd	nd	nd
	26	104	78	166	38.5	nd	nd
DMSO	0	98	107	114	-13.5	47	53
	0	102	93	141	13.5	43	57

n.d.a: Due to technical error, mutant frequency could not be determined

nd: not determined

Induced mutant frequency determined by reviewer. Induced mutant frequency = mutant frequency (treated) – mean mutant frequency (solvent controls)

Conclusions:

Trimethoxy(methyl)silane has been tested using mouse lymphoma L5178Y cells in a valid study conducted according to OECD 476 and in compliance with GLP. The results without metabolic activation were considered ambiguous: an increase of the mutant frequency was observed at a concentration of 26 µg/ml test substance, but the result would not be considered a positive response if assessed using the criteria set out in the draft guideline for in vitro mammalian cell gene mutation assays using the thymidine kinase gene (OECD 2014). In the presence of metabolic activation, a dose-related increase in the mutant frequency was observed  at concentrations above 560 µg/ml. Only the highest concentration induced an increase in mutant frequency which exceeded the global evaluation factor for the microwell method of 160 E-06. A relative increase in smaller colonies relative to larger colonies was observed at the two highest concentrations. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the substance is positive for mutagenicity to mammalian cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo Mammalian Alkaline Comet Assay (inhalation): Negative (OECD Test Guideline 489 and in compliance with GLP) (Charles River, 2017).

Mouse micronucleus assay (oral gavage): Negative (OECD Test Guideline 474 and in compliance with GLP) (Research Toxicology Centre, 2002).

The selected key studies were chosen because they are the most reliable and recent available studies for the registered substance. They were conducted according to appropriate OECD guidelines and in compliance with GLP.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-09-27 to 2002-11-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy
- Age at study initiation: no information
- Weight at study initiation: 20.8-27.2 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: no
- Housing: 5 animals per cage, polycarbonate with stainless steel mesh lid and floor
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 55
- Air changes (per hr): no information
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: not given To: 2002-10-04

Route of administration:

oral: gavage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: prepared immediately before use in corn oil

Frequency of treatment:

Single treatment

Post exposure period:

Samples taken at 24 or 48 hours

Dose / conc.:

500 mg/kg bw/day

Remarks:

expressed in terms of material as received. Solutions prepared on w/v basis without correction for displacement

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

expressed in terms of material as received. Solutions prepared on w/v basis without correction for displacement

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

expressed in terms of material as received. Solutions prepared on w/v basis without correction for displacement

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- mitomycin C
- Justification for choice of positive control(s): none given, standard control
- Route of administration: ip injection
- Doses / concentrations: 3.0 mg/kg body weight

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: preliminary toxicity assay

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): no additional information

DETAILS OF SLIDE PREPARATION: air dried and stained with May-Gruenwald and Giesma

METHOD OF ANALYSIS: 200 PCEs per animal examined for presence of micronuclei at high power (x100, oil immersion)

OTHER:

Evaluation criteria:

A test item is considered positive if it induces a statistically significant increase, exceeding the historical range of negative control values, in the incidence of micronucleated PCEs (p<0.05) in pooled data for both sexes or for either sex considered separately.

Statistics:

Original observations of polychromatic cells from control and treated groups were compared using a modified Chi-squared evaluation

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Animals from the high dose treatment group showed reduced activity,  ataxia and hunched posture.  Animals from the intermediate dose treatment  group showed hunched posture and an animal died after treatment.   Clinical signs and mortality were observed and provided evidence of  bioavailability of the test substance and adequate exposure time.  Following treatment with the test substance, no statistically significant  increase in the incidence of micronucleated PCE's over the control value  was observed at any dose-level, at any sampling time.  Following  treatment with the positive control Mitomycin-C, statistically  significant increases in the incidence of micronucleated PCEs over the  control values were observed, indicating the correct functioning of the  test system.  

The test was conducted at the limit dose of 2000 mg/kg bw at which signs  of toxicity were seen (mortality, reduced activity, ataxia and hunched  posture) indicating bioavailability of the test substance.  When tested  at the limit dose with no or minimum signs of toxicity and with the  positive and negative controls responding appropriately, the test is  considered valid.


The ratio of mature to immature to immature erythrocytes and the  proportion of immature erythrocytes to among total erythrocytes were  analyzed to evaluate the bone marrow cell toxicity. No inhibitory effect  on erythropoetic cell division was observed for either sex at any  sampling time. 

The report contains the values for PCE and NCE separately, as well as  ratios of NCE/PCE and PCE/(NCE+PCE). P/N ratios were not calculated in  the report.

Summary of incidence of micronucleated cells per 1000 cells, both sexes, 24 and 48 hours

Dose	Incidence of micronucleated cells/1000 PCE	PCE	NCE	NCE/PCE ratio	PCE/(NCE+PCE)
Sampling time 24 hours
0	1.0	20527	25105	1.22	0.45
500	1.0	20601	22891	1.11	0.47
1000	1.3	18525	21590	1.17	0.44
2000	0.5	20504	26333	1.28	0.44
Positive control	7.0	20847	35393	1.69	0.37
Sampling time 48 hours
0	0.9	20430	16561	0.81	0.55
2000	0.7	20491	24866	1.22	0.45

Conclusions:

Trimethoxy(methyl)silane has been tested in a valid mouse mutagenicity study conducted according to OECD 474 and in compliance with GLP. No increase in the incidence of micronucleated PCE was observed resulting from exposure to the test substance by oral gavage up to limit concentrations. Appropriate positive and vehicle controls were included and gave expected results. It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the test.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 July 2017 to 10 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Section 4, Health Effects. No.403, Acute Inhalation Toxicity

Version / remarks:

2009

Deviations:

yes

Remarks:

the study design was based on the study objectives (assessment of DNA damage), OECD 489 and OECD 403

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature container flushed with nitrogen
- Stability under test conditions: The stability of the bulk test item was not determined during the course of this study. Information to support the stability of each lot of the bulk test item was provided by the Sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: The test item was used as delivered.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was used as delivered.

Species:

rat

Strain:

other: Crl: WI(Han)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany OR Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: Approx. 9 weeks old
- Weight at study initiation: 150 to 330 g CONFIRM WHEN REPORT RECEIVED
- Assigned to test groups randomly: Animals were assigned to the study at the discretion of the coordinating biotechnician according to body weights, with all animals within ± 20% of the sex mean. Animals in poor health or at extremes of body weight range were not assigned to the study.
- Fasting period before study: none
- Housing: The animals were group housed together (up to 5 animals of the same sex and same exposure group together) in polycarbonate cages. Animals were separated during designated procedures/activities.
- Diet (e.g. ad libitum): Pelleted rodent diet was provided as libitum, except during designated procedures.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: at least 4 days days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21°C
- Humidity (%): 53 to 71%
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hours light and 12-hours dark

Route of administration:

inhalation: vapour

Vehicle:

- Vehicle(s)/solvent(s) used: Test item used as received.

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The design of the exposure chamber is based on the directed flow nose-only inhalation chamber. The chamber consists of animal sections with eight animal ports each. Each animal port has its own test atmosphere inlet and exhaust outlet. The number of animal sections and number of open inlets was adapted to the air flow in such a way that at each animal port the theoretical air flow is at least 1 L/min. The main inlet of the test atmosphere is located at the top section and the main outlet is located at the bottom section. The direction of the flow of the test atmosphere guarantees a freshly generated atmosphere for each individual animal. All components of the exposure chamber in contact with the test item are made of stainless steel, glass, rubber or plastic.
- Method of holding animals in test chamber: The animals were placed in polycarbonate restraining tubes, which were connected to the exposure chamber. Animals were allowed to acclimatize for at least fifteen minutes after the last animal has been placed.
- Source and rate of air: The test atmosphere was generated by passing a stream of pressurized air through the test item. The saturated vapour was diluted with pressurized air to obtain the desired concentration. Subsequently the test atmosphere was passed through the exposure chamber. For the exposure to 20 mg/L, a vapour was generated by nebulization of the test item with pressurized air. For this, the test item was transferred to a Collison nebulizer by means of a rotating pump in order to maintain a constant level of freshly available test item. The primary vapour was diluted with pressurized air before it entered the exposure chamber. For the exposure to 10 and 5 mg/L, passing pressurized air through the test item generated a vapour. The primary vapour was diluted with pressurized air and subsequently let through the exposure chamber.
- Method of conditioning air: not specified
- Temperature, humidity, pressure in air chamber: The temperature and relative humidity were measured with a humidity and temperature indicator and were recorded after the animals were placed in the experimental set up and regularly, preferably in 30 minute intervals after initiation of the generation of the test atmosphere. The probe of the indicator was inserted in one of the free animal ports of the exposure chamber. The temperature in the exposure chamber was maintained at 22 ± 3°C. The relative humidity was maintained between 40 and 70%, preferably 50 to 60%.
- Air flow rate: At each animal port the theoretical air flow is at least 1 L/min. The mean total airflow was 30 L/min for the 20 mg/L exposure group. The negative controls were exposed to pressurized air only, with a mean airflow of 20 L/min. using the same type of exposure chamber. The mean total airflows were 16 and 29 L/min for the 10 and 5 mg/L exposure groups respectively. The negative controls were exposed to pressurized air only, with a mean airflow of 22 L/min. using the same type of exposure chamber.
- Air change rate: not specified.
- Treatment of exhaust air: From the exposure chamber the test atmosphere was passed through a filter before it is released to the exhaust of the fume hood

TEST ATMOSPHERE
- Brief description of analytical method used: An adequate number of representative samples were taken for determination of the actual test item concentration during exposures at 20, 10 and 5 mg/L, respectively. Sample intervals were appropriately spaced over the exposure periods and additional samples were taken in case the air flow was readjusted. Samples were drawn for at least 2 minutes with a sample flow of 2 L/minute from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber. Samples were collected in glass collector bulbs which had a volume between 125 and 500 mL.
- Samples taken from breathing zone: yes. The concentration was measured analytically with a validated gas chromatographic method and the standard deviation(s) were calculated from the individual values measured during exposure.

Duration of treatment / exposure:

4 hours

Frequency of treatment:

Once daily for three consecutive days

Post exposure period:

1 hour after last exposure

Dose / conc.:

20 mg/L air

Remarks:

Session 1

Dose / conc.:

5 mg/L air

Remarks:

Session 2

Dose / conc.:

10 mg/L air

Remarks:

Session 2

No. of animals per sex per dose:

5 animals per sex per group

Control animals:

yes

Positive control(s):

Ethylmethanesulphonate
- Justification for choice of positive control(s): not specified in report. This positive control substance is acceptable according to the OECD guideline
- Route of administration: A single dose of positive control was administered to the appropriate animals by oral gavage on the second and third exposure day of the concurrent inhalation exposed groups, between 16-26 and 2-6 hours prior to removal of the tissues for the comet assay. Animals were dosed using a syringe with a plastic gavage cannula attached.
- Doses / concentrations: The dose level was 200 mg/kg body weight. The dose volume for each animal was based on the body weight measurement prior to dosing. A dose volume of 10 mL/kg body weight was used for each dose.

Tissues and cell types examined:

Following necropsy, the liver and lung were examined. A small part (0.4-0.7 grams) was dissected and put in labelled containers with cold medium for the in vivo comet assay main test for the examination on DNA damage with the alkaline comet assay. The remaining part was preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). Based on the results of the comet assay, it was concluded that there was no need to perform histotechnology and histopathology of the liver and lungs.
One femur containing the bone marrow was dissected and the bone marrow was transferred to labelled containers with cold medium for the examination on DNA damage with the alkaline comet assay. From the other femur, bone marrow smears were prepared and fixed with methanol. Based on the results of the comet assay, it was concluded that there was no need to further process and evaluate the smears.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Exposure levels were selected based on the EC and UN classification guidelines. The starting exposure level was limit concentration for vapours according to the OECD guidelines for acute inhalation studies and was selected based on the results of the MTD study

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): The animals were treated for 4 hours daily for three consecutive days. Within 4 hours after the end of the last inhalation exposure or between 2 – 6 hours after the last oral dosing, all surviving animals were sacrificed by an intra-peritoneal injection of Euthasol.

DETAILS OF SLIDE PREPARATION: To 20 µl of the cell suspension 280 µl melted low melting point agarose (LMAgarose) was added. The cells were mixed with the LMAgarose and 50-60 µl will be layered on a precoated comet slides in duplicate. Three slides per tissue per animal were prepared (in total 6 agarose circles; 3 for scoring and 3 for backup). The slides were marked with project and identification number. The slides were incubated for at least 10 minutes in the refrigerator in the dark until a clear ring appears at the edge of the comet slide area.

METHOD OF ANALYSIS: One hundred fifty comets were analyzed in total with a fluorescence microscope connected to a Comet Assay IV image analysis system. The DNA damage was quantified as percentage tail intensity (percentage of DNA migrating into the tail).

Evaluation criteria:

A test compound is considered positive in the comet assay (in a tissue) if the following criteria are met:
It induces a biologically as well as a statistically significant (Dunnett’s test, one-sided, p < 0.05) dose-dependent increase in percentage Tail Intensity. In case of other non-dose-dependent significant increases the data interpretation is on a case by case base.

Statistics:

Dunnett’s test for statistical significance

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF SESSION 1 AND SESSION 2 OF STUDY
- Dose range: not applicable
- Solubility: not applicable
- Clinical signs of toxicity in test animals: No clinical signs were noted during and after the inhalation exposures to test item or after the inhalation exposures to control air. No clinical signs were noted for the orally dosed positive control animals.
- Evidence of cytotoxicity in tissue analyzed: Red foci on the thymus were found for two animals exposed to 20 mg/L, one animal exposed to 10 mg/L and for one positive control animal. Red foci were seen on the lungs for one animal exposed to 20 mg/L. No abnormalities were found at macroscopic post mortem examination of the negative control animals.
- Rationale for exposure: The objective of this study was to evaluate the DNA damaging properties of trimethoxy(methyl)silane after 3-day nose-only inhalation exposure up to the limit exposure level by measuring the increase in DNA strand breaks in bone marrow, liver and lung tissue.
- Harvest times: Within 4 hours after the last inhalation exposure or within 2 and 6 hours after the last oral dosing, animals were subjected to macroscopic examination and lung, liver, kidney and bone marrow were taken and examined for DNA damage by using the alkaline in vivo Comet Assay.
- High dose with and without activation: not applicable

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: The inhalation route of administration was selected because this route was defined as a possible route of human exposure. Exposure levels were selected based on the EC and UN classification guidelines. The starting exposure level was limit concentration for vapours according to the OECD guidelines for acute inhalation studies and was selected based on the results of the MTD study.
- Statistical evaluation: No statistically significant increase in the mean Tail Intensity (%) was observed in bone marrow cells, liver cells and lung cells of animals treated with the test material.

Conclusions:

Trimethoxy(methyl)silane has been tested in a comet assay for genetic toxicity in a study, conducted according to OECD Test Guideline 489 and in compliance with GLP. Trimethoxy(methyl)silane did not induce any genotoxic alterations in bone marrow, liver and lung cells after inhalation exposure up to the limit exposure level of 20 mg/L. Negative and positive controls gave the expected results.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Trimethoxy(methyl)silane has been tested for mutagenicity to bacteria in a study which was conducted according to OECD Test Guideline 471 and in compliance with GLP (BioReliance, 2004a). No evidence of a test substance related increase in the number of revertants was observed with or without activation in the initial or the repeat experiments using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test. Additional information on mutagenicity to bacteria is provided by two other studies (Dow Corning Corporation, 1977, TNO 2002b). No evidence for genetic toxicity was observed in either of these studies.

Trimethoxy(methyl)silane has been tested for cytogenicity in mammalian cells in a study conducted according to OECD Test Guideline 473 and in compliance with GLP (BioReliance, 2004b). A statistically significant dose related increase in the number of structural aberrations in the presence of activation was evident, which was not seen in the absence of metabolic activation. It is concluded that the test substance is positive for the induction of chromosome aberrations in the presence of activation under the conditions of the study.

Trimethoxy(methyl)silane has been tested for mutagenicity in mammalian cells in a study conducted according to OECD Test Guideline 476 and in compliance with GLP (TNO, 2002a). A dose-related increase in the mutant frequency was observed with metabolic activation in L5178Y mouse lymphoma cells. The study is concluded to be positive for mutagenicity to mammalian cells under the conditions of the test.

Trimethoxy(methyl)silane has been tested in a mouse micronucleus study, which was conducted according to OECD Test Guideline 474 and in compliance with GLP (Research Toxicology Centre, 2002). No increase in the indicence of micronucleated PCE was observed following exposure to the test substance by oral gavage, administered up to limit concentrations. It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the test. Clinical signs of toxicity were observed that suggested systemic availability, though no toxicity to target tissue was observed.

A dose-range finding study was conducted to determine the maximum tolerated dose (MTD) for a Comet assay (Charles River, 2017). The objective of this study was to establish the MTD to be used in the in vivo Comet assay and to identify any sex-specific difference in toxicity. Trimethoxy(methyl)silane was administered as a vapour by nose only inhalation for 4 hours on three consecutive days to one group of three male and three female Wistar rats. Clinical signs and body weights were recorded during the observation period. On the last day of exposure the animals were sacrificed and macroscopic examination was performed. No mortality was observed during the observation period. Shallow breathing was observed during exposure, but no significant clinical signs were noted in any of the animals post exposure. Reduced body weight gain was noted for all males and one female and body weight loss was noted for two females. These changes were considered to be within the range expected for rats of this strain and age. A small increase in lung/body weight ratio was seen in males, while in females, individual increases and decreases were seen. Overall, it was considered that the changes were not indicative for evident toxicity. No relevant changes were noted for total cell count and cell differentiation of the bronchial alveolar fluid. The maximum tolerated dose value of trimethoxy(methyl)silane was established to be 20 mg/L. No sex-specific toxicity was observed.

Trimethoxy(methyl)silane has been tested in an in vivo mammalian alkaline comet assay conducted according to OECD Test Guideline 489 and in compliance with GLP (Charles River, 2017). The test substance was administered by the inhalation route. The study was conducted in two sessions:

Session 1: A test item concentration of 20 mg/L was tested including a negative control (control air) and a positive control (EMS 200 mg/kg, administered orally).

Session 2: Test item concentrations of 5 and 10 mg/L were tested including a negative control (control air) and a positive control (EMS 200 mg/kg).

After treatment, single cell suspensions from the bone marrow, liver and lung tissue were prepared. Comet slides were prepared and analyzed.

The results revealed that no statistically significant increase in the mean Tail Intensity (%) was observed in bone marrow, liver and lung cells of trimethoxy(methyl)silane treated female animals at 5, 10 and 20 mg/L compared to the vehicle treated animals. The Tail Intensity was 9.13%, 7.03% and 12.29% at 5, 10 and 20 mg/L in bone marrow, 6.80%, 10.06%, and 3.66% at 5, 10 and 20 mg/L in liver, and 4.52%, 6.33% and 8.42% at 5, 10 and 20 mg/L in lung. All these Tail Intensities in bone marrow, liver and lung are within the historical control data range of the negative control of 1.92-17.26% for bone marrow, 0.13-27.12% for liver and 0.69-37.02% for lung.

The assays passed the acceptance criteria:

- The negative control Tail Intensity (%) in bone marrow, liver and lung was within the historical control data range in the two sessions with exception of bone marrow and liver in session 1. The mean Tail Intensity in bone marrow and liver in session 1 were with 18.43% and 35.76% just above the upper limit of the ranges of 17.26% and 27.12%, respectively. Since the values were only just above the upper range of the historical data and the positive control caused severe DNA damage with a Tail Intensity of 87.94% and 95.29% in bone marrow and liver, respectively, the sensitivity of these assays was considered not to be affected and both assays were considered acceptable.

- EMS, the positive control item, induced a statistically significant increase in all assays in all tissues (Students t-test one-sided, p <0.05).  

 Exposure of the target tissue is supported by evidence of systemic exposure observed in the dose range-finding study. It was therefore concluded that the test substance does not cause DNA damage in lung, liver or bone marrow after inhalation exposure of female Wistar Han rats.

Information is available from reliable studies for all the required in vitro endpoints. Where there was more than one result for an endpoint the most reliable study available was chosen as key study. Where there was more than one reliable study, the most recent study was selected. The results of all the bacterial studies were negative. The results of testing in mammalian cells showed evidence of genetic toxicity in the presence of metabolic activation. There was evidence for clastogenicity (causing chromosomal aberrations) in the presence of metabolic activation in vitro. There was evidence of mutagenicity to mammalian cell in the presence of metabolic activation.

An in vivo micronucleus study did not support the positive result of the in vitro chromosome aberration assay, so it is concluded that the in vitro result does not reflect an ability to cause chromosome aberrations in vivo. Clinical signs of toxicity were observed that suggested systemic availability, although it is noted that the test substance did not affect the NCE/PCE ratio.

At the request of ECHA, further investigation of the ability of trimethoxy(methyl)silane to damage DNA was carried out in an in vivo mammalian alkaline comet assay conducted according to OECD Test Guideline 489 (inhalation route). The results of the study were negative and indirect evidence of exposure of the target tissue comes from the dose range-finding and main studies carried out to investigate subchronic inhalation toxicity (Dow Corning Corporation, 2007, Dow Corning Corporation, 2008). In these studies, mortalities and clinical effects were observed in animals exposed to trimethyoxy(methyl)silane at 22.2 and 44.4 mg/L for 14 days, and at 2.2 and 8.9 mg/L for 90 days. It is therefore concluded that the test substance is not clastogenic or mutagenic in vivo and no further investigation of germ cell mutagenicity is required.

Justification for classification or non-classification

The available information for trimethyoxy(methyl)silane indicates that when tested in vitro, trimethoxy(methyl)silane (CAS 1185-55-3) does not induce mutations in bacterial cells. The substance does cause chromosomal aberrations in vitro in the presence of metabolic activation. An in vivo mouse micronucleus study and an in vivo Mammalian Alkaline Comet Assay (inhalation) did not confirm the potential for damage to chromosomes or to DNA so it is concluded that trimethoxy(methyl)silane is not clastogenic and does not have DNA damaging potential. Based on the available information trimethoxy(methyl)silane does not require classification for genetic toxicity according to Regulation (EC) No. 1272/2008.