Amino functional alkoxysilanes

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 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 (OECD Test Guideline 471 (1997)) (BSL BIOSERVICE, 2012)
Cytogenicity in mammalian cells: positive with metabolic activation (OECD Test Guideline 473 (1997)) (BSL BIOSERVICE, 2013); registration substance
Mutagenicity in mammalian cells: Testing is not required as a positive result was obtained in the cytogenicity study

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:

2012-04-26 to 2012-05-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

His

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: compatible with bacterial survival and S9 activity.

Untreated negative controls:

yes

Remarks:

A. dest., BSL Lot No. 120417, 120504

Negative solvent / vehicle controls:

yes

Remarks:

acetone, Merck Lot No. K4258814

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

Positive control without metabolic activation: sodium azide for tester strains TA100, TA1535; 4-nitro-o-phenylene-diamine for TA98, TA1537; methylmethanesulfonate for TA102. Positive control with metabolic activation: 2-aminoanthracene for all strains

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 60 min; 100 µl of the test item preparation was pre-incubated with the tester strains (100 µl) and sterile buffer or the metabolic activation system (500 µl) for 60 min at 37 °C prior to adding the overlay agar (2000 µl) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used. A pre-experiment for toxicity was carried out. The experiment was repeated: the first main experiment used the plate incorporation method, the second experiment included pre-incubation.


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

METABOLIC ACTIVATION: Phenobarbital and beta-naphthoflavone induced rat liver S9: S9 mix included glucose-6-phosphate and NADP as co-factors. S9 mix contained 5% S9, and 0.5 ml S9 mix was added to a total of 2.7 ml top agar, giving a final concentration of approximately 1% S9. The activity of the S9 had been checked by the supplier for promutagen activity using 2-aminoanthracene and benzo[a]pyrene.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I, 5000 µg/plate (without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

Remarks on result:

other: all strains/cell types tested

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiments I and II with the exception of a toxic effect observed in tester strain TA 1537 at a concentration of 5000 µg/plate in experiment I.

Table 1 Pre-experiment for toxicity: mutation factor

Concentration μg/plate	TA 98		TA 100
	-MA	+MA	-MA	+MA
0*	1.0	1.0	1.0	1.0
3.16	0.7	1.4	1.0	1.2
10.0	0.7	1.1	1.0	1.2
31.6	0.9	1.5	0.8	1.3
100	1.0	1.1	0.9	1.1
316	1.1	1.0	1.0	1.2
1000	0.5	1.1	1.0	1.0
2500	0.9	1.2	0.9	1.1
5000	0.9	1.7	1.0	1.1
Positive control	19.9	58.9	7.9	6.4

* Solvent control with acetone

Table 2 Experiment 1 plate incorporation: revertants per plate (mean of 3 plates)

Concentration μg/plate	TA 98		TA 100		TA 1535		TA 1537		TA 102
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
0**	21	22	111	114	12	9	8	13	242	311
0*	20	17	128	107	12	8	10	12	279	256
31.6	17	25	108	137	10	10	9	11	272	298
100	20	19	112	114	7	9	9	9	275	294
316	21	17	126	129	11	9	8	10	273	262
1000	10	19	122	104	12	7	8	13	265	274
2500	17	20	121	122	10	8	7	9	248	313
5000	18	29	125	115	8	7	1.0	15	256	287
Positive control	391	1001	1007	683	1447	27	103	1435	517	517

* Solvent control with acetone

**Distilled water

Table 3 Experiment 2 pre-incubation: revertants per plate (mean of 3 plates)

Concentration μg/plate	TA 98		TA 100		TA 1535		TA1537		TA 102
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
0**	23	29	112	99	12	8	12	9	249	285
0*	23	31	104	91	8	12	7	8	229	258
31.6	19	26	100	109	10	9	9	7	213	298
100	19	28	112	108	12	4	5	8	235	312
316	27	23	118	99	8	9	10	7	262	304
1000	28	25	105	107	11	11	7	7	245	319
2500	26	25	116	116	10	12	11	6	244	347
5000	26	29	106	91	10	8	5	9	222	294
Positive control	659	1816	947	2229	1051	84	115	109	2029	596

* Solvent control with acetone

**Distilled water

Conclusions:

Trimethoxy(methyl)silane and its reaction products with 3-aminopropyltriethoxysilane and [3-(2,3-epoxypropoxy)propyl]trimethoxysilane have been tested in a study conducted according to OECD Test Guideline 471 and in compliance with GLP (BSL BIOSERVICE, 2012). No test-substance related increase in the number of revertants was observed with or without metabolic activation in either the initial plate incorporation assay or the repeat experiment using the pre-incubation method in Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 up to limit concentrations. Toxicity was observed in one strain in the absence of metabolic activation at the highest concentration tested. Appropriate positive and solvent controls were included and gave expected results, apart from the solvent and positive controls for TA 1535 with metabolic activation in experiment 1, where the mean values for both controls were just below the range of historical controls. 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:

02 May 2012 to 10 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimum essential medium) supplemented with 10% FBS (foetal bovine serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no information

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital / β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-experiment: 0.008 - 5.0 μl/ml; main experiment: 1.0, 2.5 and 5.0 μl/ml (-MA); 0.2, 0.3 and 0.4 μl/ml (+MA)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone;
- Justification for choice of solvent/vehicle: used at Sponsor´s request, and compatible with survival of cells and activity of S9. To reach a final concentration of 0.25% solvent v/v the solution was diluted in cell
culture medium (MEM). Precipitation of the test item could not be observed by the unaided eye. Based on the results of the solubility test, 5 μl/ml was chosen as the highest dose group of the test item for the pre-experiment.

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation; final concentration 600 μg/ml

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation; final concentration 0.83 μg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

Cells were seeded into Quadriperm dishes containing microscope slides (2 chambers per dish). Two days after seeding of the cells, the culture medium was replaced with serum-free medium containing the test item alone or the test item with 50 μl/ml S9 mix with metabolic activation. 4 h after treatment the cultures were washed twice with phosphate buffered saline and cultured in complete medium for the remaining culture time.

DURATION

- Exposure duration: 4 hours (with and without metabolic activation)

- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid was added to the culture 17.5 hours after the start of treatment. 2.5 hours later the cells were treated on the slides in the chambers with hypotonic solution (0.4% KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid (v/v).

STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: Duplicate cultures were not used. For each concentration, one Quadriperm dish containing four microscopic slides was seeded. At the end of the 20 hour preparation interval, the slides were divided into sets of two. At least one slide from each set was counted..

NUMBER OF CELLS EVALUATED: At least 200 well spread metaphases were evaluated per concentration.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; other: relative cell density

PRE-EXPERIMENT FOR TOXICITY
- 0.008, 0.016, 0.03, 0.06, 0.13, 0.25, 0.5, 1.0, 2.5 and 5.0 μl/ml, +/- MA.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreduplication: report states that endoreduplication should be reported when seen.

METABOLIC ACTIVATION:
Phenobarbital / β-naphthoflavone induced rat liver S9 (protein content 42 mg/l) was mixed with cofactor solution to result in a final protein concentration of 0.75 mg/ml in the cultures. Cofactors: 8 mM MgCl2; 33 mM KCl; 5 mM Glucose-6-phosphate; 5 mM NADP

Evaluation criteria:

A positive result is determined by: a clear and dose-related increase in the number of cells with aberrations; a biologically relevant response for at least one dose group, higher than laboratory negative control range (0-4.0% +/- MA). Note, the report does not state whether the historical negative control values are for solvent controls or medium controls.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.4 μl/ml with metabolic activation

Vehicle controls validity:

other: considerable variation was observed between the slides for the single culture used as vehicle control

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not determined

- Effects of osmolality: not determined

- Water solubility: soluble in acetone.

- Precipitation: non observed by unaided eye

- Other confounding effects: inconsistency of results between slides of the same concentration (single cultures used).

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: average values for controls were within the range of historical controls, though only one culture was used and there was considerable variation between the slides.

Main experiment. Structural chromosome aberrations, 4 h treatment, 20 h fixation

Concentration μl/ml	Count	Cells scored	Polyploid cells	Aberrant cells		Chromatid aberrations				Chromosome types			Other	MI %	Mean aberrant cells
				inc gaps	exc gaps	b	f	d	ex	ib	if	cx	ma		inc gaps	exc gaps
Without metabolic activation
0 (medium)	1	100	0	6	4	3	1	0	0	0	0	2	0	96	5.0	2.5
	2	100	0	4	1	1	0	0	0	0	0	0	0
	total	200	0	10	5	4	1	0	0	0	0	2	0
0 (acetone)	1	100	0	5	3	1	0	1	1	0	0	0	0	100	5.0	3.5
	2	100	0	5	4	2	1	0	0	0	0	1	0
	total	200	0	10	7	3	1	1	1	0	0	1	0
1.0	1	100	0	3	3	3	0	0	0	0	0	0	0	107	5.0	3.0
	2	100	1	7	3	1	1	1	0	0	0	0	0
	total	200	1	10	6	4	1	1	0	0	0	0	0
2.5	1	100	1	5	2	2	0	1	0	0	0	0	0	84	6.0	2.5
	2	100	0	7	3	1	0	1	0	0	1	0	0
	total	200	1	12	5	3	0	2	0	0	1	0	0
5.0	1	100	0	8	5	6	0	0	0	0	0	0	0	78	8.5	6.0
	2	100	0	9	7	4	2	0	2	0	0	0	0
	total	200	0	17	12	10	2	0	2	0	0	0	0
Positive control	1	100	0	7	6	3	1	1	1	0	0	0	0	91	12.5	8.0
	2	100	1	18	10	5	4	1	1	0	0	0	0
	total	200	1	25	16	8	5	2	2	0	0	0	0
with metabolic activation
0 (medium)	1	100	1	3	1	1	0	0	0	0	0	0	0	100	2.0	0.5
	2	100	0	1	0	0	0	0	0	0	0	0	0
	total	200	1	4	1	1	0	0	0	0	0	0	0
0 (acetone)	1	100	1	2	0	0	0	0	0	0	0	0	0	100	5.0	2.7
	2	200	0	13	8	9	0	0	0	0	0	0	0
	total	300	1	15	8	9	0	0	0	0	0	0	0
0.2	1	100	1	10	9	7	0	0	2	0	4	0	0	90	9.5	7.5
	2	100	1	9	6	4	0	1	2	0	0	0	0
	total	200	2	19	15	11	0	1	4	0	4	0	0
0.3	1	200	0	12	8	5	0	0	2	1	0	0	0	73	7.3	5.3
	2	200	1	17	13	10	2	0	3	0	0	0	1
	total	400	1	29	21	15	2	0	5	1	0	0	1
0.4	1	100	0	10	7	5	0	0	1	0	0	1	1	44	11.3	8.7
	2	200	2	24	19	12	3	1	4	0	1	1	1
	total	300	2	34	26	17	3	1	5	0	1	2	2
Positive control	1	100	0	14	10	9	1	0	4	0	0	0	0	70	13.0	10.5
	2	100	0	12	11	9	0	0	4	0	0	1	0
	total	200	0	26	21	18	1	0	08	0	0	1	0

MI: mitotic index; P: polyploidy; b: break; f: fragment; d: deletion; ex:chromatid type exchange; ib:iso-break; if: iso-fragment; cx: chromosome type exchange; ma: multiple aberrations (>4 events (exc gaps) - only exchanges recorded additionally for these cells. Iso-deletions and chromosomal disintegration (pulverisation) were also scored, and none recorded for any slide, so these are not included in the table

Conclusions:

Trimethoxy(methyl)silane and its reaction products with 3-aminopropyltriethoxysilane and [3-(2,3-epoxypropoxy)propyl]trimethoxysilane has been tested for cytogenicity to mammalian cells in Chinese hamster V79 cells in a study conducted according to OECD Test Guideline 473 and in compliance with GLP. Appropriate positive controls were included and gave expected results. The experiment was conducted with and without activation, and cells were exposed for four hours. Single cultures were used, and the experiment was not repeated because a positive result was reported. There was an increase in the number of cells with aberrations above solvent control levels at the highest concentration in the absence of metabolic activation, and at all concentrations evaluated that had been tested in the presence of metabolic activation. The evaluation of the slides from two of the doses (with metabolic activation) showed different results, so more than 200 metaphases were scored at these concentrations. The author of the report considered that this may possibly be an effect of the solubility of the test item. It is noted by the reviewer that extra cells were counted for the solvent control, presumably because of the variability between slides (0% and 4% aberrant cells excluding gaps), which suggests that experimental technique might have contributed to the variability. In addition, it is noted by the reviewer that there was not a clear dose response with metabolic activation. It is concluded by the author of the report that the test substance was positive for the induction of structural chromosome aberrations (was clastogenic) under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

An in vivo micronucleus assay is proposed to follow up the positive result in the in vitro cytogenicity study.

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

The registered substance, trimethoxy(methyl)silane and its reaction products with 3-aminopropyltriethoxysilane and [3-(2,3-epoxypropoxy)propyl]trimethoxysilane, has been tested in a study conducted according to OECD Test Guideline 471 and in compliance with GLP (BSL BIOSERVICE, 2012). No test-substance related increase in the number of revertants was observed with or without metabolic activation in either the initial plate incorporation assay or the repeat experiment using the pre-incubation method in Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 up to limit concentrations. Toxicity was observed in one strain in the absence of metabolic activation at the highest concentration tested. Appropriate positive and solvent controls were included and gave expected results, apart from the solvent and positive controls for TA 1535 with metabolic activation in experiment 1, where the mean values for both controls were just below the range of historical controls. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

The registered substance trimethoxy(methyl)silane and its reaction products with 3-aminopropyltriethoxysilane and [3-(2,3-epoxypropoxy)propyl]trimethoxysilane, has been tested for cytogenicity to mammalian cells in Chinese hamster V79 cells in a study conducted according to OECD Test Guideline 473 and in compliance with GLP (BSL BIOSERVICE, 2013). Appropriate positive controls were included and gave expected results. The experiment was conducted with and without activation, and cells were exposed for four hours. Single cultures were used, and the experiment was not repeated because a positive result was reported. There was an increase in the number of cells with aberrations above solvent control levels at the highest concentration in the absence of metabolic activation, and at all concentrations evaluated that had been tested in the presence of metabolic activation. The evaluation of the slides from two of the doses (with metabolic activation) showed different results, so more than 200 metaphases were scored at these concentrations. The author of the report considered that this may possibly be an effect of the solubility of the test item. It is noted by the reviewer that extra cells were counted for the solvent control, presumably because of the variability between slides (0% and 4% aberrant cells excluding gaps), which suggests that experimental technique might have contributed to the variability. In addition, it is noted by the reviewer that there was not a clear dose response with metabolic activation. It is concluded by the author of the report that the test substance was positive for the induction of structural chromosome aberrations (was clastogenic) under the conditions of the test.

Testing for mutagenicity to mammalian cells is not required as a positive result was obtained in the in vitro cytogenicity study.

An in vivo micronucleus study is proposed to investigate the potential for clastogenicity indicated by the results of the in vitro cytogenicity study.

Justification for classification or non-classification

Results from an in vitro mammalian cell cytogenicity assay indicated potential for clastogenicity, so in vivo testing is proposed.