Reaction mass of 2-{[3-(trimethoxysilyl)propyl]amino}ethanaminium chloride and 2-{benzyl[3-(trimethoxysilyl)propyl]amino}ethanaminium chloride and N-[2-(benzylamino)ethyl]-3-(trimethoxysilyl)propan-1-aminium chloride and N-[2-(dibenzylamino)ethyl]-3-(trimethoxysilyl)propan-1-aminium chloride and N-benzyl-2-{benzyl[3-(trimethoxysilyl)propyl]amino}ethanaminium chloride and N-benzyl-N-[2-(dibenzylamino)ethyl]-3-(trimethoxysilyl)propan-1-aminium chloride.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471, GLP): negative with and without metabolic activation.
Chromosome aberration assay (OECD TG 473, GLP): negative with and without metabolic activation.
Mouse Lymphoma Assay (OECD 476, GLP): negative with and without metabolic activation.

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:

22 June - 06 July 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was conducted according to OECD TG 471 with acceptable restrictions. The restriction was that no additional strain for the detection of crosslinking agents was included into the study. The study was further conducted in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1983)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

yes

Remarks:

(no additional strain for the detection of crosslinking agents was included into the study)

Qualifier:

according to guideline

Guideline:

other: EU Method B.14 (Mutagenicity - Reverse Mutation Test Using Bacteria) (1984)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

- experiment 1: 3.3, 10, 33, 100, 200 µg/plate
- experiment 2: 3.3, 10, 33, 100, 333 µg/plate

Vehicle / solvent:

- Solvent used: DMSO (spectroscopic quality (Merck) to which molecular sieve (0.4 nm) beads (about 2 mm) were added to absorb the water)
- Justification for choice of solvent: The test article was found to be not stable in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: sodium azide (SA; 1 µg/plate;TA1535); 9-aminoacridine (9AC; 60 µg/plate; TA1537); daunomycin (DM; 4 µg/plate; TA98); methylmethanesulfonate (MMS; 650 µg/plate; TA100);+S9: 2-aminoanthracene (2AA; 5 µg/plate:TA1535 and TA1537;0.5 µg/plate:TA98/TA100)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replicates in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (In a preliminary cytotoxicity experiment with TA100 both in absence and presence of metabolic activation nine concentrations of the test item were tested for cytotoxicity. The highest concentration of the test article used in the subsequent mutagenesis assay was that which gave a reduced survival on the non-selective plates.)

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the Ames test if:
- The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
- The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considerd positive (mutagenic) in the Ames test if:
- It induces at leats a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant. If the test substance showed in the first test only a positive response at one or two concentrations, the assay was repeated with doses below and exceeding those showing positive effects in the first test.
- The positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- none

ADDITIONAL INFORMATION ON CYTOTOXICITY: The cytotoxicity of the test item was tested with TA100 in absence and presence of metabolic activation. In the presence of S9-mix the survival of strain TA100 is not reduced up to and including test substance concentrations of 33 µg/plate, slightly reduced at a concentration fo 100 µg/plate, extremely reduced at concentrations from 333 µg/plate up to and including 3330 µg/plate and absent at a concentration of 5000 µg/plate.
In the absence of S9-mix the survival of strain TA100 is not reduced up to and including test substance concentrations of 33 µg/plate, moderately reduced at a concentration fo 100 µg/plate, extremely reduced at a concentrations of 333 µg/plate and absent at test substance concentrations of 1000 µg/plate and upwards.
Based on these data, the test substance was tested up to and including a concentration of 200 µg/plate in the absence and presence of metabolic activation (experiment 1). In the first experiment no toxicity was observed, therefore it was decided to test up to and including a concentration of 333 µg/plate in the second experiment.

Tab. 1: Mutagenic response of ORGANOFUNCTIONAL SILANE A-1128 in the Ames Salmonella/microsome plate test. Given is the mean number of revertant (His+) colonies / 3 replicate plates (± SD) with different strains of S. typhimurium (experiment 1).

Dose [µg/plate]		TA1535		TA1537		TA100		TA98
		+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9
Test item	3.3	17 ± 5	16 ± 1	7 ± 3	6 ± 2	29 ± 6	21 ± 1	97 ± 7	88 ± 8
	10	20 ± 5	17 ± 3	6 ± 1	3 ± 2	27 ± 3	20 ± 2	97 ± 4	85 ± 10
	33	15 ± 5	15 ± 4	6 ± 1	4 ± 2	28 ± 1	21 ± 5	105 ± 4	85 ± 12
	100	16 ± 2	15 ± 6	9 ± 2	6 ± 1	29± 6	25 ± 3	112 ± 6	87 ± 1
	200	12 ± 3	9 ± 2	9 ± 1	6 ± 2	26 ± 6	22 ± 2	113 ± 6	75 ± 21
Solvent control		14 ± 1	19 ± 1	11 ± 1	4 ± 2	30 ± 5	22 ± 4	87 ± 6	89 ± 8
Positive control		282 ± 49	273 ± 15	902 ± 64	154 ± 22	362 ± 25	321 ± 34	585 ± 71	844 ± 99

 

Tab. 2: Mutagenic response of ORGANOFUNCTIONAL SILANE A-1128 in the Ames Salmonella/microsome plate test. Given is the mean number of revertant (His+) colonies / 3 replicate plates (± SD) with different strains of S. typhimurium (experiment 2).

Dose [µg/plate]		TA1535		TA1537		TA100		TA98
		+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9
Test item	3.3	7 ± 2	9 ± 3	8 ± 3	6 ± 1	33 ± 2	32 ± 4	88 ± 7	80 ± 8
	10	7 ± 1	10 ± 4	8 ± 5	6 ± 3	30 ± 3	25 ± 3	84 ± 5	69 ± 3
	33	11 ± 2	9 ± 2	8 ± 3	5 ± 2	30 ± 8	22 ± 7	84 ± 8	63 ± 20
	100	11 ± 3	13 ± 1	10 ± 6	7 ± 1	32± 1	28 ± 6	100 ± 19	84 ± 10
	333	12 ± 2	6 ± 5	5 ± 2	3 ± 1	25 ± 5	19 ± 9	70 ± 2	40 ± 8
Solvent control		10 ± 1	8 ± 1	6 ± 2	7 ± 2	30 ± 6	27 ± 4	92 ± 26	80 ± 10
Positive control		252 ± 59	198 ± 28	1008 ± 10	145 ± 7	493 ± 20	443 ± 68	576 ± 13	1030 ± 18

 

Conclusions:

Interpretation of results: negative with and without metabolic activation

The test item was tested for mutagenicity to bacteria according to the OECD TG 471 (1983) and in compliance with GLP. No additional strain for the detection of crosslinking agents was included into the study. No test material related increase in revertants was observed up to cytotoxic concentrations. Hence, the test item is not mutagenic under the conditions of the study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 July - 20 July 1992

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:

(1983)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1984)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes, provided by a healthy adult male volunteer (age 27)

Details on mammalian cell type (if applicable):

- Type and identity of media: F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% heat-inactivated (56 °C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicilline/strptomycine (50 U/ml respectively), sodium bicarbonate (2 g/l), and heparine (30 U/ml).

Metabolic activation:

with and without

Metabolic activation system:

S9-homogenate of male rats

Test concentrations with justification for top dose:

- without S9: 100, 133, and 180 µg/ml culture medium (24 h fixation period), and 237 µg/ml culture medium (48 h fixation period)
- with S9: 180, 237, and 333 µg/ml culture medium (24 h fixation period), and 333 µg/ml culture medium (48 h fixation period)

Vehicle / solvent:

- Solvent used: DMSO (spectroscopic quality (Merck) to which molecular sieve (0.4 nm) beads (about 2 mm) were added to absorb the water)
- Justification for choice of solvent: The test article was found to be not stable in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation: (MMC-C) 0.2 µg/ml in HBSS (24 h treatment) and 0.1 µg/ml in HBSS (48 h treatment)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation: (CP) 15 µg/ml in HBSS (3 h treatment)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 and 48 h (without metabolic activation), 3 h (with metabolic activation)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/ml medium)
STAIN (for cytogenetic assays): Giemsa solution in tap water

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: at least 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: The mitotic index was determined in a pilot study carried out for the purpose of dose selection (24 and 48 h fixation period). The tested doses were: 33, 100, 133, 180, and 237 µg/ml without metabolic activation, and 33, 100, 180, 237, and 333 µg/ml with metabolic activation.

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
- it induced a dose-related statistically significant (Chi-square test, p < 0.05) increase in the number of cells with chromosome aberrations.
- a satistically significant increase in the frequency of aberrations was observed in the absence of a clear dose-response relationship, but the results were reproducible in an independently repeated experiment.

A test substance is considered negative (non-clasogenic) in the chromosome aberration test if:
- none of the tested concentrations induced a statistically significant increase (Chi-square test, p < 0.05) in the number of cells with chromosome aberrations.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each treatment group was compared to that of the solvent control using Chi-square statistics.
If p (two-tailed) is small (< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence level.

Species / strain:

lymphocytes: cultured peripheral human lymphocytes, privided by a healthy adult male volunteer (age 27)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- none

RANGE-FINDING/SCREENING STUDIES: A pilot study was carried out for dose selection based on cytotoxicity of the test item (reduction of the mitotic index).

COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome aberrations found in the solvent control cultures fell within the laboratory historical control data range.

VALIDITY OF THE TEST: The positive control chemicals (MMC-C and CP) both produced significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were optimal and that the metabolic activation system (S9 mix) functioned properly.
Finally it is concluded that this test should be considered valid.

Tab. 1: Chromosome aberration in donor cultures treated with various concentrations of the test item in the absence of metabolic activation.

Concentration	24 h fixation period															48 h fixation period
	DMSO (0.9% v/v)			100 µg/ml			133 µg/ml			180 µg/ml			MMC-C 0.2 µg/ml			DMSO (0.9% v/v)			237 µg/ml			MMC-C 0.1 µg/ml
Culture	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B
No. of cells scored	100	100	200	100	100	200	100	100	200	100	100	200	100	100	200	100	100	200	100	100	200	100	100	200
No. of cells with aberrations (+ gaps)	6	3	9	0	1	1	2	2	4	2	1	3	44	25	69	2	2	4	2	1	3	42	51	93
No. of cells with aberrations (- gaps)	1	0	1	0	0	0	0	0	0	0	0	0	33	21	54	0	0	0	1	0	1	28	40	68
Chromatid gap	8	3			1		2	2		2	1		19	6		2	2		1	1		24	21
Chromosome gap													3	3								4
Chromatid break	1												26	16					1			17	27
Chromosome break													6	3								4	2
Chromatid deletion													1									4

Tab. 2: Chromosome aberration in donor cultures treated with various concentrations of the test item in the presence of metabolic activation.

Concentration	24 h fixation period															48 h fixation period
	DMSO (0.9% v/v)			180 µg/ml			237 µg/ml			333 µg/ml			CP 15 µg/ml			DMSO (0.9% v/v)			333 µg/ml
Culture	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B	A	B	A + B
No. of cells scored	100	100	200	100	100	200	100	100	200	100	100	200	100	100	200	100	100	200	100	100	200
No. of cells with aberrations (+ gaps)	4	1	5	4	0	4	1	2	3	4	1	5	28	32	60	1	2	3	3	3	6
No. of cells with aberrations (- gaps)	0	0	0	0	0	0	0	1	1	0	0	0	25	26	51	1	0	1	0	0	0
Chromatid gap	3	1		4			1	1		4	1		14	16			2		3	3
Chromosome gap	1			1										3
Chromatid break								1					18	26
Chromosome break														7
Chromatid deletion													1	1

Conclusions:

Interpretation of results: negative with and without metabolic activation

The test item was tested forclastogenicity to cultured human peripheral lymphocytes according to the OECD TG 473 (1983) and in compliance with GLP. The test item was concluded not clastogenic with and without metabolic activation under the experimental conditions of this test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-08-22 to 2012-11-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP. No data on test material purity is given in the report. However, a certificate of analysis is included, but the data are not sufficient to calculate the purity, since the submission substance is a multiconstituent.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(no data on test material purity given)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: IWGT Recommendations

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

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

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

- Pre-Experiment I (with and without metabolic activation): 50, 150, 500, 1000, 2000, and 5000 µg/ml
- Pre-Experiment II (24 h long-term exposure assay, without metabolic activation): 1, 5, 10, 20, 50, and 80 µg/ml
- Experiment I with metabolic activation: 1.5, 3, 7, 15, 30, 75, 150, and 200 µg/ml and without metabolic activation: 1.5, 3, 7, 15, 40, 55, 70, and 85 µg/ml
- Experiment II with metabolic activation: 8, 16, 35, 70, 160, 170, 180, and 210 µg/ml and without metabolic activation: 2, 5, 10, 20, 30, 45, 50, and 55 µg/ml

Vehicle / solvent:

Due to the nature of the test item it was not possible to prepare a solution of the test item with an appropriate solvent. Based on the results of the solubility test the best suited vehicle was RPMI cell culture medium (RPMI+5% HS). The suspension was treated with ultrasonication for 5 minutes at 37°C and after that the suspension was dispersed.
The vehicle was compatible with the survival of the cells of the S9 activity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Remarks:

+S9: benzo(a)pyrene (B[a]P) 1.5 and 2.5 µg/ml; -S9: ethylmethanesulphonate (EMS) 200 and 300 µg/ml, methylmethanesulphonate (MMS) 8 and 10 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION: 4 h (short-term exposure), 24 h (long-term exposure)
Expression time (cells in growth medium): 48 h
Selection time (if incubation with selection agent): about 14 days

SELECTION AGENT ( mutation assay) 5 µg/ml trifluorothymidine
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; cells were seeded in 4 plates and evaluated
NUMBER OF CELLS SEEDED: 2000 cells per well
DETERMINATION OF CYTOTOXICITY: relative total growth (RTG)

Evaluation criteria:

The test item is considered mutagenic if following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10^6 cells
- A dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.

Statistics:

The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

+S9: 200 µg/ml and above; -S9: 55 µg/ml and above (4 h), 30 µg/ml and above (24 h)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect observed
- Effects of osmolality: no effect observed
- Solubility: Due to the nature of the test item it was not possible to prepare a solution of the test item with an appropriate solvent. Therefore, the test iteam was applied as a suspension in RPMI cell culture medium (RPMI+5% HS).
- Precipitation: not observed

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor, which is defined as the mean of the negative / vehicle mutant frequencies gathered from 10 participating laboratories plus one standard deviation. The GEF is 126 for the applied microwell method.

Tab. 1: Main Experiment I – 4 h exposure duration with metabolic activation

Test group	Concentration [µg/ml]	RTG^1 [%]	MF^2 [mutants/1E+6 cells]	IMF^3 [mutants/1E+6 cells]	small colonies [%]
Solvent control	0	100	82.3	-	6.7
			69.9	-	12.2
Test item	1.5	88.5	103.5	27.4	n.d.
	3	81.1	96.7	20.6	n.d.
	7	109.3	70.5	-5.6	n.d.
	15	98.4	84.5	8.4	n.d.
	30	87.5	108.1	32.0	n.d.
	75	73.8	100.2	24.1	14.8
	150	71.7	65.6	-10.5	14.9
	200	11.0	98.8	22.7	14.8
B[a]P	2.5	35.6	446.0	369.9	40.8

Tab. 2: Main Experiment I – 4 h exposure duration without metabolic activation

Test group	Concentration [µg/ml]	RTG^1 [%]	MF^2 [mutants/1E+6 cells]	IMF^3 [mutants/1E+6 cells]	small colonies [%]
Solvent control	0	100	53.7	-	23.6
			62.1	-	27.3
Test item	1.5	75.5	50.5	-7.4	n.d.
	3	80.4	36.6	-21.3	n.d.
	7	78.6	41.1	-16.8	n.d.
	15	74.9	38.9	-19.0	n.d.
	40	59.4	49.0	-8.9	n.d.
	55	29.8	55.1	-2.8	13.5
	70	16.2	47.0	-10.9	33.3
	85	11.6	72.1	14.2	9.3
EMS	300	56.9	465.3	407.3	n.d.
MMS	10	32.1	462.6	404.7	50.0

Tab. 3: Main Experiment II – 4 h exposure duration with metabolic activation

Test group	Concentration [µg/ml]	RTG^1 [%]	MF^2 [mutants/1E+6 cells]	IMF^3 [mutants/1E+6 cells]	small colonies [%]
Solvent control	0	100	89.1	-	9.8
			68.9	-	4.1
Test item	8	115.5	71.0	-8.0	n.d.
	16	95.5	83.2	4.2	n.d.
	35	103.6	71.1	-7.8	n.d.
	70	94.5	91.0	12.0	n.d.
	160	97.2	67.7	-11.3	n.d.
	170	90.2	56.1	-22.9	5.4
	180	66.1	91.0	12.0	7.5
	210	18.0	70.7	-8.3	16.3
B[a]P	1.5	77.6	420.1	341.1	44.2

Tab. 4: Main Experiment II – 24 h exposure duration without metabolic activation

Test group	Concentration [µg/ml]	RTG^1 [%]	MF^2 [mutants/1E+6 cells]	IMF^3 [mutants/1E+6 cells]	small colonies [%]
Solvent control	0	100	78.8	-	8.5
			62.2	-	8.3
Test item	2	92.3	66.6	-4.0	n.d.
	5	111.4	58.6	-11.9	n.d.
	10	79.2	55.8	-14.7	n.d.
	20	76.1	60.7	-9.8	n.d.
	30	43.8	54.8	-15.7	n.d.
	45	39.3	55.9	-14.7	11.1
	50	30.9	68.9	-1.6	12.8
	55	20.1	70.9	0.4	3.9
EMS	200	27.1	2239.3	2168.8	n.d.
MMS	8	37.9	849.8	779.3	47.6

¹RTG=Relative Total Growth

²MF=Mutant Frequency={-ln[solvent control cultures/total wells (selective medium)]/-ln[solvent control cultures/total wells (non-selective medium)]}x800

³MF=Induced Mutant Frequency=mutant frequency sample-mean value mutant frequency corresponding controls

n.d.=not determined

B[a]P= Benzo[a]pyrene, EMS=Ethylmethanesulfonate, MMS=Methylmethanesulfonate

Conclusions:

Interpretation of results: negative

The test item was tested for in vitro mutagenicity in the Mouse Lymphoma Assay. The study was conducted according to the OECD test guideline 476, and in compliance with GLP. In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Reaction mass of 6 benzylated amine silanes (ELINCS 414-340-6) is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro:

An Ames test according to OECD TG 471 (adopted 1983) and in compliance with GLP was performed to assess the mutagenic properties of the test substance (NOTOX, 1992a). S. typhimurium TA 1535, TA 1537, TA 98, and TA 100 were treated with 3.3-333 µg/plate of the test substance in the presence or absence of a metabolic activation system. No increase of the mutation frequency was observed in any tester strain at any dose level. No toxicity and no precipitation were observed up to 200 µg/plate in the first of 2 experiments, thus testing with up to 333 µg/plate was conducted in the second experiment. The negative and positive controls were valid. In contrast to the actual guideline, which recommends the use of an additional strain for testing of cross-linking substances, only 4 strains were used in this test, limiting the informative value. In conclusion, the test substance is not proposed to be mutagenic under the conditions of this test.

In a second in vitro test, performed according to OECD TG 473 and in compliance with GLP, the test substance was investigated on its ability to induce chromosome aberrations (NOTOX, 1992b). Cultured peripheral human lymphocytes were treated for 3 hours with 180, 237, and 333 µg/ml culture medium (24 h fixation period), and 333 µg/ml culture medium (48 h fixation period) in the presence of metabolic activation. In the absence of metabolic activation the cells were treated with 100, 133, and 180 µg/ml culture medium (24 h fixation period), and 237 µg/ml culture medium (48 h fixation period) until fixation. No genotoxic effects were observed within the study. Appropriate solvent and positive controls were included and showed expected results. The test substance is considered to be not genotoxic under the test conditions applied.

In the third available in vitro test (BSL, 2013), performed according to OECD TG 476, and in compliance with GLP, the test item was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The selection of the concentrations used in the main experiments was based on cytotoxicity data from the pre-experiments. In experiment I 200 μg/ml (with metabolic activation) and 85 µg/ml (without metabolic activation) were selected as the highest concentrations. In experiment II 210 µg/ml (with metabolic activation) and 55 µg/ml (without metabolic activation) were selected as the highest concentrations. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay. Due to the nature of the test item it was not possible to prepare a solution of the test item with an appropriate solvent. Therefore, the test item was applied as a suspension in RPMI cell culture medium (RPMI+5% HS). No precipitation of the test item was noted in the experiments. Growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I with metabolic activation the relative total growth (RTG) was 11.0% for the highest concentration (200 µg/ml) evaluated. The highest concentration evaluated without metabolic activation was 85 µg/ml with a RTG of 11.6%. In experiment II with metabolic activation the relative total growth (RTG) was 18.0% for the highest concentration (210 µg/ml) evaluated. The highest concentration evaluated without metabolic activation was 55 µg/ml with a RTG of 20.1%. In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration. In addition no dose-response relationship was observed. Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation). Appropriate solvent and positive controls were included into the test and gave the expected results. The test item is therefore concluded to be not mutagenic to mammalian cells in vitro under the conditions of the test.

In vivo:

No data available.

Justification for classification or non-classification

The available in vitro genotoxicity data are reliable and suitable for classification. Based on this data, classification for mutagenicity according to Regulation 67/584/EEC and (EC)1272/2008 is not warranted.