Bis(triethoxysilylpropyl)amine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

Mutagenicity in bacteria: S. typhimurium strains: TA 98, TA 100, TA 1535 and TA 1537 and E. coli strain: WP2 uvrA: negative with and without metabolic activation (according to OECD 471)

Clastogenicity in mammalian cells: Human peripheral lymphocytes: negative with and without metabolic activation (according to OECD 473)

Mutagenicity in mammalian cells: mouse lymphoma L5178Y cells: negative with and without metabolic activation (according to OECD 476)

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:

08 Sep - 02 Nov 2010

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:

adopted 1997

Deviations:

yes

Remarks:

2-AA was used as sole positive control in the presence of S9-mix, which is in conflict with the OECD guideline 471 (adopted 1997). No further information on S9 characterisation was given from the supplier.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium strains: his operon
E. coli strain: trp operon

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Dose Range Finder: 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate (±S9)
Main test:
-S9: 9.78, 19.6, 39.1, 78.3, 157, 313 µg/plate (TA 100); 39.1, 78.1, 156, 313, 625, 1250 µg/plate (TA 98, TA 1535, TA 1537, E. coli)
+S9: 39.1, 78.1, 156, 313, 625, 1250 µg/plate

Vehicle / solvent:

DMSO (Lot No: K40982531)
- Justification for choice of solvent/vehicle: Based on sponsor information and the result of a preliminary solubility test.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (2-AA), 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF2)

Remarks:

+S9: 2-AA (TA 98, TA 100, TA 1535, TA 1537, WP2uvrA; 1, 2, 3, 3, 2 µg/plate); -S9: 2-NF (TA 98; 5 µg/plate); SA (TA 100, TA 1535; 1.5 µg/plate); 9-AA (TA 1537; 80 µg/plate); AF2 (WP2uvrA; 0.005 µg/plate)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 2 independent experiments with 3 plates/concentration.

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition

Evaluation criteria:

The results were considered positive when the following conditions are met:
- The number of revertant colonies in the test substance groups was increased at least twice as compared to the negative control group at one or more doses per plate in at least one strain.
- The numer of revertant colonies was increased dose dependently.
- The results were reproducible.

Statistics:

Mean values and standard deviation were calculated

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

625 µg/plate (without S9); 1250 µg/plate (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

313 µg/plate (without S9); 1250 µg/plate (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1250 µg/plate (with and without S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1250 µg/plate (with and without S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1250 µg/plate (with and without S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Deposition was observed at 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES: In the preliminary test growth inhibition was observed in all strains at 1250 µg/plate in the absence and presence of S9-mix. In addition growth inhibition was observed at 313 µg/plate in TA 100 in the absence of S9-mix.

Table 1: Test results of main test

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate in the plate incorporation test
	(μg/plate)	(average of 3 plates±standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvrA (pKM101)	TA98	TA1537
–	Solvent control	73±2	12±2	104±7	10±1	5±1
–	9.78	69±1	-	-	-	-
–	19.6	71±1	-	-	-	-
–	39.1	64±7	7±5	106±1	12±1	5±1
–	78.1 (78.3, TA 100)	68±4	9±1	114±4	11±2	4±2
–	156 (157, TA 100)	66±4	8±1	132±3	11±3	5±0
–	313	15±15	9±2	99±15	11±3	6±4
–	625	-	7±3	124±16	9±2	3±2
–	1250	-	4±1	90±12	7±3	5±2
Positive controls, –S9	Name	sodium azide	sodium azide	AF2	2-nitrofluorene	9-amino-acridine
	Concentrations (μg/plate)	1.5	1.5	0.005	5	80
	Average of 3 plates±sd	294±36	425±8	905±250	550±79	642±90
+	Solvent control	81±5	8±3	128±13	17±4	12±
+	39.1	85±12	7±1	130±33	19±1	10±3
+	78.1	91±9	5±1	54±6	21±0	9±2
+	156	81±6	6±1	127±42	21±2	10±4
+	313	82±13	7±1	142±48	19±1	10±4
+	625	80±7	7±1	148±1	18±1	14±2
+	1250	59±8	5±1	107±18	12±3	8±3
Positive controls, +S9	Name	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene
	Concentrations (μg/plate)	2	3	2	1	3
	Average of 3 plates±sd	194±27	118±21	544±28	191±30	239±39

 

Table 2: Test results of main test 2.

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate in the plate incorporation test
	(μg/plate)	(average of 3 plates±standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvrA (pKM101)	TA98	TA1537
–	Solvent control	72±5	8±2	114±8	12±3	5±3
–	9.78	69±15	-	-	-	-
–	19.6	70±1	-	-	-	-
–	39.1	65±3	10±2	119±2	10±4	4±2
–	78.1 (78.3, TA 100)	65±9	9±0	117±8	10±3	6±2
–	156 (157, TA 100)	66±8	10±2	102±1	12±4	5±3
–	313	49±8	8±3	137±7	10±3	4±1
–	625	-	6±2	130±8	10±5	4±1
–	1250	-	7±4	121±13	6±1	3±1
Positive controls, –S9	Name	sodium azide	sodium azide	AF2	2-nitrofluorene	9-amino-acridine
	Concentrations (μg/plate)	1.5	1.5	0.005	5	80
	Average of 3 plates±sd	349±38	420±15	917±15	376±34	313±20
+	Solvent control	85±11	8±3	149±19	16±7	11±4
+	39.1	69±3	8±2	136±8	14±6	10±
+	78.1	71±10	6±2	148±9	16±4	12±4
+	156	70±11	7±2	154±18	14±3	9±5
+	313	79±4	11±4	149±11	16±	13±1
+	625	79±3	7±2	156±10	20±7	11±6
+	1250	62±7	7±3	155±10	17±2	7±3
Positive controls, +S9	Name	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene
	Concentrations (μg/plate)	2	3	2	1	3
	Average of 3 plates±sd	298±25	99±7	482±31	150±13	171±3

 

 

Conclusions:

negative

3-(Triethoxysilyl)-N-[3-(triethoxysilyl)propyl]-1-propanamine has been tested for mutagenicity in bacteria, in a study which was conducted according to OECD 471 and in compliance with GLP. No evidence of a test substance related increase in the number of revertants was observed with or without metabolic activation in a preincubation experiment. 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:

18 Nov 2008 - 26 Feb 2009

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Freie und Hansestadt Hamburg, Behörede für Soziales, Familie, Gesundheit und Verbraucherschutz, Hamburg, Germany

Type of assay:

other: in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
Complete medium:
Chromosome medium 1A with phytohemagglutinin (Gibco, 500 ml) supplemented with Penicillin/Streptomycin (10000 IU/ml; 5 ml)
Treatment medium:
Ham´s F10 (500 ml) with Fetal Calf Serum (13.1 ml)

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:

Dose Range Finder:
4 h, +S9: 10, 25, 100, 250, 1000, 2500, 5000 µg/ml
24 h, -S9: 10, 25, 100, 250, 1000, 2500, 5000 µg/ml
Main test:
4 h, ±S9-mix: 156.3, 312.5, 625, 1250, 2500 µg/ml
24 h, -S9-mix: 62.5, 125, 250, 500, 1000 µg/ml

Vehicle / solvent:

Ethanol
- Justification for choice of solvent/vehicle: Substance has a small solubility in DMSO and water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

-S9: Mitomycin C (0.1 and 0.2 µg/ml); +S9: Cyclophosphamide (10 and 20 µg/ml)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 and 24 h
- Expression time (cells in growth medium): 20 h (4 h incubation), 0 h (24 h incubation)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.5 ml of a 10 µg/ml solution; added 2 h before harvest of cells)
STAIN (for cytogenetic assays): Giemsa stain (1:10 in WEISE´s buffer at pH 6.8)

NUMBER OF REPLICATIONS: 2 independant experiments with and without metabolic activation each. For the repeat experiment blood from a different donor was taken.

NUMBER OF CELLS EVALUATED: 100 metaphases

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (scoring of 1000 cells), observation of haemolysis

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

Evaluation criteria:

The test item is judged to have mutagenic properties with respect to chromosomal or chromatid change, if the following criteria are fulfilled:
- the number of chromosomal aberrations is significantly (at p ≤ 0.05) increased compared with the solvent control
- the increase observed is concentration-dependent
- both duplicate cultures lead to similar results
- the increase should not occur in the severely cytotoxic range (mitotic index <0.25), as it is known that high cytotoxicity causes artefacts in the form of aberrations in in vitro chromosomal aberration tests.
- a reproducible increase in the number of cells with chromosomal aberrations.

Statistics:

The assessment was carried out by a comparison of the number of chromosome aberrations of the samples with those of the solvent control, using the exact test of R.A. FISHER (p ≤ 0.05) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, Statistical evaluation of mutagenicity test data, 1989).

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight haemolysis at 1250 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was observed at 2500 and 5000 µg/ml in the range finding assay.
RANGE-FINDING/SCREENING STUDIES: In the range finding assay toxicity (observed as haemolysis) was observed from 1000 µg/ml.

COMPARISON WITH HISTORICAL CONTROL DATA:
No difference from the results obtained and the background data (years 2006-2007) given were observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Slight haemolysis was observed at 1250 µg/ml in the experiments with and without metabolic activation. Furthermore, in the experiments without metabolic activation only 114 (4 h) and 145 (24 h) metaphases of sufficient quality could be used for evaluation.

No item-related polyploidy or endoreduplication were noted in the experiments with or without metabolic activation. Some concentrations were not further evaluated, as it was thought that they would not provide further information.

Table 1: Results from chromosome analysis in 4 and 24 h treatments in the presence and absence of metabolic activation.

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/ml	in %	with gaps	without gaps
Exposure period 4 h, fixation time 24 h, without S9 mix
Ethanol	0	100	3.0	0.5
MMC	0.1	67	18.0	11.5*
Test substance	156	96	5.5	1.0
	312	107	4.5	1.0
	625	53	3.0	0.5
	1250s	15	2.0	1.8
Exposure period 4 h, fixation time 24h, with S9 mix, Experiment 1
Ethanol	0	100	4.0	1.0
CP	10	32	23.5	16.0*
Test substance	156	97	1.0	0.5
	312	96	1.5	1.0
	625	109	3.5	0.5
	1250s	46	3.5	1.5
Exposure period 4 h, fixation time 24 h, with S9 mix, Experiment 2
Ethanol	0	100	2.0	1.0
CP	10	48	22.0	14.5*
Test substance	156	93	3.5	2.0
	312	92	1.5	0.0
	625	96	1.5	1.5
	1250s	68	3.0	0.5
Exposure period 24h, fixation time 24 h, without S9 mix
Ethanol	0	100	2.0	1.0
MMC	0.1	87	18.0	11.5*
Test substance	62.5	86	5.5	2.0
	125	98	4.5	1.5
	250	98	3.5	1.5#
	500	31	6.3	2.8

s: slight haemolysis

#: tetraploidy (excluded from evaluation)

*: significantly different from solvent control (p≤0.05)

Conclusions:

negative

3-(Triethoxysilyl)-N-[3-(triethoxysilyl)propyl]-1-propanamine has been tested for clastogenicity in cultured human peripheral lymphocytes, in a study which was conducted according to OECD 473 and in compliance with GLP. No evidence of a test substance related increase in the number of cells with aberrations was observed with or without metabolic activation in short- and long-term treatments. No test item-related polyploidy or endoreduplication were noted in the experiments with or without metabolic activation. In conclusion, the test substance did not induce chromosome aberrations in human lymphocytes under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Aug - 20 Oct 2014

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit

Type of assay:

other: mammalian cell gene mutation assay

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 1640 supplemented with 10% horse serum (HS), 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phneobarbital and β-naphthoflavone for 3 consecutive days by oral route

Test concentrations with justification for top dose:

Experiment I
Without S9 mix: 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3 mM (4 h)
With S9 mix: 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3 mM (4 h)

Experiment II
Without S9 mix: 0.1, 0.2, 0.5, 1, 1.6, 2, 2.2, 2.4 and 2.6 mM (24 h)
With S9 mix: 0.25, 0.4, 0.8, 1.6, 2, 2.4, 2.8 and 3 mM (4h)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI cell culture medium
- Justification for choice of solvent: Based on the results of the solubility test RPMI cell culture medium was used as solvent (RPMI 1640 complete + 5% HS for short term exposure, RPMI 1640 complete+ 7.5% HS for long term exposure)

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 mix: MMS in 0.9% NaCl at 8 and 10 µg/ml, EMS in medium at 200 and 300 µg/ml; +S9 mix: B[a]P in DMSO at 2.5 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
1st experiment: 4 h exposure with and without S9 mix.
2nd experiment: 4 h exposure with and 24 h without S9 mix.
- Expression time (cells in growth medium): 2 days after the end of the treatment, cells were plated in 96-well microtitre plates for determination of:
1. cloning efficiency
2. mutation frequency containing TFT selective medium.
The microtitre plates were incubated for about 6 and 14 days, respectively
- Selection time (if incubation with a selection agent): approx 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): about 16-17 days

SELECTION AGENT (mutation assays): 5 µg/ml trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: quadruplicates in two independent experiments

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

OTHER:
Small and large colonies were differentiated, as small colonies are capable to
indicate chromosomal mutations.

Evaluation criteria:

The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10E6 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.

Statistics:

The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT. For the microwell method used here the Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection. Based on the null hypothesis of the Poisson distribution, the probable number of clones/well (P) is equal to –ln(negative wells/total wells) and the plating efficiency (PE) equals P/(number of cells plated per
well). Mutant frequency then was calculated as MF = (PE(cultures in selective medium)/PE(cultures in non-selective medium)). The mutant frequency is usually expressed as “mutants per 10E6 viable cells”.
The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor. To arrive at a GEF, distributions of negative/vehicle mutant frequencies of the MLA gathered from ten laboratories were analyzed. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation. Applying this definition to the collected data, the GEF arrived to be 126 for the
microwell method.
The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent control. Mutant frequencies of the solvent/negative controls were used as reference.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

dose dependent decrease of relative total growth, with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

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 (without and with metabolic activation).

Table 1: Experiment I - 4 h exposure - With Metabolic Activation
Concentration	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Induced Mutants per 1E+06 surviving cells
[mM]
0	100.0	100.0	58.5	/
0
0.1	117.3	119.2	61.0	2.4
0.2	115.5	120.2	52.8	-5.7
0.5	97.7	98.2	76.9	18.4
1	105.3	98.7	59.5	1.0
1.5	111.9	93.9	48.3	-10.3
2	99.2	51.7	50.8	-7.7
2.5	96.3	43.2	57.8	-0.8
3	62.7	15.4	40.6	-17.9
B[a]P, 2.5 µg/ml	105.3	63.9	488.1	429.5
B[a]P = Benzo[a]pyrene
Table 2: Experiment I - 4 h exposure - Without Metabolic Activation
Concentration	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Induced Mutants per 1E+06 surviving cells
[mM]
0	100.0	100.0	124.7	/
0
0.1	101.4	107.1	121.1	-3.6
0.2	92.3	94.5	141.8	17.1
0.5	103.1	110.2	134.1	9.4
1	110.2	95.5	101.5	-23.2
1.5	90.9	68.3	130.9	6.2
2	80.7	44.0	170.4	45.7
2.5	81.9	28.6	92.4	-32.3
3	66.1	15.5	119.9	-4.8
EMS, 300 µg/ml	80.7	69.6	637.8	513.1
MMS, 10 µg/ml	79.6	72.9	510.0	385.3
EMS = Ethyl methane sulphonate
MMS = Methyl methane sulphonate
Table 3: Experiment II - 4 h Exposure - With Metabolic Activation
Concentration	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Induced Mutants per 1E+06 surviving cells
[mM]
	100.0	100.0	55.2	/
0
0.25	83.6	84.9	69.9	14.7
0.4	87.4	76.3	52.4	-2.8
0.8	97.0	88.0	63.9	8.7
1.6	117.5	75.6	67.5	12.3
2	94.1	51.9	62.0	6.8
2.4	70.4	28.8	67.2	12.0
2.8	76.7	23.4	86.4	31.2
3	69.4	16.7	82.0	26.8
B[a]P, 2.5 µg/ml	77.8	41.3	576.4	521.2
B[a]P = Benzo[a]pyrene
Table 4: Experiment II - 24 h exposure - Without Metabolic Activation
Concentration	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Induced Mutants per 1E+06 surviving cells
[mM]
0	100.0	100.0	56.4	/
0
0.1	115.4	117.7	54.9	-1.5
0.2	126.0	114.9	50.9	-5.6
0.5	109.7	99.0	45.3	-11.2
1	89.7	49.0	86.9	30.4
1.6	92.4	59.8	72.0	15.5
2	96.7	49.8	62.6	6.2
2.2	101.3	44.7	78.4	22.0
2.4	102.9	22.8	106.5	50.0
2.6	95.2	9.4	58.6	2.2
EMS, 200 µg/ml	73.2	35.2	2130.6	2074.2
MMS, 8 µg/ml	61.8	35.5	1402.8	1346.3
EMS = Ethyl methane sulphonate
MMS = Methyl methane sulphonate

 

Conclusions:

negative

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item 3-(triethoxysilyl)-N-[3-(triethoxysilyl)propyl]-1-propanamine 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

Genetic toxicity (mutagenicity) in bacteria in vitro

The mutagenicity of 3-(triethoxysilyl)-N-[3-(triethoxysilyl)propyl]-1-propanamine (CAS 13497-18-2) in bacteria was assessed in an experiment according to OECD TG 471 with Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 and the Escherichia coli strain WP2 uvrA (pKM101) (Biotoxtech Co., Ltd., 2010). A deviation to the guideline was the use of 2-aminoanthracene as the sole positive control substance in the presence of S9-mix. No further information is given, if the S9-mix has been characterised with a second substance. This deviation was supposed to have no implication on the outcome of the study results. The tester strains were treated using the preincubation method both with and without S9-mix. The concentrations tested were 39.1 - 1250 µg/plate in the presence of S9-mix as well as 9.78 – 313 (TA 100) and 39.1 -1250 µg/plate (all strains except TA 100) in the absence of S9-mix. Results achieved with vehicle (DMSO) and positive controls were valid. No genotoxicity was observed in the presence and absence of metabolic activation. Deposition of the test material was observed in the range finding assay at 5000 µg/plate. Growth inhibition was observed in all strains at 1250 µg/plate in the presence and absense of S9-mix, except for strain TA 100 it was observed at 313 µg/plate in the absence of S9-mix. In conclusion, 3-(triethoxysilyl)-N-[3-(triethoxysilyl)propyl]-1-propanamine did not induce mutations in bacteria under the test conditions applied.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

3-(Triethoxysilyl)-N-[3-(triethoxysilyl)propyl]-1-propanamine has been tested for clastogenicity in cultured human peripheral lymphocytes in a study which was conducted according to OECD TG 473 and in compliance with GLP (LPT, 2009). Human peripheral lymphocytes were cultured and treated with 3-(triethoxysilyl)-N-[3-(triethoxysilyl)propyl]-1-propanamine (diluted in ethanol) at concentrations of 156-2500 µg/ml (4 h, ±S9) or 62.5 – 1000 µg/ml (24 h, -S9) in the main experiments. Two hours before harvesting the cells (22 h after start of treatment), colcemid was added to the cultures. 24 h after start of treatment the cells were stained with Giemsa stain (10%) and 200 metaphases were scored per concentration (if available). Cytotoxicity was assessed by determination of the mitotic index (readout of 1000 cells) and by the observation of haemolysis. In a preliminary assay precipitation of the test substance was observed at 2500 and 5000 µg/mL and slight haemolysis was observed at 1000 µg/ml or at higher concentrations. No evidence of a test substance related increase in the number of cells with aberrations was observed with or without metabolic activation in short- and long-term treatments in the main experiments. No test item-related polyploidy or endoreduplication were noted in the experiments with or without metabolic activation. The results of the solvent and positive controls were within the range of the historical control data. Some concentrations were not further evaluated, as it was thought that they would not provide further information. In conclusion, the test substance did not induce chromosome aberrations in human lymphocytes under the conditions of the test.

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

In the key in vitro mammalian mutagenicity study (BSL, 2015), the test item 3-(triethoxysilyl)-N-[3-(triethoxysilyl)propyl]-1-propanamine (CAS 13497-18-2) was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The study was conducted according to OECD TG 476 and in compliance with GLP. 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. Concentrations with RTG values below 10% were not considered for evaluation of mutagenicity. No dose-response relationship was observed. 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 in the test and gave the expected results. It was therefore concluded that the test substance is not mutagenic to mammalian cells under the conditions of the test.

Justification for classification or non-classification

The available data on genetic toxicity from the test substance 3-(triethoxysilyl)-N-[3-(triethoxysilyl)propyl]-1-propanamine (CAS 13497-18-2) do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.