N,N-dimethyl-3-(trimethoxysilyl)propylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethyl-3-(trimethoxysilyl)propylamine

EC Number:

219-786-3

EC Name:

N,N-dimethyl-3-(trimethoxysilyl)propylamine

Cas Number:

2530-86-1

Molecular formula:

C8H21NO3Si

IUPAC Name:

dimethyl[3-(trimethoxysilyl)propyl]amine

Test material form:

liquid

Details on test material:

- EC No.: 219-786-3
- CAS No.: 2530-86-1
- Chemical Name: N,N-Dimethyl(aminopropyl)trimethoxysilane
- Batch No.: 1000095747
- Purity: 98.7%
- Physical State at Room Temperature: liquid
- Colour: colourless to yellowish
- Molecular Weight: 207.35 g/mol
- Storage Conditions: room temperature
- Stability in Container after Opening at Room Temperature: instable after repeated contact to air
- Stability in Water at Room Temperature: undergoes hydrolysis
- Expiry Date: 26 March 2017
- Safety Precautions: causes severe skin burns and eye damage
- Other Specification: in order to avoid degradation the test item vials will be flooded for storage with nitrogen

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- Experiment I: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
- Experiment II: 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (TA98, TA1537)

Remarks:

Without S9-mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (all strains)

Remarks:

With S9-mix

Details on test system and experimental conditions:

Samples of each tester strain were grown by culturing for 12 h at 37°C in Nutrient Broth to the late exponential or early stationary phase of growth (approx. 10E9 cells/mL). The nutrient medium consists per litre:
- 8 g Nutrient Broth
- 5 g NaCI
A solution of 125 µL ampicillin (10 mg/mL) (TA 98, TA 100) was added in order to retain the phenotypic characteristics of the strain.

The Vogel-Bonner Medium E agar plates with 2 % glucose used in the Ames Test were prepared by Eurofins Munich or provided by an appropriate supplier. Quality controls were performed. Sterilisation was performed for 20 min at 121°C in an autoclave.

The overlay agar contains per litre:

S. typhimurium:
- 7.0 g Agar Agar
- 6.0 g NaCI
- 10.5 mg L-histidine x HCI x H2O
- 12.2 mg biotin

E. coli:
7.0 g Agar Agar
6.0 g NaCl
10.2 mg tryptophan

Sterilisation was performed for 20 min at 121°C in an autoclave.

Evaluation criteria:

CYTOTOXICITY:
Cytotoxicity can be detected by a 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.

VALIDITY:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100)
- the mean values of the spontaneous reversion frequency of the control plates with and without S9 mix are within the historical control data range
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

MUTAGENICITY
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (exact values).
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 folIows:
- if in tester strains TA 98, TA 100 and E. coli WP2 uvrA 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.
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. A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

RESULTS

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

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 experiment I.

In experiment II toxic effects of the test item were noted in tester strains TA 98, TA 1535 and E. coli WP2 uvrA at a concentration of 5000 μg/plate (with and without metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 2500 μg/plate and higher (with and without metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 1000 μg/plate and higher (without metabolic activation) and at concentrations of 2500 μg/plate and higher (with metabolic activation).

The reduction in the number of revertants down to a mutation factor of 0.5 found in experiment I in tester strains TA 1535 at a concentration of 316 μg/plate (with metabolic activation) and TA 1537 (without metabolic activation) at a concentration of 31.6 μg/plate was regarded as not biologically relevant due to lack of a dose-response relationship. As well the reduction in the number of revertants down to a mutation factor of 0.5 found in experiment II in tester strain TA 1535 (with metabolic activation) at a concentration of 316 μg/plate was regarded as not biologically relevant due to lack of a dose-response relationship.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments. The negative control plates with and without S9 mix were within the historical control data range.

EXPERIMENT I (Plate-incorporation Test)

Mean revertant colonies per plate

	TA 98	TA98	TA 100	TA 100	TA 1535	TA 1535	TA 1537	TA 1537	E. coli	E. coli
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
neg. control	24	31	122	88	15	4	9	8	47	37
solvent control	23	28	97	82	14	8	9	7	54	38
test item 31.6 µg	18	30	77	72	13	4	5	8	44	35
test item 100 µg	27	32	86	73	13	8	8	9	46	30
test item 316 µg	24	29	72	94	12	4	8	8	59	38
test item 1000 µg	22	25	91	94	13	11	8	8	36	44
test item 2500 µg	19	23	109	81	23	10	7	10	36	28
test item 5000 µg	18	23	104	96	17	9	9	11	45	36
pos. control	488	1903	937	2044	936	141	117	258	427	253

EXPERIMENT II (Pre-incubation Test)

Mean revertant colonies per plate

	TA 98	TA98	TA 100	TA 100	TA 1535	TA 1535	TA 1537	TA 1537	E. coli	E. coli
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
neg. control	22	30	74	84	4	9	7	6	35	43
solvent control	25	26	51	70	5	4	5	5	38	48
test item 10 µg	30	30	52	66	8	9	5	3	33	37
test item 31.6 µg	21	27	63	75	7	6	5	6	35	38
test item 100 µg	26	28	46	73	3	6	3	5	27	33
test item 316 µg	25	25	47	78	6	2	3	4	42	37
test item 1000 µg	24	23	40	70	6	6	4	6	33	46
test item 2500 µg	21	35	63	40	8	5	5	6	25	39
test item 5000 µg	0	11	0	0	0	2	0	0	19	29
pos. control	415	683	304	1249	268	43	81	154	369	110

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

- Experiment I: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

- Experiment II: 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

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

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 experiment I.

In experiment II toxic effects of the test item were noted at concentrations of 1000 μg/plate and higher (without metabolic activation) and at concentrations of 2500 μg/plate (with metabolic

activation), depending on the particular tester strain.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the tet item is considered to be non-mutagenic in this bacterial reverse mutation assay.