N-butyl-N-[(triethoxysilyl)methyl]butan-1-amine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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-butyl-N-[(triethoxysilyl)methyl]butan-1-amine

EC Number:

810-490-5

Cas Number:

35501-23-6

Molecular formula:

C15H35NO3Si

IUPAC Name:

N-butyl-N-[(triethoxysilyl)methyl]butan-1-amine

Constituent 2

Reference substance name:

N,N-Dibutylaminomethyl-triethoxysilan

IUPAC Name:

N,N-Dibutylaminomethyl-triethoxysilan

Test material form:

other: liquid

Details on test material:

Name: N,N-Dibutylaminomethyl-triethoxysilan
CAS No: 35501-23-6
Lot No: 310113
Physical State: liquid
Colour: colourless to yellowish
Storage Conditions: at room temperature, protected from light,
protected from water/ humidity
Purity: 97.7% (GC)
Stability: rapid hydrolysis in water
Date of analysis: 31 January 2013
Expiry Date: 21 January 2015

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The S9 liver microsomal fraction was prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The following quality control determinations are performed:

a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test

A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at ≤-75 °C.
The protein concentration in the S9 preparation (Lot: 280613A, 280613B) was 33.6 and 33.4 mg/mL, respectively.

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5.0 μL/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations: 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: The test item was suspended in DMSO, processed by ultrasound for 1 min at 37 °C and diluted prior to treatment.
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

yes

Remarks:

Negative controls (A. dest., BSL BIOSERVICE Lot No. 140516, 140808) and solvent controls (DMSO, AppliChem Lot No. 4A008607) were treated in the same way as all dose groups.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Bacteria:
Five strains of S. typhimurium with the following characteristics were used:
TA 98:
his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
TA 100:
his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
TA 1535:
his G 46; rfa-; uvrB-: base-pair substitutions
TA 1537:
his C 3076; rfa-; uvrB-: frame shift mutations
TA 102:
his G 428 (pAQ1); rfa-; R-factor: base-pair substitutions

Tester strains TA 98, TA 1535 and TA 102 were obtained from MOLTOX, INC., NC 28607, USA. Tester strains TA 100 and TA 1537 were obtained from Xenometrix AG, Switzerland. They were stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen.
All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene coding for a protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chl) and biotin (bio) genes (bacteria require biotin for growth).
The tester strains TA 98, TA 100 and TA 102 contain the R-factor plasmid, pkM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms.
The properties of the S. typhimurium strains with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly according to Ames et al.. In this way it is ensured that the experimental conditions set up by Ames are fulfilled.

Preparation of Bacteria:
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. 109 cells/mL). The nutrient medium consists per litre:
8 g Nutrient Broth
5 g NaCl

A solution of 125 μL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain.

Agar Plates:
The Vogel-Bonner Medium E agar plates with 2% glucose used in the Ames Test were prepared by BSL BIOSERVICE GmbH or provided by an appropriate supplier. Quality controls were performed.
Vogel-Bonner-salts contain per litre:
10 g MgSO4 x 7 H2O
100 g citric acid
175 g NaNH4HPO4 x 4 H2O
500 g K2HPO4
Sterilisation was performed for 20 min at 121 °C in an autoclave.
Vogel-Bonner Medium E agar plates contain per litre:
15 g Agar Agar
20 mL Vogel-Bonner salts
50 mL glucose-solution (40%)
Sterilisation was performed for 20 min at 121 °C in an autoclave.

Overlay Agar
The overlay agar contains per litre:
7.0 g Agar Agar
6.0 g NaCl
10.5 mg L-histidine x HCl x H2O
12.2 mg biotin
Sterilisation was performed for 20 min at 121 °C in an autoclave.

Mammalian Microsomal Fraction S9 Mix:
The bacteria most commonly used in these reverse mutation assays do not possess the enzyme system which, in mammals, is known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system was added in the form of mammalian microsome enzyme activation mixture.

S9 Homogenate:
The S9 liver microsomal fraction was prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
The following quality control determinations are performed:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at ≤-75 °C.
The protein concentration in the S9 preparation (Lot: 280613A, 280613B) was 33.6 and 33.4 mg/mL, respectively.

Preparation of S9 Mix:
The S9 mix preparation was performed according to Ames et al..
100 mM of sodium-ortho-phosphate-buffer, pH 7.4, was ice-cold added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion:
co-factor solution 9.5 parts
liver preparation 0.5 parts

During the experiment the S9 mix was stored on ice.

S9 Mix Substitution Buffer:
The S9 mix substitution buffer was used in the study as a replacement for S9 mix, without metabolic activation (-S9).
Phosphate-buffer (0.2 M) contains per litre:
0.2 M NaH2PO4 x H2O 120 mL
0.2 M Na2HPO4 880 mL
The two solutions were mixed and the pH was adjusted to 7.4. Sterilisation was performed for 20 min at 121 °C in an autoclave.
This 0.2 M phosphate-buffer was mixed with 0.15 M KCl solution (sterile) in the following proportion:
0.2 M phosphate-buffer 9.5 parts
0.15 M KCl solution 0.5 parts
This S9 mix substitution buffer was stored at 4 °C.

Experimental Design:

1. Pre-Experiment for Toxicity:
The toxicity of the test item was determined with tester strains TA 98 and TA 100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test).
Toxicity may 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.
The test item was tested in the pre-experiment with the following concentrations:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate

2. Exposure Concentrations:
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment (see chapter 12.1.1 Pre-Experiment). 5.0 μL/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
As the results of the pre-experiment were in accordance with the criteria described above, these were reported as a part of the main experiment I.

3. Experimental Performance
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 μL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 μL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 μL Overlay agar.
For the pre-incubation method 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.
For each strain and dose level, including the controls, three plates (in a one case only two plates were evaluated) were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

Data Recording:
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH). If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.

Evaluation of Cytotoxicity:
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "B" in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

Criteria of Validity:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the control plates with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range):
- S9 + S9
min max min max
TA 98 13 48 13 61
TA 100 61 182 70 194
TA 1535 5 34 4 34
TA 1537 2 32 3 32
TA 102 136 381 91 495

- 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

Evaluation of Mutagenicity:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by 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.
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

Species / strain:

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

The test item N,N-Dibutylaminomethyl-triethoxysilan 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 TA 102.

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:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/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 in tester strains TA 98, TA 1535 and TA 1537 at a concentration of 5.0 μL/plate (without metabolic activation) No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with N,N-Dibutylaminomethyl-triethoxysilan 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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, N,N-Dibutylaminomethyl-triethoxysilan did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, N,N-Dibutylaminomethyl-triethoxysilan is considered to be non-mutagenic in this bacterial reverse mutation assay (negativ with and without metabolic activation).

Executive summary:

SUMMARY RESULTS

In order to investigate the potential of N,N-Dibutylaminomethyl-triethoxysilan for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

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:

0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/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 in noted tester strains TA 98, TA 1535 and TA 1537 at a concentration of 5.0 μL/plate (without metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with N,N-Dibutylaminomethyl-triethoxysilan 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.

Conclusion:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, N,N-Dibutylaminomethyl-triethoxysilan did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, N,N-Dibutylaminomethyl-triethoxysilan is considered to be non-mutagenic in this bacterial reverse mutation assay (negativ with and without metabolic activation).