Bis(dimethylamino)methylvinylsilane

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:

2021-08-19 to 2021-09-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997, corrected 26 June 2020

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(dimethylamino)methylvinylsilane

EC Number:

236-437-0

EC Name:

Bis(dimethylamino)methylvinylsilane

Cas Number:

13368-45-1

Molecular formula:

C7H18N2Si

IUPAC Name:

N,N,N',N',1-pentamethyl-1-vinylsilanediamine

Test material form:

liquid

Details on test material:

Bis(dimethylamino)vinylmethylsilane, Batch: 9092008001

Method

Target gene:

Four strains of S. typhimurium and one strain of E. coli WP2 uvrA (pKM101) with the following
characteristics were used:

TA98: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
TA100: his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
TA1535: his G 46; rfa-; uvrB-: base-pair substitutions
TA1537: his C 3076; rfa-; uvrB-: frame shift mutations
E. coli: WP2 uvrA (pKM101): trp-; uvrA-: base-pair substitutions

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 TA98, TA100 and E. coli 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 tester strain E. coli WP2 uvrA (pKM101) carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagens which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision repair system.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

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 is added in the form of mammalian microsome enzyme activation mixture.

S9 Homogenate:
The S9 liver microsomal fraction was prepared at Eurofins Munich. 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

The protein concentration in the S9 preparation was 34.4 mg/mL.

Preparation of S9 Mix:
The S9 mix preparation was performed according to Ames et al.
100 mM of ice-cold sodium-ortho-phosphate-buffer, pH 7.4, was 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 is 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 of purified water:
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.

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.

Pre-Experiment:
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.
As neither precipitation nor limiting toxicity of the test item was observed in either tester strain used at the maximum recommended concentration of 5.0 μL/plate (with and without metabolic activation), concentrations up to 5.0 μL/plate were selected for the main experiments.

The concentration range covered two logarithmic decades. Two independent
experiments were performed at the following concentrations:

0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
(without metabolic activation)

0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
(with metabolic activation)

Vehicle / solvent:

The test item was dissolved in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

yes

Remarks:

A. dest

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine: S. typhimurium: TA98, TA1537 -------------------------- 2-aminoanthracene: S. typhimurium: TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101)

Details on test system and experimental conditions:

Samples of each tester strain were grown by culturing for 12 h at 37 °C in S. typhimurium medium
(Nutrient Broth) and E. coli medium (Luria Bertani), respectively, to the late exponential or early stationary phase of growth (approx. 109 cells/mL).

The S. typhimurium medium (Nutrient Broth) contains per litre of purified water:
8 g Nutrient Broth
5 g NaCl

The E. coli medium (Luria Bertani) contains per litre of purified water:
10 g tryptone
10 g NaCl
5 g yeast extract

A solution of 125 μL ampicillin (10 mg/mL) (TA98, TA100, E. coli WP2 uvrA (pKM101)) 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. Quality controls were performed.
Vogel-Bonner-salts contain per litre of purified water:
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 of purified water:
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.

The overlay agar contains per litre of purified water:
S. typhimurium:
7.0 g Agar Agar
6.0 g NaCl
10.5 mg L-histidine x HCl 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.

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 is added in the form of mammalian microsome enzyme activation mixture.

S9 Homogenate:
The S9 liver microsomal fraction was prepared at Eurofins Munich. 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: 070521) was 34.4 mg/mL.

The S9 mix preparation was performed according to Ames et al.
100 mM of ice-cold sodium-ortho-phosphate-buffer, pH 7.4, was 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 is stored on ice.

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 of purified water:
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.


Pre-Experiment for Toxicity:
The toxicity of the test item was determined with tester strains TA98 and TA100 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 at the following concentrations:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate

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 or 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 is 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 were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

Evaluation criteria:

Evaluation of Cytotoxicity:
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or “B”, respectively 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.

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 TA98, TA100 and E. coli WP2 uvrA (pKM101) the number of reversions is at
least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control.
According to the 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 TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 2.5 µl w/o activation (Experiment I) and with and w/o activation (Exp II), respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 2.5 µl w/o activation (Experiment I) and with and w/o activation (Experiment II), respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 5 µl w/o activation (Experiment I), at 2.5 µl w/o activation (Experiment II) and at 5 µl with activation (Experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 5 µl w/o activation (Experiment I) and with and w/o activation (Experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 5 µl w/o activation, Experiment I only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Test results are attached as pdf file

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

The test item Bis(dimethylamino)methylvinylsilane 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 TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101). 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:
0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate (without metabolic activation)
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate (with metabolic activation)
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
The microbial contamination observed in a few plates in experiment I did not affect the quality or integrity of the results as the microbial contamination could be clearly distinguished from the Salmonella typhimurium revertants and E. coli revertants and thus did not affect the evaluation.
In experiment I, toxic effects of the test item were observed in tester strains TA98 and TA100 at concentrations of 2.5 μL/plate and higher (without metabolic activation). In tester strains TA1535, TA1537 and E. coli WP2 uvrA (pKM101), toxic effects of the test item were noted at a concentration of 5.0 μL/plate (without metabolic activation). No toxic effects were observed in all tester strains with metabolic activation.
In experiment II, toxic effects of the test item were noted in tester strains TA98 and TA100 at concentrations of 2.5 μL/plate and higher (with and without metabolic activation). In tester strain TA1535, toxic effects of the test item were also observed at concentrations of 2.5 μL/plate and higher (without metabolic activation) and at a concentration of 5.0 μL/plate (with metabolic activation). In tester strain TA1537, toxic effects of the test item were noted at a concentration of 5.0 μL/plate (with and without metabolic activation). No toxic effects were observed in tester strain E. coli WP2 uvrA (pKM101) without and with metabolic activation.
The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in experiment II, in tester strain TA1537 at a concentration of 0.316 μL/plate (with metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship and lack of concomitant clearing of the background lawn.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Bis(dimethylamino)methylvinylsilane at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. The slightly higher mutation factors noted in the negative control and the test item concentrations of tester strain TA1535 in experiment I (with metabolic activation) are attributed to the number of revertants present in the solvent control being at the lower end of the historical control data range (DMSO). Furthermore, all revertant colony numbers of the test material concentrations are within the laboratory historical data.
All criteria of validity were met