4,4,19,19-tetraethoxy-3,20-dioxa-8,15-dithia-4,19-disiladocosaneand S-(6-{[3-(triethoxysilyl)propyl]thio}hexyl) ethanethioate

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:

03.09.2020 - 23.10.2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

The OECD Guideline for Testing of Chemicals, Section 4, No. 471 – Bacterial Reverse Mutation Test - recommends using a combination of S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101).

Deviations:

yes

Remarks:

test concentrations: A 100 µL dose of the solvent control was incompatible with the survival of the bacteria. Therefore, higher stock solutions of the test items were prepared to ensure the highest test concentration of 5.0 µL/plate

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4,19,19-tetraethoxy-3,20-dioxa-8,15-dithia-4,19-disiladocosane and S-(6-{[3-(triethoxysilyl)propyl]thio}hexyl) ethanethioate

EC Number:

951-696-6

Molecular formula:

C17H36O4S2Si and C24H54O6S2Si2

IUPAC Name:

4,4,19,19-tetraethoxy-3,20-dioxa-8,15-dithia-4,19-disiladocosane and S-(6-{[3-(triethoxysilyl)propyl]thio}hexyl) ethanethioate

Test material form:

liquid

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).

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.
The S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Gießen, Germany. Male Sprague Dawley rats were induced with phenobarbital/β-naphthoflavone.
The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)
A stock of the supernatant containing the microsomes was frozen in aliquots of 5 mL and stored at
-75 °C
The protein concentration in the S9 preparation was 40.2 mg/mL (Lot: 4272) and was adjusted to 30 mg/mL.

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 at the following concentrations:

Experiment I:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate

Experiment II:
0.0158, 0.050, 0.158, 0.50, 1.58 and 5.0 µL/plate

As the results of the pre-experiment were in accordance with the criteria of validity
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

Vehicle / solvent:

The test item was dissolved in THF (Tetrahydrofuran) 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:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD; 4-nitro-o-phenylene-diamine; 2-AA; 2-aminoanthracene

Remarks:

dissolved in A.dest or DMSO

Details on test system and experimental conditions:

For the plate incorporation method, the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
25 µL Test solution at each dose level and the solvent control,
100 µL 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 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:

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

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 substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, the test substance 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 II) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coliWP2 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:

Experiment I:

0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate

Experiment II:

0.0158, 0.050, 0.158, 0.50, 1.58and 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 and II.

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.

All criteria for validity were met.