Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
There were no deviations from the study plan

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Purity/composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Stability at higher temperatures: Yes, maximum temperature: 150aC
Chemical name (lUPAC), synonym or trade name: A mixture of 3-Methacryloxypropyltris {[tris(trimethylsiloxy)silyl]ethyldimethylsiloxy} silane and 3-Methacryloxypropy{bis[tris(trimethylsiloxy)silyl]ethyIdimethylsiloxy}(3-methacryloxypropyItris {[tris(trimethylsiloxy)silyl]ethyldimethylsiloxy}silylpropane-I,3-diyloxycarbonylpropane-2,3-diyldimethylsiloxy)silane

Method

Target gene:
The objective of this study was to evaluate the test item for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains, and in a gene oftryptophan-requiring Escherichia coli bacterial strain resulting in a tryptophan-independent strain
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames) (TAI535: 2006, TA1537: 2016, TA98: 2015, TAI00: 2015) and (Master culture from The National Collections ofIndustrial and Marine Bacteria, Aberdeen, UK) (WP2uvrA, 2008)
- Methods for maintenance in cell culture if applicable: The Salmonella typhimurium strains were regularly checked to confirm their histidinerequirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UVsensitivity and the number of spontaneous revertants. The Escherichia coli WP2uvrA strain was regularly checked to confirm the tryptophanrequirement, UV -sensitivity and the number of spontaneous revertants. Stock cultures of the five strains werestored in liquid nitrogen (-196°C).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/mI). Freshly
grown cultures of each strain were used for testing.
- Properly maintained: [yes]
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
First experiment - Seven concentrations of the test item, 5.4, 17, 52, 164, 512, 1600 and 5000 ).!g/plate were tested in triplicate in the tester strains TA 1535, TA 1537, TA98, TA 100 and WP2uvrA in a dose range finding testlfirst experiment.
Second experiment - Based on the results of the first mutation assay, five doses (increasing with approximately half-log steps) of the test item were selected and tested in triplicate in each strain in the, second experiment. The highest concentration of the test item used in the second mutation assay was 5 mg/plate.
The test item was tested both in the absence and presence ofS9-mix in each strain, in two independent experiments
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [acetone]
- Justification for choice of solvent/vehicle: recommended by various regulatory agencies
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR 91
Remarks:
The positive control item used for all tester strains was 2-aminoanthracene (2AA) (Sigma).
Details on test system and experimental conditions:
The test item was tested both in the absence and presence ofS9-mix in each strain, in two independent experiments.
The vehicle control and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (l O'cells/ml) of one of the tester strains, 0.05 ml ofa dilution of the test item in acetone, and either 0.5 ml S9mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the 'condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Rationale for test conditions:
recommended procedures as liad out in the guidelines
Evaluation criteria:
A Salmonella typhimurium reverse Mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated atCharles River Den Bosch.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mglplate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA 1 00 or WP2uvrA is not greater than two
(2) times the concurrent vehicle control, and the total number of revertants in tester strains TA 1535, TA 1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test item is considered positive (mutagenic) in the test if:
a) • The total number of revertants in tester strain TA 1 00 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA 1535, TA 1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
Statistics:
Standard statistical methods were employed

Results and discussion

Test resultsopen allclose all
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:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
not valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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:
not applicable
Positive controls validity:
valid
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:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
First mutation experiment
The test item was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with concentrations of 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix.
Precipitattion of the test item on the plates was observed at the start of the incubation period at concentrations of 1600 arid 5000 µg/plate and at the end of the incubation period at concentrations of 512 µg/plate and upwards.
To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction ofthe revertant colonies were examined.
Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA 1537 in the absence of S9-mix at the highest concentration tested.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of rev~rtants were observed in any other tester strain.
Mutagenicity No increase in the number ofrevertants was observed upon treatment with the test item under all conditions tested.

Second mutation assay
To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence and presence of 10% (v/v)S9-mix. Based on the results ofthe first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate.
Precipitate - Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at concentrations of 1600 and 5000 µg/plate.
Toxicity - The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
Mutagenicity - No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.
Remarks on result:
other:
Remarks:
All bacterial strains showed negative responses over the entire, dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments

Applicant's summary and conclusion

Conclusions:
All bacterial strains showed negative responses over the entire, dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. '
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Evaluation of the mutagenic activity of the test item in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

The test item was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TAI535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WPuvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by induced by Aroclor 1254).

Batch 0008624816 of the test item was a colourless liquid. The test item was dissolved in acetone.

In the first mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 512 µg/plate and upwards. Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA1537 in the absence of S9-mix (first mutation assay) at the highest tested concentration.

In the second mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 1600 and 5000 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester.strain WPuvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in the second mutation experiment.

In this study, the negative and strain-specific positive control values were within the laboratory histor.ical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.