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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
Study period:
From May 22, 2007 to June 21, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant with international guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
476-490-9
EC Name:
-
Cas Number:
286938-65-6
Molecular formula:
C21H50O2Si3
IUPAC Name:
Trisiloxane, 1,1,1,3,5,5,5-Heptamethyl-3-Tetradecyl-
Test material form:
liquid: viscous
Details on test material:
- Name of test material: Myristyl Trisiloxane
- Molecular formula:C21H50O2Si3
- Molecular weight: 419 g/mol
- Smiles notation: CCCCCCCCCCCCCC[Si](O[Si](C)(C)C)(O[Si](C)(C)C)C
- InChI=1/C21H50O2Si3/c1-9-10-11-12-13-14-15-16-17-18-19-20-21-26(8,22-24(2,3)4)23-25(5,6)7/h9-21H2,1-8H3
- Substance type:organic
- Physical state:viscous liquid

Method

Target gene:
The Salmonella typhimurium histidine (bis) reversion system measures his - → his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 100, TA 1535, TA 102) and fra neshift (TA 98, TA 1537) mutations.

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 (pAQ1I); rfa- ; R-factor: base-pair substitutions
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Pretest: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Main test 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: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.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
The Salmonella tester strains TA 98, TA 100, TA 1535 and TA 102 were obtained from Xenometrix, San Diego, CA., USA. Tester strain TA 1537 was obtained from MOLTOX, INC, NC 28607, USA. They are stored as stock cultures in ampoules wich nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen.

Agar Plates
The Vogel-Bonner Medium E agar plates wich 2% glucose used in the Ames test were prepared by BSL BIOSERVICE 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 K2HP04
Sterilisation was performed 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-solvent (40%)
Sterilisation was perforined 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 HC1 x H20
12.2 mg Biotin
Sterilisation was performed at 121 °C in an autoclave.

DURATION
- Preincubation period:Samples of each tester strain were grown by culturing for 12 h at 38.5 °C in nutrient broth to the late exponential or early stationary phase of growth (approx. 10^9 cells/ml).
- Exposure duration:
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:3

NUMBER OF CELLS EVALUATED:10^9 cells/ml

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
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.

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

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:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed 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.
- Other confounding effects:

ADDITIONAL INFORMATION ON CYTOTOXICITY: 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. The reduction in the number of revertants down to mutation factors of 0.5 (without metabolic activation) and 0.4 (with metabolic activation) found in tester strain TA 102 in experiment II at a dose of 31.6 µg/plate was regarded as not biologically relevant due to the lacking dose-response relationship.
Remarks on result:
other: strain/cell type: base pair changes or frameshifts in the genome of the tester strains used.
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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, Myristyl Trisiloxane is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In order to investigate the potential of test item 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: 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 and II. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 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, myristyl trisiloxane is considered to be non-mutagenic in this bacterial reverse mutation assay.