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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 February 2012 to 5 November 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1998
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Dodecamethylpentasiloxane
EC Number:
205-492-2
EC Name:
Dodecamethylpentasiloxane
Cas Number:
141-63-9
Molecular formula:
C12H36O4Si5
IUPAC Name:
2,2,4,4,6,6,8,8,10,10-decamethyl-3,5,7,9-tetraoxa-2,4,6,8,10-pentasilaundecane

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:
MEM medium supplemented with:
10 % foetal bovine serum (FBS)
100 U/100 µg/mL Penicillin/Streptomycin solution
2 mM L-glutamine
0.25 mg/mL Amphotericin
25 µM HEPES

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
0-10 mM
Vehicle / solvent:
ethanol
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
no
Remarks:
with metabolic activation
Details on test system and experimental conditions:
ACTIVATION: Phenobarbital (80 mg/kg bw) and β naphthoflavone (100 mg/kg bw) induced rat liver S9 was included in the S9 mix to a final protein concentration of 0.75 mg/ml. Cofactors were added to the following concentrations: 8 mM MgCl2; 33 mM KCl; 5 mM Glucose-6-phosphate; 5 mM NADP.

METHOD OF APPLICATION: in medium;

DURATION

- Exposure duration: 4 hours (Experiment I, +/- MA, Experiment II +MA); 20 hours (Experiment II -MA).

- Expression time (cells in growth medium): 20 hours (Experiment I +/- MA and Experiment II +MA).

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Experiments I and II: Stock cultures in exponential growth were seeded into Quadriperm dishes containing at four slides. Two days after seeding the culture medium was replaced with test item suspension (with S9 mix as appropriate). The slides were divided into sets of two and at least one slide from each set was counted. The experiment was repeated. In experiment III duplicate cultures were treated at each concentration and 100 metaphases were scored per culture.

NUMBER OF CELLS EVALUATED: At least 200 well spread metaphases (100 per slide), containing 22+/- 1 centromeres, per concentration and validity controls were scored for cytogenetic damage.

NUMBER OF CELLS EVALUATED: At least 200 well spread metaphases (100 per slide in experiments I and II, 100 per culture in experiment III), containing 22+/- 1 centromeres, per concentration and validity controls were scored for cytogenetic damage.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative cell density

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
A positive result is determined by: a clear and dose-related increase in the number of cells with aberrations; a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells)

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
increased percentage of cells with aberrations, not dose dependent or reproducible
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
some reduction in Mitotic index observed at limit concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Results of chromosome aberration assay

Concentration mM Set  cells scored Chromatid types Chromosome types Polyploid cells (mean) MI % (mean) mean aberrant cells
b f d ex if id cx inc gaps exc gaps
Experiment I, without metabolic activation; 4h treatment, 20 h fixation
Negative control 1 100 2 0 0 0 1 0 0 0.5 100 3.5 3
2 100 1 0 0 0 0 0 2
Solvent control 1 100 1 0 0 0 0 0 0 0.5 100 1.5 0.5
2 100 0 0 0 0 0 0 0
2.5 1 100 0 1 0 0 0 0 0 1 90 2 1.5
2 100 0 0 0 0 0 0 1
5* 1 100 2 1 1 0 0 0 0 0 89 5 3.5
2 100 1 0 1 0 0 0 1
10 1 100 1 0 0 0 0 0 1 0.5 98 3.5 1.5
2 100 0 0 1 0 0 0 0
Positive controls 1 100 6 0 0 1 0 0 0 0.5 72 10 8
2 100 6 0 1 3 0 0 0
Experiment I, with metabolic activation; 4h treatment, 20 h fixation
Negative control 1 100 2 0 0 1 0 0 0 1 96 3 2
2 100 0 0 1 1 0 0 0
Solvent control 1 100 1 0 0 0 0 0 0 2.5 100 1.5 1
2 100 0 0 0 0 0 0 1
2.5 1 100 0 0 0 0 0 0 0 0 78 1 0
2 100 0 0 0 0 0 0 0
5 1 100 0 0 0 0 0 0 0 0 85 4 1.5
2 100 3 0 0 1 0 0 0
10 1 100 0 0 0 0 0 0 1 0 76 1.5 1
2 100 1 0 0 0 0 0 0
Positive controls 1 100 7 0 0 5 0 0 0 0.5 79 10.5 10
2 100 16 0 0 4 0 0 0
Experiment II, without metabolic activation; 20h treatment, 20 h fixation
Negative control 1 100 2 1 0 0 1 0 0 0.5 111 6.5 3
2 100 2 0 0 0 0 0 0
Solvent control 1 100 0 0 0 0 0 0 0 0 100 3.5 0.5
2 100 1 0 0 0 0 0 0
0.125 1 100 3 0 0 0 0 0 0 0 95 5 2
2 100 0 0 1 0 0 0 0
7.5 1 200 1 0 1 1 0 0 0 0.5 103 3.5 2.3
2 200 5 0 1 0 0 0 0
10 1 200 4 3 1 2 1 0 0 0.75 108 4.3 3.3
2 200 2 11 0 0 0 0 0
Positive controls** 1 100 9 1 0 2 0 0 1 0.5 88 12.5 10.5
2 100 4 1 0 6 0 0 0
Experiment II, with metabolic activation; 4h treatment, 20 h fixation
Negative control 1 100 1 1 0 0 0 0 0 0 96 5.5 2.5
2 100 2 0 0 0 0 0 1
Solvent control 1 100 2 0 0 0 0 0 0 1 100 2.5 1
2 100 0 0 0 0 0 0 0
4 1 100 7 0 0 1 0 0 0 0 72 8.5 6
2 100 4 0 0 0 0 0 1
7 1 200 6 0 0 0 0 0 0 0.75 83 5.8 3.3
2 200 4 1 2 0 1 0 0
10* 1 200 7 0 0 0 0 0 0 0.5 84 6.8 4.8
2 200 11 2 0 1 0 0 0
Positive controls 1 100 5 0 0 9 0 0 0 0.5 88 12.5 11
2 100 4 1 0 4 0 0 0

Experiment III, with metabolic activation; 4h treatment, 20 h fixation

Negative control

1

100

0

0

0

0

0

0

0

0

69

4.0

1.0

2

100

4

0

0

1

0

0

0

Solvent control

1

100

0

0

0

1

0

0

0

0

63

2.5

1.0

2

100

1

0

0

0

0

0

0

2.5

1

100

1

0

0

0

0

0

0

0

81

3.5

1.0

2

100

1

0

0

0

0

0

0

5.0

1

100

1

0

0

0

0

0

1

0

75

3.0

1.5

2

100

1

0

0

0

0

0

0

10

1

100

0

0

1

1

0

0

0

0

67

3.0

1.5

2

100

0

0

1

0

0

0

0

Positive controls**

1

100

4

1

0

1

0

0

0

0

90

9.0

7.5

2

100

6

0

0

4

0

0

0

* one cd at this concentration; ** one ma at this concentration; *** one ib at this concentrationb/ib break/isobreak f/if fragment/isofragment d/id deletion/isodeletion ex chromatid exchange cx chromosome exchange cd chromosome deletion ma multiple aberration (>4 events including gaps)

Applicant's summary and conclusion

Conclusions:
Dodecamethylpentasiloxane has been tested according to OECD 473 and in compliance with GLP. An increase in the number of aberrations was observed in the second experiment with metabolic activation but the response was not clearly dose related or reproducible. In a third experiment with metabolic activation there was no increase in the number of aberrations. Therefore, dodecamethylpentasiloxane was considered to be non-clastogenic under the conditions of the test.