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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:
08 February 2012 to 28 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

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:
Experiment I: 2.5, 5.0 and 10.0 mM (-MA); 0.25, 2.5, 5.0 and 10.0 mM (+MA). Experiment II: 0.025, 0.035 and 0.040 mM (-MA); 0.4, 8.0 and 10.0 mM (+MA)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol;

- Justification for choice of solvent/vehicle: The test item was dissolved in ethanol and diluted with cell culture medium (MEM) followed by ultrasonication for
around 5 min (final concentration of ethanol 1.0% v/v). After that the test item was well suspended. The solvent was compatible with the survival of the cells and the S9 activity at a concentration of 1.0% (v/v) ethanol in the culture medium
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
600 µg/ml, without metabolic activation
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
0.83 µg/ml, 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.

Test substance: a solution in ethanol was prepared (344.7 mg/ml; m. wt. 344.66 g/mol). This solution was diluted in MEM, then treated with ultrasonication for around 5 min, to improve the homogeneity of the resulting suspension.

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: 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. Duplicate cultures were not used. The experiment was repeated.

NUMBER OF CELLS EVALUATED: At least 200 well spread metaphases (100 per set), 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
- Determination of endoreplication: the draft report states that "it is important to report polyploidy and/or endoreduplication when this is seen", but no further mention of endoreplication is made.

OTHER: Pre-Experiment for Toxicity I: with and without metabolic activation (exposure time 4 h): 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.50, 1.0, 2.5, 5.0 and 10.0 mM
Pre-Experiment II: without S9 metabolic activation (exposure time 20 h):
0.0005, 0.0010, 0.0025, 0.005, 0.010, 0.025, 0.050, 0.075 and 0.100 mM
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 resultsopen allclose all
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
genotoxicity was observed only in the second experiment
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
mitotic index reduced to 68% at 10 mM in first experiment; mitotic index reduced to 63% at 0.035 mM -MA, experiment II.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
M. I. 55% at 10 mM in experiment I; 49% at 0.04 mM in experiment II
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: not reported
- Evaporation from medium: no information
- Water solubility: not soluble in water
- Precipitation: yes

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:



ADDITIONAL INFORMATION ON CYTOTOXICITY:

Any other information on results incl. tables

Full tables of results are attached to this EPSR

Results Experiments I and II

Concentration mM

MI %

Cells counted

mean aberrant cells

inc gaps

exc gaps

Without metabolic activation, 4 h exposure, Experiment I

0 (medium)

100

200

3.5

3.0

0 (ethanol)

100

200

1.5

0.5

2.5

80

200

4.0

3.0

5

89

200

2.0

1.0

10

55

200

1.0

0.5

Positive control

100

200

10.0

8.0

With metabolic activation, 4 h exposure, Experiment I

0 (medium)

96

200

3.0

2.0

0 (ethanol)

100

200

1.5

1.0

0.25

82

200

1.5

1.0

2.5

90

200

2.0

1.0

5

102

300

3.7

2.3

10

68

200

4.0

3.0

Positive control

79

200

10.5

10.0

Without metabolic activation, 20 h exposure, Experiment II

0 (medium)

106

200

1.5

0.0

0 (ethanol)

100

200

1.5

2.0

0.025

105

200

2.0

2.0

0.035

63

200

3.0

2.5

0.040

12.5

200

1.5

0.0

Positive control

94

200

10.5

9.5

With metabolic activation, 4 h exposure, Experiment II

0 (medium)

 96

200

5.5

1.0

0 (ethanol)

 100

200

2.5

1.0

0.4

 99

400

5.3

3.3

8

 101

400

5.3

3.8

10

 88

200

8.0

6.5

Positive control

 

200

12.5

10

Applicant's summary and conclusion

Conclusions:
2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane has been tested according to OECD 474 and in compliance with GLP, using Chinese hamster V79 cells. No evidence for induction of numerical chromosome aberrations was observed in either the initial or the repeat assay. Without metabolic activation, no biologically relevant increase in the proportion of cells with structural aberrations was observed in either of the two experiments. With metabolic activation, the negative result obtained in the first experiment was not reproduced in the second experiment. Replicate slides were counted in each experiment, but only single cultures were exposed to the controls and to each concentration the test item. It is noted that there is considerable variation between the replicate slides (for example, high dose metabolic activation group replicates providing results of 0 and 8 in experiment I; mid-dose replicates in experiment II results 4 and 1 without MA, 5 and 10 with MA). It is assumed that this lack of reproducibility was the reason that extra cells were counted. Appropriate positive, solvent and medium controls were included and gave results within the range of the historical controls. It was concluded by the authors of the draft report that the test substance is positive for the induction of structural chromosome aberrations under the conditions of the test. The reviewer notes that the differences between replicate slides; the lack of dose response in experiment 1; the unexplained differences between the solvent and medium controls; the different results in experiments 1 and 2 all call into question the biological relevance of the results. In addition, the differences between replicates mean that the absence of duplicate cultures may have had an impact on the results.