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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:
26 July 2016 to 17 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2014
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: chromosome aberration in mammalian cells

Test material

Constituent 1
Chemical structure
Reference substance name:
Octaphenylcyclotetrasiloxane
EC Number:
208-904-9
EC Name:
Octaphenylcyclotetrasiloxane
Cas Number:
546-56-5
Molecular formula:
C48H40O4Si4
IUPAC Name:
octaphenyl-1,3,5,7,2,4,6,8-tetraoxatetrasilocane
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 603152
- Expiration date of the lot/batch: 31 March 2018
- Purity test date: WILL BE INCLUDED IN THE FINAL REPORT

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: the test substance was soluble and stable in cell culture medium
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: precipitation occurred in the highest tested concentration of 2 mg/mL

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was dissolved in cell culture medium up to the maximum recommended concentration of 2 mg/mL.
- Final dilution of a dissolved solid, stock liquid or gel: From the test item stock solution of 500 µg/mL separate dosing solutions of the test item were prepared for each of the concentrations by serial dilution.
- Final preparation of a solid: The osmolality and pH of the highest concentration were measured. The osmolality was 315 mOsmol/kg and the pH was within the physiological range.

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: No data
- Suitability of cells: widely used to examine the ability of chemicals to induce cytogenic changes and thusidentify potential carcinogens or mutagens
- Cell cycle length, doubling time or proliferation index: doubling time is 12 - 14 h, plating efficiency is more than 50 %
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: diploid number, 2n = 22
- Normal (negative control) cell cycle time: no data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (minimal essential medium) medium supplemented with 10 % fetal bovine serum (FBS), 100 U/ 100 µh/mL penicillin/ streptomycin solution, 2 mM L-glutamine, 2.5 µg/mL amphotericin, 25 mM HEPES. Thawed cultures were set up in 75 cm² cell culture plastic flasks at 37°C in a 5% carbon dioxide atmosphere (95% air)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) induced rat liver metabolic activation system
Test concentrations with justification for top dose:
Pre-experiment: 0.5, 1, 2, 5, 10, 20, 50, 100, 200 and 500 µg/mL were tested with and without metabolic activation. Test concentrations in the main experiment I and II were chosen based on cytotoxity data from the pre-experiment.
Experiment I, with and without metabolic activation: 50, 100, 200 µg/mL
Experiment II, without metabolic activation: 5, 10 and 20 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: cell culture medium
- Justification for choice of solvent/vehicle: a solubility test was performed with different solvents up to the maximum recommended concentration of 2 mg/mL in cell culture medium. According to the results from the solubility test the test item was dissolved in cell culture medium.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: complete culture medium without FBS
- Cell density at seeding: 5 x 10⁵ cells per flask were seeded in 15 mL of MEM (minimal essential medium) supplemented with 10 % FBS and subcultures were made 3-4 days after seeding. Then, 1 x 10⁴ cells/ mL were seeded into cell culture flasks with complete culture medium.

ACTIVATION: The S9 supernatant was thawed and mixed with S9 co-factor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. The final percentage of S9 min in the cell culture medium is 5% (v/v). The added co-factors were: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-phosphate-buffer pH 7.4.

DURATION
- Exposure duration:
Experiment I: short-term exposure 4 h (with and without metabolic activation)
Experiment II: long-term exposure 21 h (without metabolic activation)
- Expression time (cells in growth medium): 17 hours for Experiment I, no expression time after Experiment II
- Fixation time (start of exposure up to fixation or harvest of cells): at 21 hours post-incubation following Experiment I and immediately after treatment following Experiment II


SPINDLE INHIBITOR (cytogenetic assays): incubation with Colcemid (0.2 µg/ mL culture medium) for 2.5 h

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate cultures were treated at each concentration

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Following incubation with a spindle inhibitor, the cells were trypsinated and resuspended in about 9 mL complete culture medium. Then, the cultures were transferred into tubes and incubated with hypotonic solution for 15-20 min followed by fixation with methanol and glacial acetic acid, and spread onto slides. Then the slides were stained with Giemsa and coverslipped. Afterwards, they were air dried.

NUMBER OF CELLS EVALUATED: 150 metaphases per culture were scored

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

DETERMINATION OF CYTOTOXICITY
- Method: relative increase in cell count (RICC)


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no


Rationale for test conditions:
Test concentrations in the main experiment I and II were chosen based on cytotoxity data from the pre-experiment.
Evaluation criteria:
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is dose-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data
Statistics:
Statistical significance (p value); Fischer´s exact test; χ² test for trend

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: Precipitate of the test item was noted at concentrations of 200 and 500 µg/mL after treatment with the test item in Experiment I. In Experiment II without metabolic activation, precipitation was observed at a concentration of 20 µg/mL and higher.
- Definition of acceptable cells for analysis:

RANGE-FINDING/SCREENING STUDIES: Precipitation of the test item was noted at concentrations of 50 µg/mL and higher. The highest dose group evaluated in the pre-experiment was 2000 µg/mL. The relative increase in cell count (RICC) was used as parameter for toxicity. The concentrations evaluated in the main experiment based on the results obtained in the pre-experiment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: within the ranges of the historical control data
- Negative (solvent/vehicle) historical control data: within the ranges of historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: in Experiment I and II, with and without metabolic activation, no biologically relevant decrease of the relative increase in cell count was noted in all tested concentrations up to 500 µg/mL. These results indicated that the test item has no cytotoxic effects in this test system up to the highest concentration tested.
- Polyploid cells: no biologically relevant increase in the frequencies of polyploid cells was observed after treatment with the test item.

Any other information on results incl. tables

Table 1: Summary: Experiment I and II, with and without metabolic activation

 

Dose

Group

µg/mL

RICC

(%)

Mean aberrant cells including gaps (%)

Mean aberrant cells excluding gaps (%)

Precipitation

Statistical significance

Experiment I and II, without metabolic activation

 

 

 

 

 

 

Experiment I

4-hour treatment, 21-hour preparation interval

Negative control (treatment medium) 0

100

3.3

2.3

-

-

 

50

100

5.0

3.0

-

-

 

100

103

5.0

3.7

-

-

 

200

109

3.3

1.7

+

-

 

EMS 900

95

10.2

8.9

-

+

Experiment II

21-hour treatment, no preparation interval

Negative control (treatment medium ) 0

100

2.3

1.3

-

-

 

5

97

3.0

1.7

-

-

 

10

103

2.3

0.7

-

-

 

20

98

5.7

1.7

+

-

 

EMS 400

92

11.0

7.7

-

+

Experiment I, with metabolic activation

 

 

 

 

 

 

Experiment I

4-hour treatment, 21-hour preparation interval

Negative control (treatment medium) 0

100

2.7

1.7

-

-

 

50

107

3.0

1.3

-

-

 

100

111

2.7

1.3

-

-

 

200

98

3.7

2.7

+

-

 

CPA 0.83

99

8.0

5.3

-

+

EMS: ethylmethanesulphonate

CPA: cyclophosphamine

RICC: relative increase in cell count

+ : with precipitation; statistical significance

- : without precipitation; no statistical significance

Applicant's summary and conclusion

Conclusions:
Octaphenylcyclotetrasiloxane has been tested for ability to cause chromosome aberrations in Chinese hamster V79 cells according to OECD TG 473 and under GLP. No increase in the number of cells with aberrations was observed either with or without metabolic activation when tested up to a precipitating concentration. Appropriate negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of the test.