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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:
2015-08-27 to 2016-01-13
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:
2016

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
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: minimum essential medium (MEM)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) induced rat liver S9
Test concentrations with justification for top dose:
Experiment I:
20, 50, 100 and 200 μg/mL without metabolic activation
50, 100, 200 and 500 μg/mL with metabolic activation
Experiment II:
100, 200 and 500 μg/mL without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: According to the solubility test, the test item was soluble in ethanol. The solvent was compatible with the survival of the cells and the S9 activity.
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:
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:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

ACTIVATION: S9 supernatant was mixed with S9 co-factor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. The co-factors added were: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-phosphate-buffer pH 7.5.

DURATION

- Exposure duration:
Experiment I: 4 hours, with and without metabolic activation
Experiment II: 21 hours, without metabolic activation
- Expression time (cells in growth medium): Experiment I, 16 +/- 2 hours with and without metabolic activation
- Fixation time (start of exposure up to fixation or harvest of cells): 21 hours after start of exposure

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 μg/mL culture medium) was added to the cultures about 2.5 hours before preparation of the cells
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 300 metaphases per concentration were scored for chromosome analysis

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and relative increase in cell count (RICC) (%)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
The result is considered positive when:
- at least one of the test concentrations exhibits a statistically significant increase compared to 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:
Fisher´s exact test was used to verify the results in the experiment

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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Experiment I: at 100 μg/mL and higher without metabolic activation, and at 200 μg/mL and higher with metabolic activation
Experiment II: at 500 μg/mL without metabolic activation





Any other information on results incl. tables

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

 

Dose

Group

Concentration

µg/mL

Relative Mitotic Index

 (%)

RICC

(%)

Mean % Aberrant Cells

Historical Laboratory Negative Control Range

Precipitation

Including

Gaps

Excluding Gaps

Experiment I and II, without metabolic activation

Experiment I

4 hour treatment, 21 hour preparation interval

C

0

100

105

4.0

2.0

0.0% - 4.0 %

aberrant cells

-

S

0

100

100

3.3

1.7

-

3

20

101

94

2.3

0.3

-

4

50

95

105

4.3

1.3

-

5

100

101

89

1.7

1.0

+

6

200

99

100

2.3

1.7

+

EMS

900

117

120

9.7

8.3

-

 

Experiment II

21 hour treatment, 21 hour preparation interval

C

0

99

119

1.7

0.3

0.0 % - 4.0 %

aberrant cells

-

S

0

100

100

3.3

2.0

-

4

100

101

110

3.3

1.7

-

5

200

105

93

2.3

1.0

-

6

500

109

92

1.7

0.7

+

EMS

400

78

78

12.3

9.0

-

Experiment I, with metabolic activation

Experiment I

4 hour treatment, 21 hour preparation interval

C

0

109

114

2.3

1.0

0.0 % - 4.3 %

aberrant Cells

-

S

0

100

100

1.3

0.7

-

3

50

106

99

1.7

1.0

-

4

100

80

77

5.7

1.7

-

5

200

91

82

2.7

1.0

+

6

500

74

79

1.7

1.0

+

CPA

1.5

98

150

8.7

7.3

-

C: Negative Control (Culture Medium)

S: Solvent Control (EtOH 0.5 % v/v)

EMS: Ethylmethanesulfonate

CPA: Cyclophosphamide

+ : With precipitation

- : Without precipitation

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative with and without metabolic activation

[2-(Perfluorohexyl)ethyl]triethoxysilane has been tested for ability to cause chromosome aberrations in Chinese hamster V79 cells according to OECD TG 473 and in compliance with GLP (Eurofins BioPharma, 2016). No increase in the number of cells with aberrations was observed either with or without metabolic activation when tested up to precipitating concentrations. Appropriate solvent, negative (treatment medium) and positive controls were included and gave the expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of this study.