4-[fluoro(dimethyl)silyl]butanenitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 29, 2016 - February 10, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 26 September, 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

4-[fluoro(dimethyl)silyl]butanenitrile

EC Number:

812-925-4

Cas Number:

1639345-42-8

Molecular formula:

C6H12FNSi

IUPAC Name:

4-[fluoro(dimethyl)silyl]butanenitrile

Test material form:

liquid

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes: cultured human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

MEDIA USED
- Type and identity of media:
a) Complete Culture Medium
RPMI 1640 medium supplemented with:
15 % fetal bovine serum (FBS)
100 U/100 μg/mL penicillin/streptomycin solution
0.24 g/mL PHA-L

Also used for the long-term treatment and the post incubation.

b)Treatment Medium (short-term exposure)
Complete culture medium without FBS.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver microsomal fraction was prepared from the livers of male Wistar rats which were induced ß-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.

Test concentrations with justification for top dose:

Experiment I (4 h treatment, 24 h preparation interval):
- without metabolic activation: 0.5, 1.0, 2.5, 5.0 and 7.5 mM
- with metabolic activation 2.5, 5.0, 7.5 and 9.0 mM

Experiment II (24 h treatment, 24 h preparation interval):
-without metabolic activation: 0.75, 1.0 and 1.5 mM

The concentrations evaluated in the main experiment are based on the results obtained in a pre-experiment for toxicity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to 10 mM. According to the results of the solubility test the test item was dissolved in DMSO and diluted prior to treatment.

Controls

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

waterfree DMSO, Sigma: Lot No. SZBE2800V; Merck: K46959552

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

ethylmethanesulfonate: Final concentrations 400 and 600 μg/mL cyclophosphamide Final concentration 5.0 μg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in medium

DURATION:
- Exposure duration: 4 and 24 h

SPINDLE INHIBITOR:
- colcemid (final concentration 0.2 μg/mL).

STAIN:
- The cells were stained with giemsa and according to the Fluorescent plus Giemsa technique .

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED:
- Per culture 150 metaphases were scored for structural chromosomal aberrations

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

METABOLIC ACTIVATION:
- As clear negative result were obtained in the presence of metabolic activation, the repetition of the experiment was not considered necessary. Therefore, long term treatment without metabolic activation was performed.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:

a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data.

When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data.

The test chemical is then considered unable to induce chromosomal aberrations in cultured human
peripheral blood lymphocyte cells in this test system.

Statistics:

Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test.
Statistical significance at the 5% level (p < 0.05) was evaluated by the χ² test for trend

Results and discussion

Test results

Test results 1

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

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 noted without and with metabolic activation at the concentrations evaluated.

Any other information on results incl. tables

Toxicity (Relative Mitotic Index)

In experiment I without metabolic activation, a biologically relevant decrease of the relative mitotic index (decrease below 70% rel. MI) was noted in the concentrations 2.5 mM (48% rel. MI), 5.0 mM (58% rel. MI) and 10 mM (42% rel. MI). However, the concentrations 7.5 mM (94% rel. MI) and 9.0 mM (85% rel. MI) showed no reduction of the rel. MI, although a decline in cell density was observed in the concentrations 2.5 mM and higher during microscopic evaluation of the prepared slides. Therefore, the mitotic index did not seem to reflect the actual toxic effects correctly.

In experiment I with metabolic activation, a biologically relevant decrease of the relative mitotic index was seen in the concentrations 7.5 mM (47% rel. MI), 9.0 mM (61% rel. MI) and 10 mM (34% rel. MI). These results were in line with the microscopic observations of the cell density on the object slides. A concentration-dependent decrease of the cell density was noticed at the concentration 7.5 mM and higher.

In experiment II without metabolic activation, the test substance induced a decline of the rel. MI in the concentrations 1.5 mM (56% rel. MI), 2.0 mM (33% rel. MI) and 3 mM (17% rel. MI). Hardly any cells could be detected for the concentrations 5.0 mM and 7.0 mM, so that the mitotic index was 0%.

Toxicity (Proliferation Index)

The BrdU-technique was used for determining the proliferation index to detect a possible effect on the proliferation rate after treatment with the test item and thus indicating cell cycle delay. Cell cycle delay might be the consequence of chromosomal damage as highly damaged cells will reach metaphase more slowly than their less damaged counterparts.

In the experiment I, the values of the proliferation index of the negative and solvent controls were 1.36 and 1.26, respectively (without metabolic activation) and 1.26 respectively 1.27 (with metabolic activation). The proliferation index of the highest test item concentration evaluated was 1.24 (2.5 mM) (without metabolic activation) and 1.11 (7.5 mM) (with metabolic activation).

In the experiment II, the values of the proliferation index of the negative and solvent controls were 1.59 respectively 1.54 (without metabolic activation). The proliferation index of the highest dose group evaluated was 1.16 (1.5 mM). A biologically relevant decrease of the proliferation index was indicated in experiment II as a decrease of 25% was determined compared to the solvent control.

Clastogenicity

There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in the number of cells with chromosome aberrations for at least one of the dose groups, which is higher than the laboratory negative control range.

In experiment I without metabolic activation the aberration rates of the negative control (1.7%), solvent control (2.3%) and the dose groups treated with the test item 0.5 mM (2.0%), 1.0 mM (1.7%) and 2.5 mM (1.0%) were within the historical control data of the testing facility (0.0% – 3.0%). The test item concentration 5 mM (6.3%) was increased, however the next higher dose of 7.5 mM (1.9%) did not show a rise in chromosomal aberrations. As the mitotic index was already decreased to 48% by the concentration 2.5 mM, and the mitotic index did not seem to reflect the actual toxicity of the concentrations 5 mM and 7.5 mM, these values were not included in the

assessment.

With metabolic activation the aberration rates of the negative control (1.3%), solvent control (1.7%) and the dose groups 2.5 mM (2.1%), 5.0 mM (0.7%) and 7.5 mM (3.4%) were within the historical control data of the testing facility (0.0% – 3.7%). The next higher concentration 9.0 mM was increased to 4.7%, but as only 64 cells instead of 300 cells could be evaluated due to cytotoxic effects, this result is not regarded as valid.

The Fisher´s exact test was performed to verify the results in the experiment I without and with metabolic activation. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted. Furthermore, a concentration-related increase in chromosomal aberrations was not observed.

In experiment II without metabolic activation, the aberration rates of the negative control (2.7%) and solvent control (2.0%) were within the historical control data of the testing facility (0.0% – 3.0%). The aberration rates of the test item in the concentrations 0.75 mM (9.0%), 1.0 mM (20.7%) and 1.5 mM (54.0%) were increased and therefore above the historical control data of the testing facility (0.0% – 3.0%). The Fisher´s exact test was performed to verify the results in the experiment. A statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in all evaluated dose groups of the test item in experiment II without metabolic activation.

Additionally, the χ² Test for trend revealed a concentration-related increase in chromosomal aberrations.

EMS (400 and 600 μg/mL) and CPA (5.0 μg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.

Polyploid Cells

No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

Applicant's summary and conclusion

Conclusions:

During the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item did induce structural chromosomal aberrations in human lymphocyte cells. Therefore, the test item is considered to be clastogenic in this chromosome aberration test.