3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-ene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Dec 2006 - 28 Feb 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

only 200 metaphases per dose were scored

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

only 200 metaphases per dose were scored

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-ene

EC Number:

246-791-8

EC Name:

3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-ene

Cas Number:

25291-17-2

Molecular formula:

C8H3F13

IUPAC Name:

3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-ene

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

other: Chinese hamster lung fibroblasts (CHL/IU)

Details on mammalian cell type (if applicable):

- Cell proliferation: doubling time 15 h
- Type and identity of media: L-glutamine (final concentration: 0.292 g/L) and sodium hydrogen carbonate (final concentration: approx. 1.85 g/L) were added to Eagle's minimum essential medium and basel medium (MEM) was prepared. This medium was then supplemented with 10% heat-inactivated NBCS.
- Properly maintained: yes
- Checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated i.p. with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Pre-experiment:
6 h treatment: 13.5, 27, 54.1, 108, 216*, 433*, 865*, 1730* and 3460* µg/mL without S9 mix and 13.5, 27, 54.1, 108, 216, 433*, 865*, 1730* and 3460* µg/mL with S9 mix
24 h treatment: 13.5, 27, 54.1, 108*, 216*, 433*, 865*, 1730* and 3460* µg/mL without S9 mix
*selected for scoring of chromosome aberrations

Main experiment:
6 h treatment: 108, 216, 433, 865*, 1730* and 3460* µg/mL with and without S9 mix
24 h treatment: 108, 216, 433, 865*, 1730* and 3460* µg/mL without S9 mix
*selected for scoring of chromosome aberrations

Vehicle / solvent:

- Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: The test substance was not soluble in water or DMSO. The test substance was soluble in acetone at 346 mg/mL and was not indicated any change in colour nor exothermic at room temperature within 2 h after preparation. Therefore, acetone was selected as a solvent.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

mitomycin C (MMC): 0.1 µg/mL (6 h, -S9), 0.05 µg/mL (24 h, -S9); cyclophosphamide (CPA): 6 µg/mL (6 h, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 6 h treatment: 24 h; 24 h treatment: 24 h

SPINDLE INHIBITOR (cytogenetic assays): demecolcine, 50 µL of 10 µg/mL solution

STAIN (for cytogenetic assays): 2 vol% Giemsa solution in 1/15 mol/L PBS (pH 6.8)

NUMBER OF REPLICATIONS: each dose was set in duplicate

NUMBER OF CELLS EVALUATED: 200 metaphases were evaluated per dose

DETERMINATION OF CYTOTOXICITY
- Method: cell growth rate and IC50 value

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

The findings were judged to be positive when the frequencies of cells with structural aberrations or numerical aberrations were 10% or more with a dose-related increase, or the frequencies of aberrant cells were 5% or more both in the chromosomal aberration test and the confirmation test. The other cases were judged to be negative.

Results and discussion

Test results

Key result

Species / strain:

other: Chinese hamster lung fibroblasts (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at 216 µg/mL and above at the start and end of treatment and at the end of the culture.
- Other confounding effects: Colour change of medium and corrosion of the culture dish were not observed at any doses.

RANGE-FINDING/SCREENING STUDIES: The results of the cell growth inhibition test revealed no cytotoxic properties of the test substance in the short-term treatment with and without metabolic activation and the 24 h treatment, therefore the highest dose for the main study was selected at 3460 µg/mL.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the short-term treatments with and without metabolic activation and the 24 h treatment, cytotoxic properties of the test substance were not observed. The IC50 value in all experiments was calculated to be > 3460 µg/mL.

Any other information on results incl. tables

Table 1: Results of the main experiment.

Test item	Concentration in µg/mL	Cell growth rate (%)	Frequency of cells with aberrations (%)*
			Structural aberration	Numerical aberration
Exposure period 6 h, preparation interval 24 h, without S9 mix
Acetone	0	100	0.5	0.0
MMC	0.1	ND	64.5	0.0
Test substance	865^P	89.7	3.5	2.5
	1730^P	94.0	0.0	0.5
	3460^P	90.4	1.5	1.0
Exposure period 6 h, preparation interval 24 h, with S9 mix
Acetone	0	100	0.0	0.5
CPA	6	ND	41.0	0.5
Test substance	865^P	74.9	2.0	1.0
	1730^P	90.1	1.5	0.5
	3460^P	85.3	1.0	0.5
Exposure period 24 h, preparation interval 24 h, without S9 mix**
Acetone	0	100	2.0	3.0
MMC	0.05	ND	76.0	0.5
Test substance	865^P	85.9	1.0	0.5
	1730^P	87.5	2.5	1.0
	3460^P	91.0	2.5	2.0

*: the frequency of cells with chromosomal aberrations was calculated by observing 200 metaphases per dose

**: the medium was exchanged before the demecolcine solution was added 2 h prior to cell harvest to remove the precipitation of the test substance

CPA: cyclophosphamide

MMC: mitomycin C

ND: not detected

P: precipitation of the test substance occurred

Table 2: Results of cell growth inhibition

					Frequency of cells with aberrations (%) *
Substance	Concentration in µg/mL	Treatment - recovery time (h)	S9 mix	Cell Growth rate (%)	Strucutral aberration	Numerical aberration
Acetone	0	6-18	-	100	0.0	0.0
Test item	13.5	6-18	-	98.8	n.o.	n.o.
	27.0	6-18	-	95.6	n.o.	n.o.
	54.1	6-18	-	91.4	n.o.	n.o.
	108	6-18	-	88.1	n.o.	n.o.
	216	6-18	-	78.9	0.0	0.0
	433	6-18	-	75.4	2.0	2.0
	865	6-18	-	68.0	0.0	0.0
	1730	6-18	-	75.6	4.0	2.0
	3460	6-18	-	84.1	2.0	2.0
Acetone	0	6-18	+	100	0.0	0.0
Test item	13.5	6-18	+	91.1	n.o.	n.o.
	27.0	6-18	+	89.7	n.o.	n.o.
	54.1	6-18	+	99.6	n.o.	n.o.
	108	6-18	+	93.5	n.o.	n.o.
	216	6-18	+	88.9	n.o.	n.o.
	433	6-18	+	80.7	0.0	0.0
	865	6-18	+	84.0	4.0	2.0
	1730	6-18	+	87.5	0.0	2.0
	3460	6-18	+	74.7	0.0	0.0
Acetone	0	24-0	-	100	0.0	0.0
Test item	13.5	24-0	-	95.1	n.o.	n.o.
	27.0	24-0	-	92.0	n.o.	n.o.
	54.1	24-0	-	92.1	n.o.	n.o.
	108	24-0	-	84.9	0.0	0.0
	216	24-0	-	74.2	0.0	0.0
	433	24-0	-	82.1	0.0	0.0
	865	24-0	-	84.0	few meta
	1730	24-0	-	71.0	few meta
	3460	24-0	-	78.6	few meta

n.o.: not observed

few meta: the frequency of metaphases was extremely few

* The frequency of cells with chromosomal aberrations was calculated by observing 50 metaphases per dose. The highest dose was set at 3460 µg/mL equivalent to 10 mmol/L, as the maximum dose in case of no cytotoxicity on the guidelines, the dose levels based on a geometric progression of 2 were selected.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative