Potassium 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanesulphonate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Chromosome aberration test in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-octane-1-sulfonic acid

Cas Number:

913252-35-4

IUPAC Name:

3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-octane-1-sulfonic acid

Test material form:

liquid

Details on test material:

- Purity: 35.6% wt

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, Manassas, VA
- Suitability of cells: In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 20. The frozen lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination.
- Normal (negative control) cell cycle time: Average cell cycle time of 10-14 hours with a modal chromosome number of 20

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

12.5, 25, 50, 100, 150, 200, 250 and 300 μg/mL

Selection of dose levels for the chromosome aberration assay was based on cell growth inhibition relative to the solvent control. Substantial toxicity (i.e., at least 50% cell growth inhibition, relative to the solvent control) was observed at dose levels ≥428 μg/mL in all three treatment groups. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 12.5 to 300 μg/mL for all three treatment groups.

Vehicle / solvent:

Water was used as the test substance solvent based on the solubility of the test substance and compatibility with target cells. The test substance was soluble in water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 5e5 cells/ 25 cm2 flask

DURATION
- Preincubation period: 16-24 hours
- Exposure duration: 4 hours in presence and absence of S9 activation and 20 hours continuously in the absence of S9 activation
- Expression time (cells in growth medium): After the exposure period, the treatment medium was removed, the cells washed with CMF-PBS, refed with complete medium and returned to the incubator. Two hours prior to the scheduled cell harvest, Colecmid® was added to duplicate flasks at a final concentration of 0.1 µg/mL and the flasks returned to the incubator until cell collection.
- Collection of Metaphase Cells: Approximately 2 after addition of Colecmid®, metaphase cells were harvested for both the non-activated and S9-activated studies by trypsinization. Cells were collected approximately 20 hours after initiation of treatment. The cells were collected by centrifugation at approximately 800 rpm for 5 minutes. The cell pellet was resuspended in 2-4 mL 0.075 M potassium chloride (KCl) and allowed to stand at room temperature for 4-8 minutes. The cells were collected by centrifugation, the supernatant aspirated and the cells fixed with 2 washes of approximately 2 mL Carnoy’s fixative (methanol:glacial acetic acid, 3:1, v/v). The cells were stored in fixative at approximately 2-8°C.

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were prepared and an aliquot of the cells suspension was dropped from an appropriate distance by means of a Pasteur pipet on clean microscope slides and allowed to air dry at room temperature. If necessary, the following techniques may have been used to achieve optimum spreading of metaphases: dip slide in cold water or ice, use of slide warmer, or additional centrifugation steps, as needed. The dried slides were stained with 5% Giemsa, air dried and permanently mounted.

NUMBER OF CELLS EVALUATED: Whenever possible, a minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Whenever possible, a minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations. The number of metaphase spreads that were examined and scored per duplicate flask was reduced within the percentage of aberrant cells reached as significant level (at least 10%) before 100 cells were scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a bread (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but instead were considered part of the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely damaged cells (≥10 aberrations) were also recorded. Chromatid gaps (as aligned achromatic region in one chromatid, the size of which is equal to or smaller than the width of the chromatid) and isochromatid gaps (an aligned, achromatic region in both chromatids, the size of which is equal to or smaller than the width of the chromatids) were recorded but not included in the analysis. The XY coordinates for each cell with chromosomal aberrations were recorded using the microscope stage. Polyploid and endoreduplicated cells were evaluated from each treatment flask per 100 metaphase scored.

DETERMINATION OF CYTOTOXICITY: In the preliminary toxicity assay, a concurrent toxicity test was conducted in both the non-activated and the S9-activated test systems. After cell harvest an aliquot of the cell suspension was removed from each culture and counted using a Coulter counter. The presence of test substance precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control.

Rationale for test conditions:

Selection of doses for microscopic analysis was based on toxicity (the lowest dose with at least 50% reduction in cell growth and the next two lower doses) in the non-activated 4-hour exposure group.

Evaluation criteria:

The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control and are presented for the toxicity and aberration studies. The number and types of aberrations found, the percentage of structurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell were calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.

Criteria for Valid Test: The frequency of cells with structural chromosome aberrations in the solvent control must be within the range of the historical solvent control. The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p≤0.05, Fisher's Exact test) relative to the solvent control.

Statistics:

Statistical analysis of the percent aberrant cells was performed using the Fisher's Exact test. Fisher's Exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's Exact test at any test substance dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test substance was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (p≤0.05). However, values that are statistically significant but do not exceed the range of historical solvent controls may be judged as not biologically significant. Test substances not demonstrating a statistically significant increase in aberrations will be concluded to be negative.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at dose levels ≥ 428 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Initial Chromosome Aberration Assay
In the initial chromosome aberration assay, the test substance was soluble in water and in the treatment medium at all dose levels tested at the beginning and conclusion of the treatment period. The pH of the highest concentration of test substance in treatment medium was approximately 7.4 Due to lack of a dose level with close to 50% reduction in cell growth or mitotic index, the chromosome aberration assay was repeated in the non-activated 4-hour exposure group at dose levels ranging from 12.5 to 300 μg/mL. The mitotic index at the highest dose level evaluated for chromosome aberrations, 300 μg/mL, was 59% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 50, 150 and 300 μg/mL. The percentage of cells with structural aberrations in the S9-activated 4-hour exposure group was statistically increased (21.0%) above that of the solvent control at 300 μg/mL (p≤0.01, Fisher's Exact test). The Cochran-Armitage test was also positive for a dose response (p≤0.05). The percentage of cells with numerical aberrations was statistically increased above that of the solvent control at dose levels 50 and 150 μg/mL (p≤0.01 and p≤0.05, respectively, Fisher's Exact test). However, the percentages of cells with numerical aberrations at 50 and 150 μg/mL (6.5% and 6.0%, respectively) were within the historical solvent control range of 0.0% to 10.0%. Therefore, they were not considered to be biologically significant. The percentage of structurally damaged cells in the CP (positive control) treatment group (18.0%) was statistically significant. Toxicity of the test substance (cell growth inhibition relative to the solvent control) in CHO cells when treated for 20 hours in the absence of S9 activation was 55% at 250 μg/mL, the highest test concentration evaluated for chromosome aberrations in the non-activated 20-hour continuous exposure group. The mitotic index at the highest dose level evaluated for chromosome aberrations, 250 μg/mL, was 59% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 50, 150 and 250 μg/mL. The percentage of cells with structural and numerical aberrations in the non-activated 20-hour exposure group was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher's Exact test). The percentage of structurally damaged cells in the MMC (positive control) treatment group (18.0%) was statistically significant.

Repeat Chromosome Aberration Assay
In the chromosome aberration assay, the test substance was soluble in water and in the treatment medium at all dose levels tested at the beginning and conclusion of the treatment period. The pH of the highest concentration of test substance in treatment medium was approximately 7.0. Toxicity of the test substance (cell growth inhibition relative to the solvent control) in CHO cells when treated for 4 hours in the absence of S9 activation was 74% at 300 μg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 300 μg/mL, was 61% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 50, 100 and 300 μg/mL. The percentage of cells with structural aberrations in the non-activated 4-hour exposure group was statistically increased (23.0%) above that of the solvent control at 300 μg/mL (p≤0.01, Fisher's Exact test). The Cochran-Armitage test was also positive for a dose response (p≤0.05). The percentage of cells with numerical aberrations in the test substance-treated group was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher's Exact test). The percentage of structurally damaged cells in the MMC (positive control) treatment group (21.0%) was statistically significant.

Any other information on results incl. tables

Initial Assay Summary
Treatment µg/mL	S9 Activation	Treatment time	Mean Mitotic Index	Cells Scored		Aberrations per Cell		Cells with Aberrations
				Numerical	Structural	Mean	± SD	Numerical (%)	Structural (%)
Water	+S9	4	9.3	200	200	0.005	0.071	1.5	0.5
Test substance
50	+S9	4	11.3	200	200	0.005	0.071	6.5**	0.5
150	+S9	4	11.3	200	200	0.020	0.140	6.0*	2.0
300	+S9	4	3.8	200	100	0.440	1.305	1.0*	21.0**
CP
10	+S9	4	2.1	200	100	0.340	0.913	0.0	18.0**
Water	-S9	20	10.9	200	200	0.000	0.000	1.5	0.0
Test substance
50	-S9	20	10.4	200	200	0.000	0.000	0.5	0.0
150	-S9	20	9.2	200	200	0.000	0.000	2.0	0.0
300	-S9	20	4.5	200	200	0.000	0.000	1.5	0.0
MMC
0.1	-S9	20	6.7	200	100	0.240	0.712	0.0	18.0**
Treatment: Cells from all treatment conditions were harvested 20 hours after the initiation of the treatments Aberrations per Cell: Severely damaged cells were counted as 10 aberrations. Percent Aberrant Cells: * p≤0.05; ** p≤0.01; using Fisher’s Exact test.

 

 

Repeat Assay Summary
Treatment µg/mL	S9 Activation	Treatment time	Mean Mitotic Index	Cells Scored		Aberrations per Cell		Cells with Aberrations
				Numerical	Structural	Mean	± SD	Numerical (%)	Structural (%)
Water	-S9	4	10.6	200	200	0.005	0.071	0.5	0.5
Test substance
50	-S9	4	8.4	200	200	0.010	0.100	1.5	1.0
150	-S9	4	8.2	200	200	0.005	0.071	1.0	0.5
300	-S9	4	4.1	200	100	0.290	0.608	0.5	23.0**
MMC
0.2	-S9	20	5.9	200	100	0.230	0.468	0.5	21.0**
Treatment: Cells from all treatment conditions were harvested 20 hours after the initiation of the treatments Aberrations per Cell: Severely damaged cells were counted as 10 aberrations. Percent Aberrant Cells: * p≤0.05; ** p≤0.01; using Fisher’s Exact test.

Applicant's summary and conclusion

Conclusions:

The test substance was concluded to be positive for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations in CHO cells in both non-activated and S9-activated test systems.

Executive summary:

The test substance was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced S9 activation system in accordance with OECD Guideline 473. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. The dosing solution concentrations were adjusted to compensate for the purity (30%) of the test substance using a correction factor of 3.33. The Sponsor later provided a Certificate of Analysis with a purity of 35.6%.

 

Water was used as the test substance solvent based on the solubility of the test substance and compatibility with target cells. The test substance was soluble in water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility. In the preliminary toxicity assay, the maximum dose tested was 4280 μg/mL (10 mM). The test substance was soluble in water at all concentrations tested. Visible precipitate was observed in the treatment medium at dose levels ≥ 1284 μg/mL and dose levels ≤ 428 μg/mL were soluble in the treatment medium at the beginning and conclusion of the treatment period. Selection of dose levels for the chromosome aberration assay was based on cell growth inhibition relative to the solvent control. Substantial toxicity (i.e., at least 50% cell growth inhibition, relative to the solvent control) was observed at dose levels ≥ 428 μg/mL in all three treatment groups. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 12.5 to 300 μg/mL for all three treatment groups. In the initial chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. The test substance was soluble in water and in the treatment medium at all dose levels tested at the beginning and conclusion of the treatment period. Selection of doses for microscopic analysis was based on toxicity (the lowest dose with at least 50% reduction in cell growth and the next two lower doses) in the S9-activated 4-hour and the non-activated 20-hour exposure groups.

 

The percentage of cells with structural aberrations in the S9-activated 4-hour exposure group was statistically increased (21.0%) above that of the solvent control at 300 μg/mL (p≤0.01, Fisher's Exact test). The Cochran-Armitage test was also positive for a dose response (p≤0.05). The percentage of cells with numerical aberrations was statistically increased above that of the solvent control at dose levels 50 and 150 μg/mL (p≤0.01 and p≤0.05, respectively, Fisher's Exact test). However, the percentages of cells with numerical aberrations at 50 and 150 μg/mL (6.5% and 6.0%, respectively) were within the historical solvent control range of 0.0% to 10.0%. Therefore, they were not considered to be biologically significant. The percentage of cells with structural or numerical aberrations in the non-activated 20-hour exposure group was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher's Exact test).

 

Due to lack of a dose level with close to 50% reduction in cell growth or mitotic index, the chromosome aberration assay was repeated in the non-activated 4-hour exposure group. Although the dose levels selected for the repeat assay were 25, 100, 300, 325, 350, 375, 400 and 415 μg/mL, the repeat assay was inadvertently tested at the same doses used in the initial assay (12.5, 25, 50, 100, 150, 200, 250 and 300 μg/mL). Since the toxicity data from the repeat assay showed a shift in toxicity (50% reduction in cell growth and mitotic index was achieved), the Study director concluded that the alternate doses tested in the repeat assay had no adverse effect on the outcome of the study.

 

In the repeat chromosome aberration assay, the cells were treated for 4 hours in the non-activated test system. All cells were harvested 20 hours after treatment initiation. The test substance was soluble in water and in the treatment medium at all dose levels tested at the beginning and conclusion of the treatment period. Selection of doses for microscopic analysis was based on toxicity (the lowest dose with at least 50% reduction in cell growth and the next two lower doses) in the non-activated 4-hour exposure group.

 

The percentage of cells with structural aberrations in the non-activated 4-hour exposure group was statistically increased (23.0%) above that of the solvent control at 300 μg/mL (p≤0.01, Fisher's Exact test). The Cochran-Armitage test was also positive for a dose response (p≤0.05). The percentage of cells with numerical aberrations in the test substance-treated group was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher's Exact test). Based on the findings of this study, the test substance was concluded to be positive for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations in CHO cells in both non-activated and S9-activated test systems.