3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctan-1-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at the time of study conduct.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctan-1-ol

EC Number:

211-477-1

EC Name:

3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctan-1-ol

Cas Number:

647-42-7

Molecular formula:

C8H5F13O

IUPAC Name:

3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctan-1-ol

Details on test material:

- Purity: 99.7%

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fisher's medium for leucemic cells of mice
- 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:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary Toxicity Assay: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 3650 µg/mL
Initial Mutagenesis Assay (cloning): 30, 35, 40, 50, 75 µg/mL (without activation); 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 µg/mL (with activation)
Extended Treatment Assay (cloning): 20, 25, 30, 35, 40, 50 µg/mL (without activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on solubility of test substance and compatibility with the target cells

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: Methyl methanesulfonate and 7,12-dimethyl-benz(a)anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Treatment was carried out in conical tubes containing cells, F0P medium or S9 activation mixture, and test or control substance. Treatment tubes were gassed with 5±1% CO2 in air, capped tightly, and incubated with mechanical mixing for 4 (-S9, +S9) or 24 (-S9) hours at 37±1°C. The preparation and addition of test substance dosing solutions were carried out under amber lighting and the cells were incubated in the dark during the exposure period. After the treatment period, the cells were washed twice with F0P or F0P supplemented with horse serum, L-glutamine, penicillin, and streptomycin (F10P). After the second wash, the cells were resuspended in F10P, gassed with 5±1% CO2 in air, and placed on the droller drum apparatus at 37±1°C.

DURATION
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 2 days for 4-hour exposure; 3 days for 24-hour exposure
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 3e5 cells/mL (at end of treatment)


DETERMINATION OF CYTOTOXICITY
- Method: Exposure in the absence and presence of S9 activation for 4 hours, and without activation for 24 hours. For the 4-hour exposure, the cell population density was determined 24 and 48 hours after the exposure; the cultures were adjusted to 3e5 cells/mL after 24 hours only. For the 24-hour exposure, cell population density was determined 24, 48, and 72 hour after exposure. The cell population was adjusted to 3e5 cells/mL immediately after test substance removal and 24 hours after test substance removal. Cultures with <3e5 cells/mL were not adjusted. Toxicity was measured as suspension growth of the treated cultures relative to the growth of the solvent control cultures after 48 hours,

OTHER: Scoring Procedures: After the incubation, the VC plates were counted for the total number of colonies per plate and the total relative growth determined. The TFT-resistant colonies were then counted for each culture with ≥20% total relative growth (including at least one concentration with ≥10% but ≤20% total growth). The diameters of the TFT-resistant colonies for the positive and solvent controls and, in the case of a positive response, the test-substance-treated cultures were determined over a range of approximately 0.2 to 1.1 mm.

Evaluation criteria:

A result was considered positive if a concentration-related increase in mutant frequency was observed in the treated cultures and one or more treatment conditions with 10% or greater total growth exhibited mutant frequencies of ≥90 mutants per 10e6 clonable cells over the background level (based on the average mutant frequency of duplicate cultures). If the average solvent control mutant frequency was >90 mutants per 10e6 clonable cells, a doubling of mutant frequency over the background was also required. A result was considered negative if the treated cultures exhibited mutant frequencies of <90 mutants per 10e6 clonable cells over the background level (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency. There are some situations where a chemical would be considered negative where there was no culture showing between 10-20% survival: (1) There was no evidence of mutagenicity (e.g., no dose response or increase in mutant frequencies between 45 and 89 mutants per 10e6 above control) in a series of data points within 100% to 20% survival AND there was at least one negative data point between 20% and 25% survival. (2) There was no evidence of mutagenicity in a series of data points between 100% to 25% survival AND there was also a negative data point between 10% and 1% survival. In this case it would be acceptable to count the TFT colonies of cultures exhibiting <10% growth.

Statistics:

The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control for suspension growth over 2 days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection). The mutant frequency (number of mutants per 10e6 surviving cells) for each treatment condition was determined by dividing the average number of colonies in the three TFT plates by the average number of colonies in the three corresponding VC plates and multiplying by the dilution factor (2e4) then multiplying by 10e6. For simplicity, this is described as (Average # TFT colonies/average # VC colonies)x200.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Visible precipitate was present only at 3650 µg/mL in the treatment medium. Suspension growth relative to the solvent controls was 0% without activation with 4- and 24-hour exposures at ≥ 50 μg/mL (except for 3650 μg/mL with 4-hr exposure) and 0% with S9 activation with a 4-hour exposure at ≥ 15 μg/mL (except for 3650 μg/mL).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Under the conditions of this study, the test substance was concluded to be negative in the absence and presence of S9 metabolic activation in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay.

Executive summary:

The test substance was tested in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay in the absence and presence of Aroclor-induced rat liver S9. The preliminary toxicity assay was used to establish the concentration range for the mutagenesis assay. The mutagenesis assay was used to evaluate the mutagenic potential of the test substance. In the preliminary toxicity assay, the maximum concentration of the test substance in treatment medium was 3650 μg/mL. Visible precipitate was present only at 3650 μg/mL in treatment medium. Substantial toxicity was observed at concentrations ≥ 50 μg/mL without activation with 4- and 24-hour exposures and ≥ 5.0 μg/mL with S9 activation. Based on the results of the preliminary toxicity assay, the concentrations tested in the initial mutagenesis assay ranged from 5.0 to 75 μg/mL for the non-activated cultures and 1.0 to 25 μg/mL for the S9-activated cultures with a 4-hour exposure. No visible precipitate was present at any concentration in treatment medium. The concentrations chosen for cloning were 30, 35, 40, 50, and 75 μg/mL without activation and 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 μg/mL with S9 activation. No cloned cultures exhibited mutant frequencies ≥ 90 mutants per 10e6 clonable cells over that of the solvent control. There was no concentration-related increase in mutant frequency. Based on the results of the preliminary toxicity assay, the concentrations tested in the extended treatment assay ranged from 5.0 to 75 μg/mL for non-activated cultures with a 24-hour exposure. No visible precipitate was present at any concentration in treatment medium. The concentrations chosen for cloning were 20, 25, 30, 35, 40, and 50 μg/mL. No cloned cultures exhibited mutant frequencies ≥ 90 mutants per 10e6 clonable cells over that of the solvent control. There was no concentration-related increase in mutant frequency. The trifluorothymidine-resistant colonies for the positive and solvent control cultures from the mutation assay were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the positive controls yielded the expected increase in small colonies and large colonies. All criteria for a valid test were met. Under the conditions of this study, the test substance was concluded to be negative in the presence and absence of S9 metabolic activation in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay. The assay was negative.