Alcohols, C8-14, γ-ω-perfluoro

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:

key study

Study period:

2005-08-26 to 2005-12-06

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-Perfluoralkylethanol

IUPAC Name:

2-Perfluoralkylethanol

Test material form:

other: solid (waxy)

Details on test material:

- Name of test material (as cited in study report): perfluoralkylethanols (2-Perfluoralkylethanol)

- Physical state: solid (waxy), yellowish
- Analytical purity: Sum perfluoralkylethanols 94.8%

- Purity test date: 2005-06-07
- Lot/batch No.: DEGB 0166711
- Expiration date of the lot/batch: 2006-06-07
- Storage condition of test material: At room temperature, protected from light

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/b-Naphthoflavone induced rat liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

Experiment I
without S9 mix: 7.8; 15.5; 31.0; 46.5; and 62.0 microgram/mL
with S9 mix: 1.9; 3.9; 7.8; 15.5; and 23.3 microgram/mL
Experiment IA
without S9 mix: 15.5; 31.0; 47.1; 62.0; and 108.5 microgram/mL
with S9 mix: 3.9; 7.5; 15.0; 22.6; and 30.0 microgram/mL
Experiment II
without S9 mix: 31.3; 62.5; 125.0; 187.5; 250 microgram/mL

Vehicle / solvent:

ethanol (On the day of the experiment (immediately before treatment), the test item was completely
molten at 50°C, homogenised, and then dissolved in ethanol (E. MERCK, D-64293
Darmstadt; purity: 99.8 %). The final concentration of ethanol in the culture medium did
not exceed 0.5 % (v/v). The solvent was chosen to its solubility properties and its relative
nontoxicity to the cell cultures.)

Controls

Negative solvent / vehicle controls:

yes

Remarks:

ethanol; On the day of the experiment (immediately before treatment), the test item was completely molten at 50°C, homogenised, and then dissolved in ethanol (E. MERCK, D-64293 Darmstadt; purity: 99.8 %)

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Remarks:

MMS: used in experiments without etabolic activaton; CPA used with metabolic activation

Details on test system and experimental conditions:

In the mutation experiment 1x10exp7 cells/flask (80 cm2 flasks) suspended in 10 mL RPMI medium with 3 % horse serum (15 % during 24 h treatment) were exposed to various concentrations of the test item either in the presence or absence of metabolic activation. After 4 h (after 24 h in the second experiment) the test item was removed by centrifugation (425 g, 10 min) and washing twice in "saline G". Subsequently the cells were resuspended in 30 mL complete culture medium and incubated for an expression and growth period of totally 72 h. In the second experiment the expression time without metabolic activation was 48 hours (RPMI medium with 15 % horse serum).
The cell density was determined each day and adjusted to 3x10exp5 cells/mL, if necessary. Relative suspension and total growth (RSG and RTG) of the treated cell cultures were calculated after 48 h (72 h following continuous treatment) according to the method of Clive and Spector. One sample of the cells was taken at the end of the treatment (4 h and 24 h, respectively), diluted and seeded into microtiter plates (about 2.0 cells/well), to determine the survival of the cells after treatment (cloning efficiency 1). After the expression period the cells were seeded into microtiter plates. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4x10exp3 cells in selective medium with TFT (SERVA, 0-69042 Heidelberg). The viability (cloning efficiency 2) was determined by seeding about 2.0 cells per well into 2 microtiter plates (same medium without TFT). The plates are incubated at 37 °C in 4.5 % CO2 and 95.5 % humidified air for 10 - 15 days to determine the cloning efficiency and to evaluate mutagenicity. Then the plates were evaluated manually.
The numbers of colonies were counted manually. In accordance with their size the colonies were classified into two groups. The colony size distribution was determined in the controls and at all concentrations of the test item. Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the density of the small colonies was considerably higher).

Evaluation criteria:

A test item is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points.
A test item producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered nonmutagenic in this system.
A significant response is described as follows:
The test item is classified as mutagenic if it reproducibly induces a mutation frequency that is at least two times higher than the spontaneous mutation frequency (negative or solvent control) in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants.
A positive response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.
Results of test groups are rejected if the relative total growth, the relative suspension growth and/or cloning efficiency 1 is less than 10 % of the solvent control or the cloning efficiency 2 after the expression period is less than 20 %.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a clastogenic effects are indicated.

Results and discussion

Any other information on results incl. tables

The study was performed to investigate the potential of perfluoroalkylethanols to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in three independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. Since one of the two parallel cultures did not meet the acceptance criteria (non-responding positive controls with and without metabolic activation), the experiment was repeated as experiment IA. The concentration range of experiment IA was adjusted to the toxicity observed in the first experiment.

The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours.

The highest applied concentration in the pre-test on toxicity (1000µg/mL) was limited by the solubility of the test item. The dose range of the main experiments was limited by toxicity and solubility of the test item.

Toxicity indicated by a cloning efficiency I or a relative total growth of less than 50 % in both parallel cultures was noted at 23.3 µg/mL in the first experiment with metabolic activation and at 62.0 µg/mL and above in experiment IA without and at 15.5 µg/mL and above with metabolic activation. In the second experiment no relevant toxic effects were observed up to the maximum concentration.

The threshold of twice the mutation frequency of the corresponding solvent control was just reached in the first culture of experiment IA at 30.0 µg/mL with metabolic activation. However, the absolute value of the mutation frequency (146 colonies per 10exp6 cells) was rather low and remained well within the historical range of negative and solvent controls and the control range of this study. Since no increase was observed in the parallel culture under identical conditions, this isolated effect was judged as a biologically irrelevant fluctuation. In the second experiment the mutation frequency exceeded the historical control range in culture I at 125 µg/mL. However, the threshold of twice the mutation frequency of the corresponding solvent control was not reached and no comparable increase was observed in the parallel culture under identical conditions nor in both cultures at even higher concentrations. In this study the range of the solvent controls was from 44 up to 174 mutant colonies per 10exp6 cells; the range of the groups treated with the test item was from 36 up to 280 mutant colonies per 10exp6 cells.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

The tested concentrations are described in table II (see attached). The evaluated experimental points and the results are summarised in table I (see attached).

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Therefore, perfluoroalkylethanols is considered to be non-mutagenic in this mouse lymphoma assay.

Executive summary:

The study was performed to investigate the potential of perfluoroalkylethanols to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in three independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. Since one of the two parallel cultures did not meet the acceptance criteria (non-responding positive controls with and without metabolic activation), the experiment was repeated as experiment IA. The concentration range of experiment IA was adjusted to the toxicity observed in the first experiment. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours. The highest applied concentration in the pre-test on toxicity (1000µg/mL) was limited by the solubility of the test item. The dose range of the main experiments was limited by toxicity and solubility of the test item. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.