Carboxymethyldimethyl-3-[[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)sulphonyl]amino]propylammonium hydroxide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

Carboxymethyldimethyl-3-[[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)sulphonyl]amino]propylammonium hydroxide

EC Number:

252-046-8

EC Name:

Carboxymethyldimethyl-3-[[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)sulphonyl]amino]propylammonium hydroxide

Cas Number:

34455-29-3

Molecular formula:

C15H19F13N2O4S

IUPAC Name:

N-(carboxymethyl)-N,N-dimethyl-3-{[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)sulfonyl]amino}propan-1-aminium hydroxide

Details on test material:

-Purity: 87%

Method

Target gene:

L5178Y/TK+/- mouse lymphoma cells are heterozygous at the normally diploid thymidine kinase (TK) locus.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: cells were prepared in 50% conditioned F0P supplemented with 10% horse serum and 2 mM L-glutamine (F10P) and 50% Fischer's Media for Leukemic Cells of Mice with 0.1% Pluronics F-68 (F0P). All media contained antibiotics.
- Properly maintained: yes, Each batch of frozen cells was tested and found to be free of mycoplasma contamination.
- Periodically "cleansed" against high spontaneous background: yes, Prior to use in the assay, L5178Y/TK+/- cells were cleansed to reduce the frequency of spontaneously occurring TK-/- cells.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary Toxicity Assay: 0.977 to 500 μg/mL
Mutagenicity Assay: 0.977 to 500 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The vehicle of choice, ethanol (EtOH), permitted preparation of a workable suspension at a concentration of 50 mg/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- For the PRELIMINARY TOXICITY ASSAY only, after a 4-hour treatment in the presence and absence of S9 activation, cells were washed with culture medium and cultured in suspension for two days post-treatment, with cell concentration adjustment on the first day. After a 24-hour treatment in the absence of S9 activation, cells were washed with culture medium and immediately readjusted to 3x10e5 cells/mL. Cells were then cultured in suspension for an additional two days post-treatment with cell concentration adjustment on the first day.
- For the DEFINITIVE ASSAY only, at the end of the exposure period, the cells were washed with culture medium and collected by centrifugation. The cells were resuspended in 20 mL F10P on Day 1 and in 10 mL F10P on Day 2, and incubated at 37 ± 1°C for two days following treatment. Cell population adjustments to 3x105 cells/mL were made as follows:
* 4 hour treatment – 1 and 2 days after treatment.
* 24 hour treatment – immediately after test substance removal, and 2 and 3 days after treatment.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxic effects of each treatment condition were expressed relative to the vehicle-treated control for suspension growth over two days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection).

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 exhibit induced mutant frequencies of ≥90 mutants per 10e6 clonable cells (based on the average mutant frequency of duplicate cultures). If the average vehicle control mutant frequency was >90 mutants per 10e6 clonable cells, a doubling of mutant frequency over the vehicle would also be required.

A result was considered negative if the treated cultures exhibit induced mutant frequencies of less than 90 mutants per 10e6 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- the test substance did not have an adverse impact on the pH or osmolality of the cultures.

Any other information on results incl. tables

Based on the results of the preliminary toxicity assay, cultures were treated in the mutagenicity assay at concentrations of 31.3, 62.5, 125, 250, 375 and 500 μg/mL (4-hour treatments with and without S9), and 0.977, 1.95, 3.91, 7.81, 15.6, 31.3, 46.9, 62.5 and 93.8 μg/mL (24-hour treatment without S9). No visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 62.5, 125, 250, 375 and 500 μg/mL (4-hour treatments with and without S9), and 15.6, 31.3, 46.9 and 62.5 μg/mL (24-hour treatment without S9) exhibited 64 to 94%, 37 to 96%, and 19 to 79% RSG (relative suspension growth), respectively, and were cloned (cultures treated at other lower concentrations were discarded prior to cloning because a sufficient number of higher concentrations was available; cultures treated at other higher concentrations were excluded from evaluation of mutagenicity due to excessive toxicity). Relative total growth of the cloned cultures ranged from 66 to 107% (4-hour treatment with S9), 33 to 72% (4-hour treatment without S9) and 15 to 58% (24-hour treatment without S9). No increases in induced mutant frequency ≥90 mutants per 10e6 clonable cells were observed under any treatment condition.

Applicant's summary and conclusion

Conclusions:

The results indicate that the test substance was negative in the L5178Y/TK+/- Mouse Lymphoma Assay under the conditions, and according to the criteria, of the test protocol.

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).

Executive summary:

The test substance was evaluated to determine its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of Aroclor-induced rat liver S9 in accordance with OECD guideline 476. The test substance was prepared in ethanol and evaluated in a preliminary toxicity assay at concentrations from 1.95 to 500 μg/mL using 4-hour treatments with and without S9, and at concentrations from 0.977 to 250 μg/mL using a 24-hour treatment without S9. Based on the results, cultures were treated in the mutagenicity assay at concentrations of 31.3, 62.5, 125, 250, 375 and 500 μg/mL (4-hour treatments with and without S9), and 0.977, 1.95, 3.91, 7.81, 15.6, 31.3, 46.9, 62.5 and 93.8 μg/mL (24-hour treatment without S9). No visible precipitate was observed at the beginning or end of treatment.

No increases in induced mutant frequency ≥90 mutants per 10e6 clonable cells were observed under any treatment condition. All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met. These results indicate the test substance was negative in the L5178Y/TK+/- Mouse Lymphoma Assay, under the conditions, and according to the criteria, of the test protocol.