Reaction products of C16-18 (even numbered), C18 unsaturated alkylamines with C10-13 alkylbenzenesulfonic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 April 2017 - 21 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

1

Reference substance name:

Reaction products of C16-18 (even numbered), C18 unsaturated alkylamines with C10-13 alkylbenzenesulfonic acid

EC Number:

701-164-2

IUPAC Name:

Reaction products of C16-18 (even numbered), C18 unsaturated alkylamines with C10-13 alkylbenzenesulfonic acid

Test material form:

liquid: viscous

Details on test material:

Chemical registery number : 701-164-2
Chemical name : Reaction products of C16-18 (even numbered), C18 unsaturated alkylamines with C10-13 alkylbenzenesulfonic acid

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agreed by each registrant.

Method

Target gene:

TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

2% S9 Mix

Test concentrations with justification for top dose:

The test item is a UVCB compound; therefore the maximum recommended dose level was set at 5000 µg/mL with no correction for purity. However the maximum achievable concentration was set at 1250 µg/mL due to the toxicity of THF to L5178Y cells.

Vehicle / solvent:

Solvent tetrahydrofuran used as vehicle controls.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.

Cell Culture: The stocks of cells are stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in RPMI 1640 medium. The cells have a generation time of approximately 12 hours and were subcultured accordingly. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Cleansing: Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours.

Test Item Preparation: the test item was accurately weighed and formulated in THF prior to serial dilutions being prepared. The test item is a UVCB compound; therefore the maximum recommended dose level was set at 5000 µg/mL with no correction for purity. However the maximum achievable concentration was set at 1250 µg/mL due to the toxicity of THF to L5178Y cells. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

Evaluation criteria:

A mutation assay is considered acceptable if it meets the following criteria:
1.The majority of the plates, for both viability (%V) and 5-TFT resistance, are analysable for each experiment.
2. The absolute viability (%V) at the time of mutant selection of the solvent controls is 65 to 120%.
3. The total suspension growth of the solvent control following 4-hour exposure, calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold increase in cell number, should be in the range of 8 to 32. Following 24-hour exposure the total suspension growth should be in the range of 32 to 180.
4. The in-house vehicle control mutant frequency is in the range of 50 – 170 x 10-6 cells.
5. Positive controls meets at least one of the following two acceptance criteria developed by the IWGT workgroup:
The positive control should demonstrate an absolute increase in total MF.
The positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent untreated/solvent control.
6. The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures.
7. For non-toxic test materials the upper test material concentrations are 10 mM, 2 mg/mL or 2-µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCBs) the upper dose level may need to be higher and the maximum concentration is 5 mg/mL.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test material is considered to be non-mutagenic in this assay.

Executive summary:

The maximum dose level used was predominantly limited by test material induced toxicity, however precipitate was also considered. No precipitate of the test material was observed throughout the main test. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. Optimum levels of toxicity were considered to have been achieved in both 4-hour exposure groups and very near to optimum toxicity was achieved in the 24-hour exposure group. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test material did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

The test material did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it is considered to be non-mutagenic in this assay.