4-ethynyl-2,6-difluoro-aniline

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:

May 15 - July 21, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

May 30, 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 29, 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

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

Test material

Test material information

Constituent 1

Reference substance name:

4-ethynyl-2,6-difluoro-aniline

EC Number:

865-131-5

Cas Number:

753501-37-0

Molecular formula:

C₈H₅F₂N

IUPAC Name:

4-ethynyl-2,6-difluoro-aniline

Test material form:

solid: crystalline

Method

Target gene:

TK gene

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit in Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
- Suitability of cells: The L5178Y cell line has been used successfully in in vitro experiments for many years. L5178Y cells are characterized by a high proliferation rate (doubling time 10 - 12 h in stock cultures) and cloning efficiency of untreated cells of usually more than 50 % both necessary for the appropriate performance of the study.
- Normal cell cycle time (negative control): 10 – 12 h

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Methods for maintenance in cell culture: Thawed stock cultures were propagated in plastic flasks in RPMI 1640 complete culture medium. The cells were subcultured two times prior to treatment. The cell cultures were incubated at 37 ± 1.5°C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5% ambient air.
- Cell cycle length, doubling time or proliferation index: 10 – 12 h doubling time
- Modal number of chromosomes: diploid (40 +/- 2)
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
RPMI 1640 medium supplemented with 15 % horse serum (HS) (3 % HS during 4 hour treatment), 100 U/100 μg/mL Penicillin/Streptomycin, 220 μg/mL Sodium-Pyruvate, and 0.5 – 0.75 % Amphotericin used as antifungal agent.
The cell cultures were incubated at 37 ± 1.5°C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5% ambient air.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital/β-naphthoflavone induced rat liver S9
- method of preparation of S9 mix: according to the currently valid version of the ICCR-Roßdorf SOP for rat liver S9 preparation.
- concentration or volume of S9 mix and S9 in the final culture medium: 29 mg/L

Test concentrations with justification for top dose:

Experiment I (- S9): 25, 50, 100 and 200 µg/mL
Experiment I (+ S9): 50, 100, 200, 250 and 300 µg/mL
Experiment II (- S9): 12.5, 25, 50, 100, 150, 200 and 250 µg/mL

The highest dose selected based on the recommendation of OECD TG 490.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen based upon its solubility properties and its non-toxicity to the cells.

- Justification for percentage of solvent in the final culture medium: 0.5% (v/v)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4h and 24h (second experiment without S9)
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48 h
- Selection time (if incubation with a selective agent): 10 – 15 days
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: 5 µg/mL TFT, incubation for 10-15 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: After the expression period the cultures were seeded into microtiter plates. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4E+3 cells in selective medium (see below) with TFT. The viability (cloning efficiency) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT).
- Criteria for small (slow growing) and large (fast growing) colonies: Criteria to determine colony size were the absolute size of the colony (more than 25% of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth (RTG); relative suspension growth (RSG, pre-experiment)
METHODS FOR MEASUREMENTS OF GENOTOXICIY: 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 25% of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).

Evaluation criteria:

A test item is considered to be clearly mutagenic when all of these criteria are met:
a) the IMF reproducibly (in both parallel cultures) exceeds a threshold of 126 colonies per 106 cells above the corresponding solvent control and
b) a relevant increase of the MF is dose-dependent.
c) any of the results (mean values) are outside the distribution of the historical negative control data (e.g. Poisson-based 95% confidence limit)

A test item is considered to be clearly non-mutagenic if, in all experimental conditions examined:
a) there is no concentration-related response or,
b) if there is an increase in MF, the IMF does not reproducibly exceed a threshold of 126 colonies per 106 cells above the corresponding solvent control.
c) all results (mean values) are inside the distribution of the historical negative control data (based 95% confidence limits).

Statistics:

The statistical analysis was performed in the mean values of culture I and II.
A linear regression will be performed using a validated test script of "R", a language and environment for statistical computing and graphics (p < 0.05), to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item will be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance will be considered together.

Results and discussion

Test results

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 7.49 (solvent control), 7.50 (test item 1530 µg/mL)
- Data on osmolality: 372 mOsm (solvent control), 357 mOsm (test item 1530 µg/mL)
- Precipitation and time of the determination: Precipitation occurred at 800 µg/mL at the end of treatment in experiment I (4h exposure) with and without metabolic activation

RANGE-FINDING/SCREENING STUDIES (if applicable): The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 12.0 μg/mL and 1530.0 μg/mL (equal to a molar concentration of approximately 10 mM) were used.
Relevant toxic effects indicated by a relative suspension growth (RSG) below 50% were observed at 328.5 μg/mL and above in the presence and absence of metabolic activation after 4 h and 24 h treatment.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation occurred at 765.0 μg/mL and above in the presence and absence of metabolic activation following 4 hours treatment. After continuous treatment (24 hours) precipitation was noted at 1530.0 μg/mL.
The following doses were used in the main experiment:
Experiment I (- S9 mix, 4 h): 25, 50, 100, 200 µg/mL
Experiment I (+S9 mix, 4 h): 50, 100, 200, 250, 300 µg/mL
Experiment II (- S9 mix, 24 h): 12.5, 25, 50, 100, 150, 200, 250 µg/mL

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Please refer to the attached background material.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value
Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) and cloning efficiency: Please refer to the attached background material.

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: 1E+7 (3E+6 during 24 h exposure) cells/flask (80 cm2 flasks) were exposed to various concentrations; 3E+5 cells/mL were sub-cultured
o Number of cells plated in selective and non-selective medium: 4E+3 cells/well of a microtiter plate (in selective medium), 2 cells/well of a microtiter plate (non-selective medium)
o When using the thymidine kinase gene on L5178Y cells: colony sizing for the negative and positive controls and if the test chemical is positive, and related mutant frequency: Please refer to the attached background material

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please refer to the attached background material.
- Negative (solvent/vehicle) historical control data: Please refer to the attached background material.

Applicant's summary and conclusion

Conclusions:

The test item is considered non-mutagenic in this mouse lymphoma thymidine kinase locus assay using the cell line L5178Y.

Executive summary:

The study was performed to investigate the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours.  The maximum concentration (1530 μg/mL) applied in the pre-experiment was chosen with regard to the molecular weight of the test item corresponding to a molar concentration of about 10 mM. The maximum concentration of the main experiments was limited by cytotoxic effects. The test item was dissolved in DMSO.  

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, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.