1H-Imidazole-4,5-dicarbonitrile, 2-(trifluoromethyl)-, lithium salt (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November 2019 - January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

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:

other: gene mutation in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

lithium(1+) 4,5-dicyano-2-(trifluoromethyl)-1H-imidazol-1-ide

EC Number:

691-963-1

Cas Number:

761441-54-7

Molecular formula:

C6 F3 N4. Li

IUPAC Name:

lithium(1+) 4,5-dicyano-2-(trifluoromethyl)-1H-imidazol-1-ide

Test material form:

solid

Method

Target gene:

The mutation assay method used in this study is based on the identification of L5178Y colonies which have become resistant to a toxic thymidine analogue trifluorothymidine (TFT).This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides, which are used in nucleic acid synthesis resulting in the death of TK-competent cells.TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot metabolise trifluorothymidine and thus survive and grow in its presence. In the L5178Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on chromosome 11. Cells which are heterozygous at the TK locus (TK+/-) may undergo a single step forward mutation to the TK-/- genotype in which little or no TK activity remains. The cells used, L5178Y TK+/-, are derived from one of the two clones originated from a thymic tumour induced in a DBA/2 mouse by methylcholanthrene. The use of the TK mutation system in L5178Y mouse lymphoma cells has been well characterised and validated (D. Clive et al., 1979) and is accepted by most of the regulatory authorities. The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant colonies designated as small or large. It has been evaluated that point mutations and deletions within the active allele (intragenic event) produce large colonies. Small colonies result in part from lesions that affect not only the active TK allele but also a flanking gene whose expression modulates the growth rate of cells.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI medium supplemented with Horse serum.
- Properly maintained: yes; Permanent stocks of the L5178Y TK+/- cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory.
- Prior to use, cells were cleansed of pre-existing mutants.

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction: Species: Rat; Strain: Sprague Dawley; Tissue: Liver Inducing Agents: Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Number: 3971

Test concentrations with justification for top dose:

Based on solubility data, the maximum practicable concentration of the test item in the final treatment medium was 1920 µg/mL (corresponding to 10mM) using RPMI 1640Minimal as solvent. This concentration is the upper limit to testing as indicated in the Study Protocol.

Based on the results obtained in the preliminary trial, the assy for mutation at the TK locus was performed using the following dose levels:
Main Assay I (+S9, 3 hour treatment): 1920, 960, 480, 240 and 120 ug/mL.
Main Assay I (-S9, 3 hour treatment): 1920, 960, 480, 240 and 120 ug/mL.

Negative results were obtained in Main Assay I, thus a second experiment (Main Assay II) in the absence of S9 metabolism was performed, using a longer treatment time (24 hours) and the dose levels described in the following table:
Main Assay II (-S9, 24 hour treatment): 1500, 1200, 960, 768, 307 and 123 ug/mL.

Vehicle / solvent:

Test item solutions were prepared using complete medium (RPMI 1640 Minimal medium).

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

Cytotoxicity assay
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined. Treatments were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in 20mL of complete medium (10%). Cell concentrations were adjusted to 8 cells/mL using complete medium (20%) and, for each dose level, 0.2mL was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 7-8 days. Wells containing viable clones were identified by eye using background illumination and then counted.

Mutation assay
The mutation assay was performed including vehicle and positive controls, in the absence and presence of S9 metabolising system. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. In the first experiment (Main Assay I), the cells were exposed to the test item for a short treatment time (3 hours). Clear negative results were obtained, thus a second experiment (Main Assay II) in the absence of S9 metabolism was performed, using a longer treatment time (24 hours). After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2×10^5 cells/mL. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.
During the expression period (two days after treatment), the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determined and adjusted to give 2×10^5 cells/mL. After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 µg/mL) and an estimated 2 × 10^3 cells were plated in each well of four 96-well plates.
Plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored.
After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted.

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×10^-6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines (RobinsonW.D., 1990).

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:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Survival after treatment
In the first experiment, in the absence of S9 metabolic activation, mild toxicity was noticed at the highest concentration tested reducing the Relative Total Growth (RTG) to 54% of the concurrent negative control. Slight or no relevant reduction of RTG was observed at the remaining concentrations tested. In the presence of S9 metabolism, test item treatment at 1920 µg/mL yielded mild toxicity reducing RTG to 55% of the concurrent negative control value. Slight or no relevant toxicity was observed over the remaining concentrations tested.
In the second experiment, in the absence of S9 metabolic activation using the long term treatment, dose-related cytotoxicity was observed. Moderate toxicity, reducing RTG to 28% of the concurrent negative control was noticed at the highest dose level (1500 µg/mL), while at the next two lower concentrations (1200 and 960 µg/mL) test item treatment yielded a mild cytotoxicity (45% and 43% RTG). Slight or no relevant toxicity was observed over the remaining concentrations tested.

Mutation results
No statistically significant or biologically relevant increase in mutant frequency values was observed at any concentration tested, in the absence of S9 metabolic activation, using the short or long treatment. A statistically significant increase was noticed at the highest dose level in the presence of S9 metabolism and a linear trend was also indicated. However, the observed increase was lower than the Global Evaluation Factor, thus it was considered of no biological relevance.

Any other information on results incl. tables

see Final Report

Applicant's summary and conclusion

Conclusions:

It is concluded that LiTDI does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item LiTDI was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. Based on solubility data, the maximum practicable concentration of the test item in the final treatment medium was 1920 µg/mL (corresponding to 10mM) using RPMI 1640 Minimal as solvent. This concentration is the upper limit to testing as indicated in the Study Protocol. On the basis of this result, a preliminary cytotoxicity assay was performed both in the absence and presence of S9 metabolic activation, using themaximumdose level of 1920 µg/mL and a wide range of lower dose levels: 960, 480, 240, 120, 60.0, 30.0, 15.0 and 7.50 µg/mL. No precipitation of the test item was noted upon addition of the test item to the cultures and at the end of the 3 and 24 hour treatment periods. In the absence of S9 metabolic activation, using the 3 hour treatment time, mild reduction of Relative Survival (RS: 55%) was observed at the highest concentration, while slight or no relevant toxicity was noted at the remaining dose levels. In the presence of S9 metabolism the test item yielded a slight toxic effect, reducing RS to 66% of the concurrent negative control value, only at the highest dose level. Using the 24 hour treatment time, a dose dependent cytotoxicity was observed, reducing RS to 26% and 0% at 960 and 1920 µg/mL, respectively.

Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed using the following dose levels:

Main Assay I - 3 hours without S9 : 1920, 960, 480, 240 and 120 µg/mL

Main Assay I - 3 hours with S9 : 1920, 960, 480, 240 and 120 µg/mL

Main Assay II - 24 hours without S9 : 1500, 1200, 960, 768, 307 and 123 µg/mL

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The cloning efficiencies at Day 2, the suspension growth over 2 days and the mutant frequencies in the solvent control cultures fell within the normal range. Both positive controls, methylmethanesulphonate and benzo(a)pyrene, induced a clear increase above the spontaneous background in the small colony mutation frequency, higher than 150×10-6. The study was accepted as valid. Using the short treatment time in the absence of S9 metabolism, mild toxicity was noticed at the highest concentration tested reducing the Relative Total Growth (RTG) to 54% of the concurrent negative control. Slight or no relevant reduction of RTG was observed at the remaining concentrations tested. In the presence of S9 metabolism, the test item yielded 55% RTG at the top concentration, while slight or no relevant toxicity was seen at the remaining dose levels. Using the long treatment time in the absence of S9 metabolic activation, a dose dependent cytotoxicity was observed, reducing RTG to 28% of the concurrent negative control at 1500 µg/mL.

No relevant increase in mutant frequencies was observed following treatment with the test item in the absence or presence of S9 metabolism, in any experiment.

It is concluded that LiTDI does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

ERBC