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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro testing was conducted in accordance with relevant guidelines, to investigate the genotixicity of the registered substance. Negative results were obtained in the presence and absence of metabolic activation.

Link to relevant study records

Reference

Reference 1

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

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

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 / 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).

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).

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.

see Final Report

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

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Study start: 18 November 2019; End of experimental phase: 18 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Adopted July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

The test item was assayed for the ability to induce micronuclei in human lymphocytes cultured in vitro, after treatment in the absence and presence of S9 metabolism.

Species / strain / cell type:

lymphocytes: human lymphocytes

Details on mammalian cell type (if applicable):

Two batches of human whole blood for Main Assay provided by Biopredic International (France), were used in this study.

Cytokinesis block (if used):

the inhibitor of actin polymerisation: cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction, provided by Trinova Biochem GmbH, from Rats pretrerated with Phenobarbital and 5,6-Benzoflavone

Test concentrations with justification for top dose:

The test item was assayed at the highest dose level of 1920 µg/mL (10mM), the maximum as indicated in the Study Protocol, and at the lower dose levels of 1280, 853, 569, 379, 253, 169, 112 and 74.9 µg/mL. The additional dose level of 49.9 µg/mL was included for the continuous treatment condition.

Following the 3 hour treatment in the absence and presence of S9 metabolism, no cytotoxicity was observed over the whole dose range.
Following the continuous treatment in the absence of S9, moderate cytotoxicity was observed at the highest dose level of 1920 µg/mL (44%). Mild cytotoxicity
(25%) was observed at the next lower dose level of 1280 µg/mL, while no remarkable cytotoxicity was noticed over the remaining dose range.


The experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point.

Vehicle / solvent:

Solutions of the test item were prepared in culture medium.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Colchicine 0.080 and 0.040 µg/mL

Details on test system and experimental conditions:

Culture media:
The culture medium for the lymphocytes had the following composition:

RPMI 1640 1x (Dutch modification) 500mL
Foetal Calf Serum 100mL
L-Glutamine (200mM) 6.25mL
Antibiotic solution 1.25mL

The foetal calf serum was heat-inactivated at 56°C for 20 minutes before use. For the initiation of the cultures, medium with the addition of phytohaemagglutin (PHA) was used in the following proportion: 10mL of PHA was added to 500mL of medium.

Preparation of the test cultures and treatment:

For each treatment series two replicate cultures were prepared at each test point including negative and positive controls.
Lymphocyte cultures were treated approximately fourty-eight hours after they were initiated.
Before treatment, cultures were centrifuged at 1000 rpm for 10 minutes and the culture medium was decanted and replaced with treatment medium.
For the short treatment series, the composition of the treatment media was as follows:

Presence of S9 metabolism
Test item solution or solvent/vehicle 0.5 mL
S9 mix 1.00mL
Culture medium (without PHA) 3.5 mL

Absence of S9 metabolism
Test item solution or solvent/vehicle 0.5 mL
Culture medium (without PHA) 4.5 mL


For the short treatment series, the treatment mediawere added to the tubes and the cultures were incubated for 3 hours at 37°C. At the end of treatment time, the cell cultures were centrifuged and washed twice with Phosphate Buffered Saline Solution. Fresh medium was added and the cultures were incubated for a further 28 hours (Recovery Period) before harvesting. At the same time, Cytochalasin-B was added to achieve a final concentration of 6 µg/mL.

For the continuous treatment series, 3 hours after beginning of treatment, Cytochalasin-B was also added and the cultures were incubated for a further 28 hours before harvesting.

Harvesting and slide preparation

The lymphocyte cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant was removed.
The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension.
A few drops of the cell suspension obtained in this waywere dropped onto clean,wet, greasefree glass slides. Three slides were prepared for each test point and each was labelled with the identity of the culture.
The slides were allowed to air dry and kept at room temperature prior to staining with a solution of Acridine Orange in PBS.

Slide evaluation

The cytokinesis-block proliferation index CBPI was calculated as follows:
CBPI =
mononucleated +2×binucleated +3×multinucleated/ total number of cells counted

where mononucleated, binucleated and multinucleated are respectively the number of mononucleated cells, binucleated cells and multinucleated cells. CBPI was used to measure the cytotoxic effect. Five hundred cells per cell culture were analysed. ForMain Assays 1 and 2, since negligible cytotoxcity was observed, the evaluation of CBPI of the lowest dose level was omitted. The highest dose level for genotoxicity assessment was selected on the basis of the cytotoxicity as calculated by the CBPI.
The percentage cytotoxicity was evaluated according to the following formula:

%Cytotoxicity =100-100 [ CBPIT -1 / CBPIC -1]

where:
T = test item treated culture
C = untreated/solvent control culture
The highest dose level for genotoxicity assessment was selected as a dose which produces a substantial cytotoxicity compared with the solvent control. Ideally the cytotoxicity should be between 50% and 60%. In the absence of cytotoxicity, the highest treatment level is selected as the highest dose level for
scoring.
Two lower dose levels were also selected for the scoring of micronuclei.
For the three selected doses, for the solvent and the positive control Cyclophosphamide, 1000 binucleated cells per cell culture were scored to assess the
frequency of micronucleated cells.
Concerning cultures treated with Colchicine, since it is a known mitotic spindle poison which induces mitotic slippage and cytokinesis block, a greater magnitude of response was observed in mononucleated cells. For this reason, 1000 mononucleated cells per cell culture were scored.

The criteria for identifying micronuclei were as follows:

1. The micronucleus diameter was less than 1/3 of the nucleus diameter
2. The micronucleus diameter was greater than 1/16 of the nucleus diameter
3. No overlapping with the nucleus was observed
4. Micronuclei were non-refractile and had the same staining intensity as the main nuclei

Rationale for test conditions:

Solubility and cytotoxicity tests were conducted ahead of the main study for dose selection.

Evaluation criteria:

Acceptance criteria:
The assay is considered valid if the following criteria are met:
– The incidence of micronucleated cells of the negative control is within the distribution range of our historical control values.
– Concurrent positive controls induce responses that are compatible with those generated in our historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
– Adequate cell proliferation is observed in solvent control cultures.
– The appropriate number of doses and cells is analysed.

Criterion for outcome:
In this assay, the test item is considered as clearly positive if the following criteria are met:
– Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
– The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values.
– There is a significant dose effect relationship.

The test item is considered clearly negative if the following criteria are met:
– None of the concentrations shows a statistically significant increase in the incidence of micronucleated cells.
– There is no concentration related increase when evaluated with the Cochran-Armitage trend test.
– All the results are inside the distribution of the historical control data.

Statistics:

Statistical analysis:
For the statistical analysis, a modified X^2 test was used to compare the number of cells with micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Key result

Species / strain:

lymphocytes: human

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

Conclusions:

On the basis of the above results, it is concluded that test item does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions

Executive summary:

The test item LITHIUM 4,5-DICYANO-2-(TRIFLUOROMETYL)IMIDAZOLATE was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation.
Three treatment conditions were performed. A short termtreatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of approximately 32 hours, corresponding to approximately two cell cycle lengths, was used. A long term (continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours.
Solutions of the test item were prepared in culture medium.
The test item was assayed at the highest dose level of 1920 µg/mL (10mM), the maximum as indicated in the Study Protocol, and at the lower dose levels of 1280, 853, 569, 379, 253, 169, 112 and 74.9 µg/mL. The additional dose level of 49.9 µg/mL was included for the continuous treatment condition.
The experiment included appropriate untreated/negative and positive controls. Two replicate cell cultures were prepared at each test point.
The actin polymerisation inhibitor Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesis block
proliferation index CBPI was calculated in order to evaluate cytotoxicity.
Dose levels for the scoring of micronuclei were selected with the aim to evaluate the test item concentrations at adequate levels of cytotoxicity, covering a range from the maximum (55 ± 5%) to slight or no toxicity. In the absence of cytotoxicity the highest dose level was selected for scoring.
Based on the results obtained, the following concentrations were selected for the scoring of micronuclei for all treatment series: 1920, 1280 and 853 µg/mL
One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.
Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed.
Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system and the responses were compatible with those generated in our historical control database.
The study was accepted as valid.
Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrent solvent control value was observed at any dose level, in any treatment series. All incidences were within the distribution of historical negative control values and no concentration related increase was seen.
It is concluded that LITHIUM 4,5-DICYANO-2-(TRIFLUOROMETYL)IMIDAZOLATE does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 January 2014 -- 07 March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

312.5, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: water for injections, batch No. 3F1542.
- Justification for choice: according to solubility assays performed at CiToxLAB France, the test item was soluble in the vehicle at 50 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or in the absence of a rat liver metabolizing system.

Executive summary:

The potential of Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate to induce reverse mutations was evaluated in Salmonella typhimurium. The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B.13/14) and in compliance with the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity study. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item was dissolved in water for injections.

The number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid. Since the test item was freely soluble and non-cytotoxic in the preliminary test, the highest dose-level selected for the main experiment was 5000 µg/plate, according to the criteria specified in the international guidelines. The treatment-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains, in both experiments, with and without S9 mix. No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels, in both experiments with and without S9 mix. No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted towards any of the tested strains in both experiments without S9 mix and in the first experiment with S9 mix. A moderate to strong toxicity (thinning of the bacterial lawn) was noted in the second experiment with S9 mix at dose-levels superior or equal to 2500 µg/plate in the TA 1537 and TA 100 strains, and at the highest tested dose-level of 5000 µg/plate in the TA 98 and TA 102 strains. The test item did not induce any noteworthy increase in the number of revertants in both experiments, either with or without S9 mix, in any of the five tested strains.

Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or in the absence of a rat liver metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

in vitro gene mutation study in bacteria
The potential of Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate to induce reverse mutations was evaluated in Salmonella typhimurium. The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B.13/14) and in compliance with the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity study. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.
The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item was dissolved in water for injections.
The number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid. Since the test item was freely soluble and non-cytotoxic in the preliminary test, the highest dose-level selected for the main experiment was 5000 µg/plate, according to the criteria specified in the international guidelines. The treatment-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains, in both experiments, with and without S9 mix. No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels, in both experiments with and without S9 mix. No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted towards any of the tested strains in both experiments without S9 mix and in the first experiment with S9 mix. A moderate to strong toxicity (thinning of the bacterial lawn) was noted in the second experiment with S9 mix at dose-levels superior or equal to 2500 µg/plate in the TA 1537 and TA 100 strains, and at the highest tested dose-level of 5000 µg/plate in the TA 98 and TA 102 strains. The test item did not induce any noteworthy increase in the number of revertants in both experiments, either with or without S9 mix, in any of the five tested strains.
Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or in the absence of a rat liver metabolizing system.

 

in vitro micronucleus study
The test item Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation.
Three treatment conditions were performed. A short termtreatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of approximately 32 hours, corresponding to approximately two cell cycle lengths, was used. A long term (continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours.
Solutions of the test item were prepared in culture medium.
The test item was assayed at the highest dose level of 1920 µg/mL (10mM), the maximum as indicated in the Study Protocol, and at the lower dose levels of 1280, 853, 569, 379, 253, 169, 112 and 74.9 µg/mL. The additional dose level of 49.9 µg/mL was included for the continuous treatment condition.
The experiment included appropriate untreated/negative and positive controls. Two replicate cell cultures were prepared at each test point.
The actin polymerisation inhibitor Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesis block
proliferation index CBPI was calculated in order to evaluate cytotoxicity.
Dose levels for the scoring of micronuclei were selected with the aim to evaluate the test item concentrations at adequate levels of cytotoxicity, covering a range from the maximum (55 ± 5%) to slight or no toxicity. In the absence of cytotoxicity the highest dose level was selected for scoring.
Based on the results obtained, the following concentrations were selected for the scoring of micronuclei for all treatment series: 1920, 1280 and 853 µg/mL
One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.
Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed.
Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system and the responses were compatible with those generated in our historical control database.
The study was accepted as valid.
Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrent solvent control value was observed at any dose level, in any treatment series. All incidences were within the distribution of historical negative control values and no concentration related increase was seen.
It is concluded that Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions

 

in vitro gene mutation study in mammalian cells / ERBC 2021 / K1 WoE / OECD 490
The test item Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate 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 Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate 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.

Justification for classification or non-classification

In vitro testing was conducted in accordance with Annexes VII and VIII of REACH to investigate the genotoxicity of the registered substance. No genotoxicity was observed in the absence and presence of metabolic activation, therefore, the registered substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008.