lithium bis(fluorosulfonyl)imide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 23 April 2010 and 10 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

lithium(1+) bis(fluorosulfonyl)azanide

EC Number:

924-516-9

Cas Number:

171611-11-3

Molecular formula:

(FSO2)2NLi

IUPAC Name:

lithium(1+) bis(fluorosulfonyl)azanide

Specific details on test material used for the study:

Abbreviation: LiFSI
Appearance; white powder
Lot No.: KS100113
Storage conditions: Containers should be sealed to avoid moisture absorption. Direct sunlight is strictly prohibited (protected from light), and attention should be paid for water and high humidity (measured temperature: 2 to 8 °C).

Method

Target gene:

histidine locus in Salmoenella strains
Tryptophan locus in E.coli strains

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 prepared from liver homogenates from Slc:SD rats treated with phenobarbital and 5,6-benzoflabone

Test concentrations with justification for top dose:

The test substance was dissolved in and diluted with Japanese Pharmacopoeia water for injection (Lot No. 8L88, Otsuka Pharmaceutical Factory, Inc.) to the prescribed concentrations. Calculations for the preparation was made on the basis that the purity of the test substance was 24.0%

Range-finding test (amount of LiFSI):
With and without S9 mix: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

In the range-finding test inhibition of cell growth or precipitation of the test substance was not detected in any test strains in both the non-activated and activated assays; therefore, the highest concentration in the main test was set at 5000 μg/plate

Definitive test (amount of LiFSI):
With and without S9 mix: 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

Water was chosen as the vehicle because test test substance is soluble in water.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.01, 0.01 and 0.1 µg/plate for strains TA100, E.coli WP2uvrA and TA98 respectively

Positive control substance:

other: 2-(2-Furyl)-3-( 5-nitro-2-furyI)acrylamide

Remarks:

without S9-mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for strain TA1535

Positive control substance:

sodium azide

Remarks:

without S9-mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

80 µg/plate for strain TA1537

Positive control substance:

other: 9-Aminoacridine hydrochloride hydrate

Remarks:

without S9-mix

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.5, 1, 2, 2 and 10 µg/plate for strains TA98, TA100, TA1535, TA1537 and WP2uvrA respectively

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9-mix

Details on test system and experimental conditions:

Subculture of test strains
Each thawed test strain suspension (12 μL) was inoculated into an L-shaped tube (capacity approximately 40 mL) containing 12 mL of the culture medium for subculture (nutrient broth medium). The mixture in the L-shaped tube was cooled with ice until the start of incubation, for 7.5 h in the dose-finding test, 7.4 h in the main test, and 7.0 h in the confirmatory test. Then, the mixture was incubated for 10 h in a shaking water bath (Personal- 11·EX, Taitec Corporation) set at 37°C, 40 mm in stroke, and at a rate 100 times/min. At the termination of incubation, OD660nm of the culture obtained was measured using a spectrophotometer (mini photo 518, Taitec Corporation) to calculate the number of viable cells by the correlation equation between the number of viable cells and OD660nm· A culture in which the number of viable cells was above 1 x 10^9 cells/mL was used in the test.

Treatment with the test and control preparations
The pre-incubation method was used for treatment with the test and control preparations. To a polyethylene tube (capacity of 5 mL) with a cap containing 0.1 mL of the test or control preparation, 0.5 mL of 0.1 mol/L sodium-phosphate buffer (pH 7.4) was added and mixed for the non-activated assay, and 0.5 mL of S9 mix was added and mixed for the activated assay. To these mixtures, 0.1 mL of each culture was added, which were incubated (pre-incubation) for 20 min in a shaking water bath (Personal-11 ·EX, Taitec Corporation) set at 37°C, 40 mm in stroke, and at a rate 100 times/min. After the termination of pre-incubation, 2 mL of top agar containing 0.05 mmol/L L-histidine and 0.05 mmol/L D-biotin was added to the mixtures of S. typhimurium, and 2 mL of top agar containing 0.05 mmol/L L-tryptophan was added to the mixtures of E.coli, which were mixed and spread over each plate. After the top agar was solidified on a flat place, the plates were placed in an incubator (MIR-262, SANYO Electric Co., Ltd.) set at 37°C and incubated for 49 h. In each of the dose-finding, main, and confirmatory tests, a sterility test was performed to check bacterial contamination in the test preparation of the highest concentration and in the S9 mix used in the test.

Each test strain was tested using 3 plates in the negative control group, 2 plates per dose in the test substance groups, and 2 plates in the positive control group.

Observation
The plates in the negative control group, test substance groups and positive control group of each test strain were observed with a stereoscopic microscope (SZ6045TR, Olympus Optical Co., Ltd.) for inhibition of cell growth, and the plates in the test substance groups were macroscopically checked for precipitation of the test substance. Then, revertants in each plate in the negative control group, test substance groups, and positive control group of each test strain were counted using a colony analyzer (CA-1 lD, System Science Co., Ltd.).

Evaluation criteria:

Confirmation of sensitivity of test strains
A test strain was judged to have appropriate sensitivity when the average number of revertants in the negative control group of the test strain was within the range of control values based on the historical control data of the test facility, and the value in the positive control group of the test strain was twice or more that in the negative control group.

Evaluation criteria of test results
The test substance was judged to be positive for potential to induce genetic mutation when the average number of revertants in the test substance groups was
twice or more that in the negative control group, and reproducibility in the dose-related increase in the number of revertants was demonstrated, in at least one
test strain. No statistical analyses were applied for evaluation of the test results.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The results of the dose-finding test (5 to 5000 μg/plate) were as follows: In S. typhimurium TA98 in the non-activated assay, the average numbers of revertants at 150 to 1500 μg/plate increased and was twice or more than in the negative control group, this effect was not dose-related. The average number of revertants in the test substance groups was less than twice that in the negative control group in the other test strainsfor both the non-activated and activated assays, and no dose-related increase in the number of revertants was noted. No inhibition of cell growth or precipitation of the test substance was detected in any test strains, for the non-activated and activated assays.

The results of the main test (78.1 to 5000 μg/plate) were as follows: the average number of revertants in the test substance groups were less than twice that in the negative control group in each test strain for the non-activated and activated assays, and no dose-related increase in the number of revertants was noted. No inhibition of cell growth or precipitation of the test substance was detected in any test strains, for the non-activated and activated assays.
As the increase in the number of revertants in S. typhimurium TA98 in the non-activated assay was not reproducible between the dose-finding test and the main test, a confirmatory test was conducted at the same doses as in the main test to assess reproducibility. This confirmatory test showed the average number of revertants in the test substance groups to be less than twice that in the negative control group, and no dose-related increase was noted, and no inhibition of cell growth or precipitation of the test substance was detected.
In the dose-finding, main, and confirmatory tests, all the average numbers of revertants in the negative control group of all test strains were within the control range based on the historical control data of the test facility. The average number of
revertants in the positive control group of each test strain clearly increased, and was twice or more that in the negative control group in all test strains.
In the sterility tests in the dose-finding, main, and confirmatory tests, no bacterial contamination was detected in the test preparation of the highest dose or in the S9 mix.

Applicant's summary and conclusion

Conclusions:

In conclusion, LiFSI is not mutagenic to the bacteria under the conditions of this test.

Executive summary:

The potential of LiFSI to induce genetic mutation in bacteria was evaluated by a reverse mutation test using Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2uvrA. The test was conducted by the pre-incubation method, in the non-activated assay (in the absence of the S9 mix metabolic activation system) and activated assay (in the presence of the S9 mix metabolic activation system).

In the dose-finding test, the highest dose of the test substance was set at 5000 μg/plate, and a total of 7 doses (5 to 5000 μg/plate) were set in approximately threefold dilution series (6-step serial dilution) for each test strain in both the non-activated and activated assays. In the main test, the highest dose of the test substance was set at 5000 μg/plate, and a total of 7 doses (78.1 to 5000 μg/plate) were set in a twofold dilution series (6-step serial dilution) for each test strain in both the non-activated and activated assays. In addition, a confirmatory test was conducted using S. typhimurium TA98 by the non-activated assay at the same doses as in the main test.

The results of the dose-finding test were as follows: In the non-activated assay using S. typhimurium TA98, the average numbers of revertants at 150 to 1500 μg/plate increased and was twice or more than in the negative control group, this effect was not dose-related. The average number of revertants in the test substance groups was less than twice that in the negative control group in the other test strains both the non-activated and activated assays, and no dose-related increase in the number of revertants was noted. No inhibition of cell growth or precipitation of the test substance was detected in any test strains, either the non-activated and activated assays.

The results of the main and confirmatory tests were as follows: the average number of revertants in the test substance groups were less than twice that in the negative control group in each test strain for both the non-activated and activated assays, and no dose-related increase in the number of revertants was noted. No inhibition of cell growth or precipitation of the test substance was detected in any test strains, under all test conditions used in these tests.

In the dose-finding, main, and confirmatory tests, all the average numbers of revertants in the negative control group of all test strains were within the control range based on the historical control data of the test facility. The average number of revertants in the positive control group clearly increased and was twice or more that in the negative control group. These results confirmed that each test strain had appropriate sensitivity to the mutagens.

On the basis of these results, the increase in the numbers of revertants in S. typhimurium TA98 in the non-activated assay in the dose-finding test was not dose-related, and showed no reproducibility; therefore, this was not considered to be positive reaction.

In conclusion, LiFSI is not mutagenic to the bacteria under the conditions of this test.