Lithium trifluoromethanesulphonate

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:

From 01-August-2016 to 05-October-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Lithium trifluoromethanesulphonate

EC Number:

251-528-5

EC Name:

Lithium trifluoromethanesulphonate

Cas Number:

33454-82-9

Molecular formula:

CHF3O3S.Li

IUPAC Name:

lithium trifluoromethanesulphonate

Test material form:

solid: particulate/powder

Details on test material:

See confidential details on test material

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL:
- At room temperature container flushed with nitrogen, desiccated

Method

Target gene:

- Salmonella typhimurium bacterial strains: a gene of histidine-requiring resulting in histidine-independent strains.
- Escherichia coli bacterial strain: a gene of tryptophan-requiring resulting in a tryptophan-independent strain.

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Dose range finding test
TA100 and WP2uvrA strains: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate with and without S9-mix tested in triplicate.

First experiment: Direct plate assay
TA1535, TA1537 and TA98 strains: 52, 164, 512, 1600 and 5000 µg/plate with and without S9-mix tested in triplicate.

Second experiment: Pre-incubation assay
All strains: 52, 164, 512, 1600 and 5000 µg/plate with and without S9-mix tested in triplicate.

According to the OECD Guideline No. 471, the recommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate or 5 µl/plate.

Vehicle / solvent:

Milli-Q water (Millipore Corp., Bedford, MA., USA)

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

Controls 2

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Controls 3

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

Without metabolic activation

Controls 4

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation

Controls 5

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation

Controls 6

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Controls 7

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation

Controls 8

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Controls 9

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Controls 10

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Controls 11

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Controls 12

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

SOURCE OF BACTERIA STRAINS:
Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535: 2006, TA1537: 2016, TA98: 2015, TA100: 2015; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA: 2008)]

PREPARATION OF BACTERIAL CULTURES:
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10E9 cells/ml). Freshly grown cultures of each strain were used for a test.

AGAR PLATES:
Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin (Merck) and 15 µg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan (Sigma).

TOP AGAR:
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

PREPARATION OF S9-MIX:
S9-mix was prepared immediately before use and kept on ice.
S9-mix contained per 10 ml: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution; 1 ml 0.33 M KCl solution. The above solution was filter (0.22 µm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.

ENVIRONMENTAL CONDITIONS:
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 35.4 - 38.8°C). Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Rationale for test conditions:

Test conditions were those recommended in the international guidelines (e.g. OECD, EC).

Evaluation criteria:

No formal hypothesis testing was done.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

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

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

In absence of S9-mix, a 5.9-fold increase in the number of revertant colonies compared to the solvent control was observed at 1600 µg/plate. For further details, see below.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In tester strain TA98 (absence of S9-mix), a 5.9-fold increase in the number of revertant colonies compared to the solvent control was observed at 1600 µg/plate. The three numbers of revertant colonies observed at this concentration were: 20, 22 and 171.
The 171 value is not only very different from the two other values obtained at 1600 µg/plate, but also very different than the values obtained at all the other doses, including the 2000 µg/plate values (12, 18 and 19).
This observation of an outlier, so far beyond the historical control data range and the two other values of the triplicate, is a rare phenomenon and is probably linked to an incidental fluctuation in the number of revertant colonies. The absence of any observed dose effect is confirming that this 171 value is an outlier. Therefore, the 5.9 times increase of revertant colonies observed at 1600 µg/plate is considered to be related to a specific outlier and to be not biologically relevant.

Any other information on results incl. tables

Dose range finding test

Concentrations selected for the direct plate assay and the pre-incubation assay were 52, 164, 512, 1600 and 5000 μg/plate.

First experiment: Direct plate assay

Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.

Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.

No reduction of the bacterial background lawn and no biologically significant decrease in the number of revertants were observed.

In the direct plate test, no increase in the number of revertants was observed upon treatment with Lithium Trifluoromethanesulfonate under all conditions tested.

Second experiment: Pre-incubation assay

Based on the results of the first mutation assay, the test item was tested up to the dose level of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.

There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

In the pre-incubation test, no increase in the number of revertants was observed upon treatment with Lithium Trifluoromethanesulfonate under all conditions tested, excepted with strain TA98 (absence of S9 -mix) at 1600 µg/plate. Therefore, the 5.9 times increase of revertant colonies observed at 1600 µg/plate is considered to be related to a specific outlier and to be not biologically relevant. For further details, see in section "Additional information on results".

Applicant's summary and conclusion

Conclusions:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic
activation system functioned properly.

Based on the results of this study it is concluded that Lithium Trifluoromethanesulfonate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The assessment of the mutagenic activity of Lithium Trifluoromethanesulfonate was carried out, under GLP compliance, using the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (plate incorporation and pre-incubation methods) based on the OECD Guideline No. 471 (adopted 21 July 1997) and the EU Method B.13/14.

Lithium Trifluoromethanesulfonate was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a preincubation assay both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254).

Lithium Trifluoromethanesulfonate did not induce a significant dose-related increase in the number of revertant (His+ ) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+ ) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that Lithium Trifluoromethanesulfonate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.