Lithium bis(trifluoromethylsulfonyl)imide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2001-11-29 to 2002-04-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD Guideline and EU Method and according to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese guideline: Notification No. 700 of the Environmental Agency, No. 1039 of the Ministry of Health and Welfare and No. 1014 of Ministry of International Trade and Industry, 9th December 1986

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Method

Target gene:

Each strains derived from Salmonella typhimurium LT2 contains one mutation in the histidine operon, resulting in a requirement for histidine. The strain of Escherichia coli contains one mutation in the tryptophan operon, resulting in a requirement for tryptophan.

The TA 1535, TA 100, TA 102 and WP2 uvrA strains are reverted by base-pair substitution mutagens and the TA 1537 and TA 98 strains by frameshift mutagens. In addition, the TA 102 strain detects oxidative mutagens.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Cofactor-supplemented post-mitochondrial fraction (S9 fraction) purchased by Moltox and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.

Test concentrations with justification for top dose:

0, 312.5, 625, 1250, 2500 and 5000 µg/plate for all mutagenicity experiments with and without S9 mix, except for the third confirmatory experiment with the TA 98 strain with S9 mix: 0, 312.5, 625 and 1250 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see Details on test system and conditions

Details on test system and experimental conditions:

METHOD OF APPLICATION: The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second and third experiments with S9 mix were performed according to the preincubation method.

POSITIVE CONTROLS :
-Without activation :
* 1 µg/plate of Sodium azide (NaN3) in DMSO: strains TA 1535 and TA 100
* 50 µg/plate of 9-aminoacridine (9AA) in DMSO: stain TA 1537
* 0.5 µg/plate of 2-nitrofluorene (2NF) in DMSO: strain TA 98
* 0.5 µg/plate of Mitomycin (MMC) in distilled water: strain TA 102
* 2 µg/plate of 4-nitroquinoline 1-oxide (4NQO) in DMSO: strain WP2 uvrA

-With activation :
* 2 µg/plate of 2-anthramine (2AM) in DMSO: strains TA 1535, TA 1537, TA 98 and TA 100
* 10 µg/plate of 2-anthramine (2AM) in DMSO: strains TA 102 and WP2 uvrA

DURATION
- Preincubation period: The test item solution (0.1 mL), the bacterial suspension (0.1 mL) and the S9 mix (0.5 mL) were incubated for 60 min at 37ºC before adding the overlay agar and pouring onto the surface of a minimum agar plate.
- Exposure duration: 48 to 72 hours
- Incubation: 37°C
- Expression time (cells in growth medium): The number of revertants is determined at the end of the exposure time.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: All revertants per plates are counted

Evaluation criteria:

CRITERIA FOR VALIDITY:
The study was considered valid if the following criteria are fully met:
- the number of revertant in the negative controls is consistent with the test site's historical data;
- the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the test site's historical data.

EVALUATION CRITERIA:
A reproducible two-fold increase 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 in the evaluation of the data obtained.

Statistics:

No statistical analysis was performed

Results and discussion

Test results

Test results 1

Species / strain:

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

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:

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 number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.
No toxicity was noted towards all the strains used, both with and without S9 mix.

In the second experiment with S9 mix (preincubation method), a 4-fold increase in the number of revertants was noted in the TA 98 strain, only at the dose-level of 625 µg/plate.
In order to check the reliability of this increase, a third confirmatory experiment was performed with this strain, under the same experimental conditions. No increase in the number of revertants was reproduced in this third experiment and therefore the effect noted in the second experiment was considered as sporadic.
The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five remaining strains.

Any other information on results incl. tables

Table 1: Summary table of the first experiment

		Salmonella typhimuriumstrains										Escherichia coli
		TA98		TA100		TA102		TA1535		TA1537		WP2 uvrA
Concentrations (µg/plate)		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Vehicle	M*^1	26	26	94	102	261	335	23	15	11	12	37	42
	SD*^2	7	4	5	18	11	75	4	6	3	1	2	6
	R*^3	-	-	-	-	-	-	-	-	-	-	-	-
312.5	M*^1	20	29	91	116	260	364	18	10	11	13	38	42
	SD*^2	4	6	7	11	38	82	2	4	1	2	11	14
	R*^3	0.8	1.1	1.0	1.1	1.0	1.1	0.8	0.7	1.0	1.1	1.0	1.0
625	M*^1	20	31	92	108	290	362	16	12	10	17	36	37
	SD*^2	4	3	1	17	3	28	6	5	2	8	7	1
	R*^3	0.8	1.2	1.0	1.1	1.1	1.1	0.7	0.8	0.9	1.4	1.0	0.9
1250	M*^1	20	24	102	95	271	349	17	11	11	15	32	46
	SD*^2	9	6	7	12	11	25	3	1	5	8	5	4
	R*^3	0.8	0.9	1.1	0.9	1.0	1.0	0.7	0.7	1.0	1.3	0.9	1.1
2500	M*^1	24	27	96	115	281	355	22	12	11	14	29	41
	SD*^2	5	4	7	17	33	34	4	4	4	3	3	7
	R*^3	0.9	1.1	1.0	1.1	1.1	1.1	1.0	0.8	1.0	1.2	0.8	1.0
5000	M*^1	20	22	80	119	262	303	22	12	10	10	28	36
	SD*^2	4	4	7	18	23	47	4	5	7	2	4	6
	R*^3	0.8	0.9	0.9	1.2	1.0	0.9	1.0	0.8	0.9	0.8	0.8	0.9
NaN3 (1 µg/plate)	M*^1	-	-	819	-	-	-	712	-	-	-	-	-
	SD*^2	-	-	106	-	-	-	46	-	-	-	-	-
	R*^3	-	-	8.7	-	-	-	31.4	-	-	-	-	-
2NF (0.5 µg/plate)	M*^1	200	-	-	-	-	-	-	-	-	-	-	-
	SD*^2	30	-	-	-	-	-	-	-	-	-	-	-
	R*^3	7.7	-	-	-	-	-	-	-	-	-	-	-
9AA (50 µg/plate)	M*^1	-	-	-	-	-	-	-	-	358	-	-	-
	SD*^2	-	-	-	-	-	-	-	-	137	-	-	-
	R*^3	-	-	-	-	-	-	-	-	32.5	-	-	-
MMC (0.5 µg/plate)	M*^1	-	-	-	-	2442	-	-	-	-	-	-	-
	SD*^2	-	-	-	-	183	-	-	-	-	-	-	-
	R*^3	-	-	-	-	9.4	-	-	-	-	-	-	-
4NQO (2 µg/plate)	M*^1	-	-	-	-	-	-	-	-	-	-	1966	-
	SD*^2	-	-	-	-	-	-	-	-	-	-	134	-
	R*^3	-	-	-	-	-	-	-	-	-	-	53.6	-
2AM (2 or 10 µg/plate)	M*^1	-	1197	-	2054	-	4927	-	215	-	121	-	645
	SD*^2	-	22	-	90	-	184	-	12	-	12	-	11
	R*^3	-	46.6	-	20.1	-	14.7	-	14.0	-	10.3	-	15.5

 

M*¹: Mean revertant colony counts

SD*²: Standard deviation

R*³: Ratio treated / solvent

Vehicle: Distilled water

NaN3: Sodium azide

2NF: 2-nitrofluorene

9AA: 9-aminoacridine

MMC: Mitomycin

4NQO: 4-nitroquinoline 1-oxide

2AM: 2-anthramine

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under these experimental conditions, the test item BIS TRIFLUOROMETHANESULFONIMIDE LITHIUM does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.

Executive summary:

The objective of this study was to evaluate the potential of the test item BIS TRIFLUOROMETHANESULFONIMIDE LITHIUM (TFSILi) to induce reverse mutation in Salmonella typhimurium and Escherichia coli.

A preliminary toxicity test was performed to define the dose-levels of TFSILi to be used for mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation sytem, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. A third confirmatory experiment was performed with S9 mix.

All experiments was performed according to the direct plate incorporation method except for the second and third tests with S9 mix, which were performed according to the preincubation method (60 minutes, 37°C).

Five strains of bacteria Salmonella typhimurium: TA1535, TA1537, TA98, TA100 and TA102 and one strain of Escherichia coli: WP2 uvrA were used. 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 thinning of the bacterial lawn.

The test item TFSILi was dissolved in distilled water.

Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.

The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate, for all mutagenicity experiments with and without S9 mix, except for the third confirmatory experiment with the TA98 strain with S9 mix where 312.5, 625 and 1250 µg/plate were tested.

No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. No toxicity was noted towards all the strain used, both with and without S9 mix. No increase in the number of revertans which could be considered as relevant was noted, both with and without S9 mix, in any of the six tester strains. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Under these experimental conditions, the test item TFSILi does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.