Trifluoromethanesulphonic anhydride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial Reverse Mutation assay (Ames test): not mutagenic in bacteria (2001)
- In vitro Mammalian chromosome aberration test (by analogy): not clastogenic (Kr. 2, 1994)
- In vitro Mammalian Cell Gene Mutation test (Mouse Lymphoma Assay)(by analogy): not mutagenic (Kr. 1, 2013)

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:

key study

Study period:

From 18 Jun 2012 to 10 Jan 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK+/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 with Horse serum, penicillin/streptomycin and sodium pyruvate
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver, S9

Test concentrations with justification for top dose:

Exp I: 1553, 777, 388, 194, 97, 49, 24 and 12 µg/mL (without S9/4 hours of exposure)
Exp I: 1563, 782, 391, 195, 98, 49, 24 and 12 µg/mL (with S9/ 4 hours of exposure)
Exp II: 1577, 789, 394, 197, 99, 49, 25 and 12 µg/mL (with and without S9/ 4 and 24 hours of exposure respectively)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Medium RPMI 1640 without supplements
- Justification for choice of solvent/vehicle: the test subtance was completely soluble, and this solvent doesn't have any effects on the viability of the cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Medium RPMI 1640 without supplements

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Medium RPMI 1640 without supplements

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: Exp I: 4 h (with and without metabolic activation), Exp II: 4h (with metabolic activation) and 24 h (without metabolic activation)
- Expression time (cells in growth medium): during 48 h after the end of the treatment period
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: not applicable.The mutant frequency (MF) is calculated as : MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: large and small colonies were scored.

Evaluation criteria:

The test item is considered to have mutagenic effects if:
- the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 106 cells above the corresponding solvent control.
- the relative increase of the mutation frequency shows a dose relationship.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
Results of test groups are generally rejected if the relative total growth is less than 10% of the solvent control.
The biological relevance of the results is always considered first. Appropriate statistical methods are used as an aid in evaluating the test results. However, the results of statistical testing were assessed with respect to dose-response relationship. Reproducibility and historical data was also taken into consideration.

Statistics:

- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies.
- Statistical significance was confirmed by means of the non-parametric X2 test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(Not mutagenic), see tables 7.6.1/3 and 7.6.1/3

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH value was determined at the maximal concentration of the test item and in the solvent control with and without metabolic activation (See Table)
- Effects of osmolality: osmolality was determined at the maximal concentration of the test item and in the solvent control with and without metabolic activation (See Table 7.6.1/1)
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no precipitation was observed in the treatments (Exp I + Exp II) with and without metabolic activation.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
the dose range of the test item used in the preliminary toxicity test was 0.014 mg/ml to 1.5 mg/ml. Based on the results of the solubility of the test item in aqueous media (0.9% NaCl solution and Medium RPMI 1640 without supplements) of the preliminary experiment, the highest concentration for the two main experiments was defined as 1.5 mg/mL. In addition, as no cytotoxicty was observed, the maximum concentration of the test item should be 0.01 mol/L (1.5 mg/ml) according to the regulatory guideline recommondations (OECD N° 473)

COMPARISON WITH HISTORICAL CONTROL DATA: yes. See Tables 7.6.1/4 and 7.6.1/5

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed at all tested concentrations (pre-experiment, Exp I and Exp II) with and without metabolic activation.

Table 7.6.1/1 Osmolarity and pH

	Osmolarity in mOsmol/kg	pH-Value
Solvent control medium without supplements	281	7.435
Solvent control 0.9 % NaCl in medium with supplements	291	n.d.
Positve control CPA in medium with supplements ,4.5 µg/mL	287	7.522
Positve control MMS in medium with supplements, 19.5 µg/mL	385	7.523
Test Item in medium with supplements; 1.577 mg/mL	294	7.025
Test Item in medium with supplements; 0.1 mg/mL	287	7.485

 Table 7.6.1/2 the cytotoxicity and mutagenicity results Exp I

Treatment	Conc. (µg/ml)	Relative Total Growth Culture A	Mutants per 10^6cells Culture A	Relative Total Growth Culture B	Mutants per 10^6cells Culture B	Relative Total Growth Mean	Mutants per 10^6cells Mean
Exp. I without metabolic activation (-S9), exposition time 4 hours
Solvent control medium	--	--	168.5	--	191	--	179.75
Positive control MMS	19.5	33.7%	583.5	20.3%	588	27.0%	585.75
Test Item	1553	86.9%	150.5	91.6%	202	89.2%	176.25
Test Item	777	105.8%	157	82.8%	221.5	94.3%	189.25
Test Item	388	94.9%	214	91.6%	267.5	93.3%	240.75
Test Item	194	96.7%	175	108.2%	232	102.5%	203.5
Test Item	97	95.2%	172	106.4%	215.5	100.8%	193.75
Test Item	49	117.0%	176	81.7%	227.5	99.3%	201.75
Test Item	24	106.1%	177.5	100.5%	225.5	103.3%	201.5
Test Item	12	113.0%	178.5	103.6%	210.5	108.3%	194.5
Exp. I with metabolic activation (+S9), exposition time 4 hours
Solvent control medium	--	--	78	--	95.5	--	86.75
Solvent control 0.9% NaCl	--	--	46.5	--	70.5	--	58.5
Positive control CPA	4.5	29.5%	797.5	28.7%	407.5	29.1%	602.5
Test Item	1563	102.2%	151	97.5%	172.5	99.8%	161.75
Test Item	782	133.6%	120	93.0%	149	113.3%	134.5
Test Item	391	100.2%	152	90.3%	131	95.2%	141.5
Test Item	195	125.2%	92.5	152.2%	134.5	138.7%	113.5
Test Item	98	141.7%	108	81.4%	136	111.6%	122
Test Item	49	119.5%	102.5	86.4%	167.5	103.0%	135
Test Item	24	161.8%	107.5	86.5%	175.5	124.1%	141.5
Test Item	12	95.4%	128	64.9%	197	80.1%	162.5

Values above threshold (see below) are given in bold characters.

 

Threshold for mutagenic effects: number of mutant colonies per 10⁶cells of the respective solvent control plus 126.

Threshold solvent contr. (medium) (-S9): 305.75 mutants/10⁶cells

Threshold solvent contr. (medium) (+S9): 212.75 mutants/10⁶cells

Table 7.6.1/3 the cytotoxicity and mutagenicity results Exp II

Treatment	Conc. (µg/ml)	Relative Total Growth Culture A	Mutants per 10^6cells Culture A	Relative Total Growth Culture B	Mutants per 10^6cells Culture B	Relative Total Growth Mean	Mutants per 10^6cells Mean
Exp. II without metabolic activation (-S9), exposition time 24 hours
Solvent control medium	--	--	118.5	--	134.5	--	126.5
Positive control MMS	19.5	16.9%	736.5	14.4%	953	15.7%	844.75
Test Item	1577	98.4%	123	106.4%	105	102.4%	114
Test Item	789	121.6%	102	112.6%	120	117.1%	111
Test Item	394	133.3%	85	136.0%	88.5	134.6%	86.75
Test Item	197	98.5%	108	98.8%	152	98.7%	130
Test Item	99	85.7%	156.5	119.3%	95.5	102.5%	126
Test Item	49	103.4%	123	108.9%	116	106.1%	119.5
Test Item	25	121.3%	85	115.3%	92	118.3%	88.5
Test Item	12	116.9%	124.5	116.2%	86	116.5%	105.25
Exp. II with metabolic activation (+S9), exposition time 4 hours
Solvent control medium	--	--	98	--	116.5	--	107.25
Solvent control 0.9% NaCl		--	114.5	--	135.5	--	125
Positive control CPA	4.5	23.1%	844	84.3%	697.5	28.7%	770.75
Test Item	1577	116.5%	112	121.3%	160.5	118.9%	136.25
Test Item	789	87.6%	146	124.8%	146.5	106.2%	146.25
Test Item	394	72.6%	179	98.9%	177.5	85.7%	178.25
Test Item	197	97.7%	128.5	113.5%	170	105.6%	149.25
Test Item	99	81.1%	142	135.7%	142.5	108.4%	142.25
Test Item	49	58.9%	143	126.3%	118	92.6%	130.5
Test Item	25	89.2%	129	132.7%	142.5	110.9%	135.75
Test Item	12	101.5%	126	124.5%	179	113%	152.5

Values above threshold (see below) are given in bold characters.

 

Threshold for mutagenic effects: number of mutant colonies per 10⁶cells of the respective solvent control plus 126.

Threshold solvent contr. (medium) (-S9): 252.5 mutants/10⁶cells

Threshold solvent contr. (medium) (+S9): 233.25 mutants/10⁶cells

Table 7.6.1/4 Historical Data for the Experiments with Metabolic Activation

Parameter	mutant frequency of positive control CPA
Mean	646.38
Standard Deviation	175.31
Range (min – max)	359.0 - 994.0
Study 12032101G880 First Experiment	602.5
Study 12032101G880 Second Experiment	770.75

 

Table 7.61/5 Historical Data for the Experiments without Metabolic Activation

Parameter	mutant frequency of positive control MMS
Mean	717.96
Standard Deviation	340.54
Range (min – max)	317.5 - 1363.0
Study 12032101G880 First Experiment	585.75
Study 12032101G880 Second Experiment	844.75

 

Conclusions:

Interpretation of results:
negative with and without metabolic activation

Under the experimental conditions of this study, triflic acid did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, Trifluoromethanesulfonic acid is considered to be non-mutagenic under the conditions of the mouse lymphoma assay.

Executive summary:

In an vitro mammalian mutation assay (Andres, 2013), performed according to the OECD guideline N° 473 and in complinace with good laboratory practice, trifluoromethanesulfonic acid (purity >= 99%) diluted in Medium RPMI 1640 without supplements was tested in the L5178Y TK +/- mouse lymphoma cell line in the presence and the absence of mammalian metabolic activation (S9 mix).

Two independent experiments were performed. In experiment I, L5178Y TK +/- mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (Medium RPMI 1640 without supplements) and positive controls (methylmethanesulphonate (MMS) or cyclophosphamide (CP) for the without and with metabolic activation respectively) using 4- hour exposure groups both in the absence and presenc of metabolic activation. In experiment II, the cells were treated with the test item at eight dose levels using a 4- hour exposure group in the presence of metabolic activation and a 24- hour exposure group in the absence of metabolic activation.

Based on the results of the solubility of the test item in aqueous media (0.9% NaCl solution and Medium RPMI 1640 without supplements) of the preliminary experiment, the highest concentration for the two main experiments was defined as 1.5 mg/mL. Based on this concentration seven further lower concentrations were also used in the assay (12 – 782 µg/ml).

None of the tested concentrations of the test item showed a cytotoxic effect on the cells. Furthermore, no substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item

 

The positive controls induced appropriate increases in mutant frequency in all mutation experiments thus demonstrating the activity of the S9 - mix and the validity of the assay.

In conclusion, it can be stated, that during the mutagenicity test described and under the experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.Therefore,Trifluoromethanesulfonic acid is considered to be non-mutagenic under the conditions of the mouse lymphoma assay.

This study is considered as acceptable and satisfies the requirements for the mammalian mutagenicity endpoint.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 1, 1994 to November 2, 1994.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

No data on pH, osmolality and precipitation

Qualifier:

according to guideline

Guideline:

other: Notification No.700 of the Planning and Coordination Bureau, Environment Agency (EA), No. 1039 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare (MHW) & No. 1014. MITI, 1986.

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

other: Chinese hamster lung fibroblasts (CHL cells, clone No.11)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's minimum essential medium, supplemented with 10% v/v newborn calf serum. The medium is hereafter referred to as 10% NCS/MEM.
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from induced rat liver (Phenobarbital/5,6-benzoflavone)

Test concentrations with justification for top dose:

Preliminary toxicity assay:
- Direct method (without S9)/24: 0, 5, 10, 30, 50, 100, 300, 500, 1000 and 1500 µg/mL and 48 hours treatment: 0, 100, 300, 500 and 1500 µg/mL
- Metabolic activation method/24 and 48 hours treatment: 0, 10, 30, 50, 100, 300, 500, 1000 and 1500 µg/mL

Chromosomal aberration test:
- Direct method (without S9)/24 and 48 hours treatment: 0, 375, 750 and 1500 µg/mL
- Metabolic activation method:
* With S9 mix (6 hours): 0, 375, 750 and 1500 µg/mL
* Without S9 mix (6 hours): 0, 375, 750 and 1500 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with S9 mix: Mitomycin C (MMC), without S9 mix: Cyclophosphamide monohydrate (CPA, purity: 98.8%)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Without metabolic activation:
- Exposure duration: 6 hours/24 hours/ 48 hours
- Expression time (cells in growth medium): 18 hours/0 hours/ 0 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours/24 hours/ 48 hours

with metabolic activation:
- Exposure duration: 6 hours
- Expression time (cells in growth medium): 18 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (final concentration 0.1 µg/mL)
STAIN (for cytogenetic assays): no data

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 metaphases per concentration (100 from each duplicate culture)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no data

Evaluation criteria:

The results were judged as follows:
The incidence of cells (mean value for two dishes) with structural aberrations including gap or numerical aberration was:
- Less than 5%: negative (-)
- 5% or more, less than 10%: suspect positive (+/-)
- 10% or more, less than 20%: positive (+)
- 20% or more, less than 50%: positive (++)
- 50% or more: positive (+++)
The test substance was judged to be positive for the induction of the chromosomal aberrations when the incidence of cells with aberrations was dose-related or reproducible.

Statistics:

Any statistical methods were not used.

Key result

Species / strain:

mammalian cell line, other: Chinese hamster lung fibroblasts (CHL cells, clone No.11)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

determined in the preliminary test (See Table 7.6.1/1)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: freely soluble, no more details
- Precipitation: no data

RANGE-FINDING/SCREENING STUDIES:
The cell growth test and the cell division inhibition test were conducted to determine the concentrations of the test substance for the main test (See Table 7.6.1/1). Because of a weak cytotoxicity of the test substance, a concentration inhibiting 50% (IC50) of the cell growth was not found at the doses below 1500 µg/mL (10 mM) for 24 and 48 hours treatments of the direct method, while IC 50 in the metabolic activation method was approximately 1400 µg/mL. The maximum concentration of the test substance sufficient for assessing chromosomal aberration was 1500 µg/mL in every treatment methods. Therefore, the following 3 doses were employed for the chromosomal aberration test (direct method and metabolic activation method):
375, 750 and 1500 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA:
Solvent and positive control are in the range of historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Determined only in the preliminary study. described in the Table 7.6.1/1 and in the "Range-finding/screening studies" part.

Conclusions:

Interpretation of results:
negative with and without metabolic activation

Under the test conditions, trifluoromethanesulphonic acid did not induce chromosome aberrations in Chinese hamster lung fibroblasts (CHL cells, clone No.11) in the presence and absence of metabolic activation.

Executive summary:

In an in vitro chromosome aberration test (Shozo Ogura, 1994), performed similarly to the OECD guideline N° 473 and in compliance with good laboratory practice, Chinese Hamster Lung fibroblasts (CHL cells, Clone N°11) were treated with trifluoromethanesulfonic acid at three dose levels and in each treatment condition, in duplicate, together with negative and positive controls. The dose range was selected on the basis of the results of a preliminary toxicity test and was 375, 750 and 1500 µg/ml for the 6 -hour treatments both with and without S9 and the 24 -hour and 48- hour continuous treatments without S9.

The negative controls gave frequencies of aberrations within the range expected for the CHL cell line. Positive controls induced the appropriate response. Chromosomal aberrations including the structural aberrations and the numerical aberrations were not induced up to 1500 µg/ml by the direct method and the metabolic activation method.

Under the test conditions, trifluoromethanesulfonic acid is not clastogenic with and without metabolic activation.

This study is considered as acceptable and satisfies the requirements for the cytogenicity endpoint.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 April 2001 to 5 June 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: See below

Version / remarks:

“Standards Stipulated by the Minister for Health, Labour and Welfare on the Basis of the Provisions of Article 57 Paragraph 3 Item 1 of the Industrial Safety and Health Act” (Ministry of Health, Labour and Welfare Notification No. 77, 1 September 1988 and Ministry of Health, Labour and Welfare Notification No. 67, 2 June 1997)

Qualifier:

according to guideline

Guideline:

other: see below

Version / remarks:

Partial revision of “Methods for Implementing Tests for New Chemical Substances, etc.” (Notification No. 287 of the Environment, Health, and Safety Bureau, Notification No. 127 of the Environmental Health Bureau, 31 October 1997 Bureau Notification No. 2, 31 October 1997)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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 used for the preparation of S9 mix was liver of rats treated with a combination of phenobarbital and 5,6-benzoflavone to induce drug-metabolizing enzymes, which was purchased from Kikkoman.

Test concentrations with justification for top dose:

In order to set the dose level for the main study, a dose finding study was carried out in which the number of revertant colonies was counted, the presence or absence of antibacterial activity was observed, and the presence or absence of precipitation of the study substance was observed. The dose level for the dose finding study was set at 6 doses with 5,000 µg/plate as the highest dose and the lower doses obtained from a geometric progression of 4.
The study substance did not show antibacterial activity, regardless of metabolic activation or not, with any of the bacterial strains or at any of the dose levels, and dose-respondent increase in the number of revertant colonies to twice that of the negative (solvent) controls was not found. Dose-dependent precipitation of the study substance was observed at 5000 µg/plate with metabolic activation, but there was no effect on the revertant colony count.
From the above results, the dose level for the main study for all bacterial strains, regardless of metabolic activation or not, was set at 5 doses with 5000 µg/plate as the highest dose and the lower doses obtained from a geometric progression of 2

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetonitrile (lot number: ELJ6328)
- Justification for choice of solvent/vehicle: The medium for the study substance was set on the basis of the results of solubility tests. As the study substance is unstable in water, the solubility tests were carried out using DMSO, acetone, tetrahydrofuran (THF), 1,4-dioxane, acetonitrile, and N,N-dimethylformamide (DMF) as media. The preparation concentrations for the solubility tests were set at 50 mg/mL for DMSO and DMF, 100 mg/mL for acetone and 1,4-dioxane, 125 mg/mL for acetonitrile, and 250 mg/mL for THF. The study substance was added to the different media to the respective concentrations, and the solutions were checked visually for reactions with heat generation or smoking, etc. and for solubility. It was found that the study substance dissolved in all of the media. However, foaming of the solution, heat generation, and coloration were found when the study substance was added to DMSO, acetone, THF, 1,4-dioxane, and DMF, suggesting reactivity of the study substance with each of these media. It was therefore judged that none of these media were suitable as media for the study substance. With acetonitrile, on the other hand, no changes at all were found that might indicate reactivity, such as heat generation, foaming, or coloration of the solution. It was therefore judged that the study substance is stable with respect to acetonitrile, and acetonitrile was chosen as the medium for the study substance. It has also been shown that acetonitrile has no adverse effects at all on the development or metabolic activation systems of bacterial or revertant colonies.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

benzo(a)pyrene

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide [1] and 2-aminoanthracene [2]

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation method

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 hours
- Incubation: 37°C
- Expression time (cells in growth medium): the number of revertant colonies that appeared was counted, the presence or absence of antibacterial activity due to the study substance was observed by stereoscopic microscope, and the presence or absence of precipitation of the study substance was observed macroscopically.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Revertant colonies were counted both by eye and by a CAD-11 colony analyzer corrected for counting loss.

OTHER EXAMINATIONS:
- Other: Visual examination of the plates was performed: precipitation was recorded and reported.

Rationale for test conditions:

These strains are generally used to investigate the ability to induce reverse mutations as they have high sensitivity to known mutagenic substances.
A dose-finding study and a main study were conducted, and the reproducibility of results from both the dose-finding study and the main study was confirmed.

Evaluation criteria:

* Acceptable standards for negative (solvent) control values and positive control values:
The numbers of revertant colonies for the negative (solvent) control and positive control plates were compared to background data and the standard values based on the background data (mean ± 2 x standard deviation), and if the numbers of revertant colonies were within the range of the standard values, or if they deviated from the standard values but from comparison with the background data this was considered to be due to an incidental factor, the corresponding study results were judged to have been obtained as a result of appropriate procedures with appropriate sterilization.

* Acceptable standards for study results:
If the following requirements were met, the corresponding study results were judged to have been obtained as a result of appropriate procedures.
1) The sterilization test showed no bacterial contamination.
2) The negative (solvent) control values met the acceptable standards.
3) The positive control values met the acceptable standards, and the positive control values were two or more times the negative (solvent) control values.

* Judgement:
The study substance was judged to have the ability to induce reverse mutation if the negative (solvent) control values, the positive control values, and the study results all met the acceptable standards; a dose-dependent increase in the number of revertant colonies with study substance treatment to twice or more the number of revertant colonies in the negative (solvent) controls was observed; and reproducibility was confirmed.

Statistics:

No statistical methods were used for the evaluation of the study results.

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

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:

valid

Positive controls validity:

valid

Additional information on results:

With the test substance, regardless of the presence or absence of activation, the number of revertant colonies did not increase dose-dependently with any of the bacterial strains, and the number of revertant colonies was less than twice the number with the negative (solvent) control. Also, the results showed reproducibility in both the dose finding study and the main study. At the same time, the sterilization tests carried out on the Nutrient broth No. 2 used in the preculture, the maximum concentration study substance solution, the S9 mix, and the 0.1 M phosphate buffer solution showed that there was no bacterial contamination. The numbers of revertant colonies with the negative (solvent) controls and the positive controls were within the range of the respective standard values based on background data. The positive controls 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminoacridine, 9-aminoacridine, and benzo[α]pyrene all showed twice or more the number of revertant colonies than the negative (solvent) controls with all bacterial strains. These results indicate that the study was conducted appropriately. In addition, no environmental factors or deviations from the study protocol were found that could be suspected of adversely impacting the reliability of the study.

Table 1: Results of the range-finding assay

Study period			From 24 March 2001 to 27 March 2001
With or without metabolic activation		Study substance dose (µg/plate)	Number of revertant colonies (colonies/plate)
			Base-pair substitution type							Frame shift type
			TA100			TA1535		WP2uvrA		TA98			TA1537
S9 mix (-)		Negative control	129 129	(129)		11 11	(11)	35 35	(35)	23 26	(25)		9 12	(11)
		5	122 124	(123)		9 9	(9)	35 34	(35)	25 24	(25)		13 10	(12)
		20	135 124	(130)		11 9	(10)	33 36	(35)	25 26	(26)		9 9	(9)
		78	126 122	(124)		9 8	(9)	35 37	(36)	25 24	(25)		11 9	(10)
		313	129 135	(132)		12 11	(12)	35 34	(35)	26 23	(25)		12 9	(11)
		1250	135 118	(127)		9 11	(10)	35 36	(36)	24 25	(25)		11 12	(12)
		5000	134 117	(126)		9 9	(9)	35 36	(36)	25 27	(26)		9 9	(9)
S9 mix (+)		Negative control	128 135	(132)		9 11	(10)	36 35	(36)	25 26	(26)		11 12	(12)
		5	136 136	(136)		11 9	(10)	37 33	(35)	28 23	(26)		9 11	(10)
		20	128 136	(132)		9 8	(9)	36 34	(35)	24 24	(24)		9 11	(10)
		78	135 137	(136)		11 9	(10)	37 38	(38)	26 27	(27)		9 10	(10)
		313	134 125	(130)		11 12	(12)	36 33	(35)	25 26	(26)		11 8	(10)
		1250	135 134	(135)		9 11	(10)	36 37	(37)	25 25	(25)		9 12	(11)
		5000	132 139	# #(136)		11 9	# #(10)	36 37	# #(37)	23 26	# #(25)		13 13	# #(13)
Positive controls	S9 mix not needed	Name	AF-2^1)			NaN3^2)		AF-2		AF-2			9AA^3)
		Dose (µg/plate)	0.01			0.5		0.01		0.1			80.0
		No. colonies/plate	687 713		(700)	1006 1013	(1010)	464 451	(458)	469 498		(484)	513 563		(538)
	S9 mix needed	Name	B[α]P^4)			2AA^5)		2AA		B[α]P			B[α]P
		Dose (µg/plate)	5.0			2.0		10.0		5.0			5.0
		No. colonies/plate	1497 1464		(1481)	376 379	(378)	1062 1016	(1039)	326 365		(346)	79 86		(83)

1) 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide  2) Sodium azide  3) 9-aminoacridine 4) Benzo[α]pyrene  5) 2-aminoanthracene

Note: # to the right of the number indicates precipitation was found

Table 2: Results of the main assay

Study period			From 17 May 2001 to 21 May 2001
With or without metabolic activation		Study substance dose (µg/plate)	Number of revertant colonies (colonies/plate)
			Base-pair substitution type							Frame shift type
			TA100			TA1535		WP2uvrA		TA98			TA1537
S9 mix (-)		Negative control	129 132	(131)		9 11	(10)	33 35	(34)	23 24	(24)		11 11	(11)
		313	128 123	(126)		9 9	(9)	35 35	(35)	24 24	(24)		10 9	(10)
		625	128 134	(131)		9 11	(10)	32 34	(33)	25 24	(25)		10 9	(10)
		1250	133 135	(134)		9 8	(9)	33 33	(33)	24 24	(24)		9 11	(10)
		2500	132 132	(132)		11 8	(10)	35 34	(35)	26 25	(26)		11 11	(11)
		5000	99 103	(101)		8 7	(8)	33 35	(34)	23 26	(25)		6 7	(7)
S9 mix (+)		Negative control	139 142	(141)		9 11	(10)	35 35	(35)	26 25	(26)		11 11	(11)
		313	145 139	(142)		9 11	(10)	30 36	(33)	25 23	(24)		9 11	(10)
		625	145 145	(145)		9 9	(9)	35 35	(35)	26 24	(25)		10 8	(9)
		1250	139 145	(142)		11 8	(10)	37 35	(36)	27 25	(26)		11 9	(10)
		2500	152 134	(143)		8 11	(10)	36 35	(36)	26 25	(26)		9 11	(10)
		5000	153 148	# #(151)		11 11	# #(11)	38 37	# #(38)	26 25	# #(26)		12 13	# #(13)
Positive controls	S9 mix not needed	Name	AF-2^1)			NaN3^2)		AF-2		AF-2			9AA^3)
		Dose (µg/plate)	0.01			0.5		0.01		0.1			80.0
		No. colonies/plate	624 659		(642)	1033 986	(1010)	436 457	(447)	462 434		(484)	653 613		(633)
	S9 mix needed	Name	B[α]P^4)			2AA^5)		2AA		B[α]P			B[α]P
		Dose (µg/plate)	5.0			2.0		10.0		5.0			5.0
		No. colonies/plate	1424 1474		(1449)	432 411	(422)	1062 1014	(1038)	326 326		(326)	83 89		(86)

1) 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide  2) Sodium azide  3) 9-aminoacridine  4) Benzo[α]pyrene  5) 2-aminoanthracene

Note: # to the right of the number indicates precipitation was found

Conclusions:

From the foregoing results, it was concluded that the study substance does not have the ability to induce reverse mutations under the present study conditions.
Furthermore, the study substance did not show antibacterial activity with any of the bacterial strains, regardless of the presence or absence of activation. Also, precipitation of the study substance occurred at a dose of 5000 µg/plate with activation.

Executive summary:

The ability of trifluoromethanesulfonic anhydride to induce reverse mutations was investigated using the base-pair substitution mutant strains Salmonella typhimurium TA100 and TA1535 and Escherichia coli WP2 uvrA and the frame shift mutant strain Salmonella typhimurium TA98. In accordance with the applicable guidelines for testing methods, a dose-finding study and a main study were carried out and the reproducibility of both the dose-finding study and the main study were confirmed. Both the dose-finding study and the main study were carried out by the preincubation method in test tubes under hermetically sealed conditions at 37ºC for 40 min. Statistical methods were not used for evaluation of the study results. The number of revertant colonies with the study substance was compared to the number of revertant colonies with the negative control, and the result was considered positive if the number of revertant colonies with the study substance increased in a dose-dependent fashion to twice or more the number with the negative control, and the results were reproducible. The study results are summarized as follows.

1.    There number of revertant colonies with the study substance did not increase in a dose-dependent fashion in any of the bacterial strains, regardless of whether or not there was metabolic activation, and the number did not increase to greater than twice the number with the negative (solvent) controls. Also, the results of both the dose-finding study and the main study were found to be reproducible.

 

2.    The number of revertant colonies with the negative (solvent) controls and the positive controls were compared to the background data, and all values were found to be appropriate. The positive controls were 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminoacridine, 2-aminoanthracene, and benzo[α]pyrene, and with all the bacterial strains the number of revertant colonies with the positive controls increased to greater than twice the number with the negative (solvent) controls. These results indicate that the study was conducted appropriately.

 

3.    From the above results, it is judged that the study substance does not have the ability to induce reverse mutations under the conditions of the present study.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

In an aqueous environment Trifluoromethanesulfonic anhydride is rapidly hydrolysed to form trifluoromethanesulfonic acid (CAS RN 1493-13-6) so when absorbed in the body, the target substance is expected to be rapidly degraded into trifluoromethanesulfonic acid.
Therefore, the systemic toxicity data obtained with trifluoromethanesulfonic acid can be used to assess the systemic toxicity profile of trifluoromethanesulfonic anhydride. As the target substance forms no other molecule than the source substance in aqueous environment, the effect concentrations observed with the source substance can be directly used for the target substance, no correction has to be applied.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mammalian cell line, other: Vhinese hamster lung fibroblasts (CHL cells, clone No.11)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Under the test conditions, trifluoromethanesulphonic acid did not induce chromosome aberrations in Chinese hamster lung fibroblasts (CHL cells, clone No.11) in the presence and absence of metabolic activation. Therefore, Trifluoromethanesulfonic anhydride is considered to be non-mutagenic under the conditions of the chromosome aberration test.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

In an aqueous environment Trifluoromethanesulfonic anhydride is rapidly hydrolysed to form trifluoromethanesulfonic acid (CAS RN 1493-13-6) so when absorbed in the body, the target substance is expected to be rapidly degraded into trifluoromethanesulfonic acid.
Therefore, the systemic toxicity data obtained with trifluoromethanesulfonic acid can be used to assess the systemic toxicity profile of trifluoromethanesulfonic anhydride. As the target substance forms no other molecule than the source substance in aqueous environment, the effect concentrations observed with the source substance can be directly used for the target substance, no correction has to be applied.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

valid

Positive controls validity:

valid

Conclusions:

Under the experimental conditions of this study, Trifluoromethanesulfonic acid did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, By analogy Trifluoromethanesulfonic anhydride is considered to be non-mutagenic under the conditions of the mouse lymphoma assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial Reverse Mutation assay:

One reverse bacterial gene mutation test (Kato, 2001) was selected as a key study, (OECD 471, Kr: 1). In this study, Trifluoromethanesulphonic anhydride is not mutagenic in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2uvrA- with and without metabolic activation, up to a 5000 µg/plate concentration.The results were negative with or without metabolic activation in all tested strains. Therefore, Trifluoromethanesulphonic anhydride showed no mutagenic action in Bacteria.

In vitro Mammalian chromosome aberration test:

One In vitro, cytogenetic study in CHL cells (OECD 473, Kr: 2) was available on trifluoromethanesulfonic acid and was selected as the key study (Shozo Ogura, 1994). Chinese Hamster Lung fibroblasts (CHL cells, Clone N°11) were treated with trifluoromethanesulphonic acid at three dose levels, in each treatment case, in duplicate, together with negative and positive controls. The dose range was selected on the basis of the results of a preliminary toxicity test and was 375, 750 and 1500 µg/ml for the 6 -hour treatments both with and without S9 and the 24 -hour and 48- hour continuous treatments without S9.

The negative controls gave frequencies of aberrations within the range expected for the CHL cell line. Positive controls induced the appropriate response. Chromosomal aberrations including the structural aberrations and the numerical aberrations were not induced up to 1500 µg/ml by the direct method and the metabolic activation method.

Under the test conditions, trifluoromethanesulphonic acid is not clastogenic with and without metabolic activation. Therefore, by analogy, trifluoromethanesulfonic anhydride is considered as not clastogenic.

In vitro Mammalian Cell Gene Mutation test:

One study was available on trifluoromethanesulfonic acid and was considered as the key study.

An in vitro mammalian mutation assay (Andres, 2013), performed according to the OECD guideline N° 473 and in compliance with good laboratory practice, trifluoromethanesulphonic acid (purity >= 99%) diluted in Medium RPMI 1640 without supplements was tested in the L5178Y TK +/- mouse lymphoma cell line in the presence and the absence of mammalian metabolic activation (S9 mix).

Two independent experiments were performed. In experiment I, L5178Y TK +/- mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (Medium RPMI 1640 without supplements) and positive controls (methylmethanesulphonate (MMS) or cyclophosphamide (CP) for without and with metabolic activation respectively) using 4- hour exposure groups both in the absence and presence of metabolic activation. In experiment II, the cells were treated with the test item at eight dose levels using a 4- hour exposure group in the presence of metabolic activation and a 24- hour exposure group in the absence of metabolic activation.

Based on the results of the solubility of the test item in aqueous media (0.9% NaCl solution and Medium RPMI 1640 without supplements) of the preliminary experiment, the highest concentration for the two main experiments was defined as 1.5 mg/mL. Based on this concentration seven further lower concentrations were also used in the assay (12 – 782 µg/ml).

None of the tested concentrations of the test item showed a cytotoxic effect on the cells. Furthermore, no substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. 

The positive controls induced appropriate increases in mutant frequency in all mutation experiments thus demonstrating the activity of the S9 - mix and validity of the assay.

Under the experimental conditions reported in this study, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.Therefore,Trifluoromethanesulphonic acid is considered to be non-mutagenic under the conditions of the mouse lymphoma assay and by analogy, trifluoromethanesulfonic anhydride is also considered to be non mutagenic.

In conclusion: based on all these studies, trifluoromethanesulphonic acid is considered as not genotoxic.

Justification for classification or non-classification

Regarding on the overall negative results from the in vitro genotoxicity studies performed on trifluoromethanesulfonic anhydride or on the close analogue trifluoromethanesulfonic acid, it is likely that trifluoromethanesulphonic anydride doesn't present genotoxic activity potential, therefore no classification is required according to the CLP Regulation.