Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

Link to relevant study records

Referenceopen allclose all

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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
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. certificate)
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
Remarks:
See below for more details
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 10cells

Culture A

Relative Total Growth Culture B

Mutants per 10cells Culture B

Relative Total Growth Mean

Mutants per 10cells 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 10cells

Culture A

Relative Total Growth Culture B

Mutants per 10cells Culture B

Relative Total Growth Mean

Mutants per 10cells 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.

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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No data on pH, osmolality and precipitation
Qualifier:
according to
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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 6, 1994 to September 6, 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: only two replicates per concentration for the test substance were performed. No justification was done in the study report.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Each strains derived from S.thyphimurium contains one mutation in the histidine operon, resulting in a requirement for histidine.
The amino acid requirement was determined as tryptophan for E. coli strain. See details in Table 7.6.1/1
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A.
Additional strain / cell type characteristics:
other: additional mutation in uvrA, uvrB and rfa genes. The plasmid pKM101 was present in TA98 and TA100 in order to increase the sensibility of these strains to some mutagens. See details in Table 7.6.1/1.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/5,6-benzoflavone-pretreated male Sprague-Dawley rat liver S9 fraction.
Test concentrations with justification for top dose:
Preliminary test: 0 (vehicle), 50, 100, 200, 500, 1000, 2000 and 5000 µg/plate (+/-S9 mix)
Mutagenicity test: 0 (vehicle), 313, 625, 1250, 2500 and 5000 µg/plate (+/-S9mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
yes
Remarks:
In the sterility test, after 0.1mL of each bacterial culture, the test substance solution, S9 mix and 0.1 M sodium phosphate buffer were smeared on a minimal glucose agar plate and incubated at 37 °C for 48 hours, bacterial contamination was checked.
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), Sodium azide (NaN3), 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine. 2HCl (ICR-191) and 2-Aminoanthracene (2AA).
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION:
- Preincubation period: 20 minutes at 37+/-0.5°C
- Exposure duration: 48 hours at 37+/-0.5°C

NUMBER OF REPLICATIONS: three plates were used for the negative control group and two plates for the test substance treatment group and for the positive control groups (+/-S9 mix).

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
A reproducible 2-fold increase in the number of revertants compared with the vehicle controls was considered as a positive result.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Observed at 5000 µg/plate in TA1535 and WP2uvrA (+/-S9 mix). The cytotoxicity was observed up to 1250 µg/plate in TA98 without S9mix and at 5000 µg/plate with S9mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation: no change was observed in the test substance solution within 2 hours after preparation except for heating of the solution when a 5 w/v% solution of the test substance was prepared.
- Other: the sterility test demonstrated that the test system was free from bacterial contamination.

RANGE-FINDING/SCREENING STUDIES: a preliminary study was performed on TA1535, TA100, WP2uvrA, TA98 and TA1537 with and without metabolic activation. The tested concentrations of the substance were 0, 50, 100, 200, 500, 1000, 2000, 5000 µg/plate. No cytotoxicty in terms of decrease of the number of revertants or effects on the bacterial lawn was observed.

COMPARISON WITH HISTORICAL CONTROL DATA: the numbers of revertant colonies in the positive controls and the negative control were within the range of background data in Hita laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: A slight growth inhibition wa observed at 5000 µg/plate for TA1535 and WP2uvrA (+/-S9mix) in the main test. The cytotoxicity was observed up to 1250 µg/plate in TA98 without S9mix and at 5000 µg/plate with S9mix. However, no statistically significant decreasing of revertant colonies was found.

Table 7.6.1/3: Number of revertants per plate (mean of duplicates) in the absence of metabolic activation in finding test (preincubation method)

PFC-MS

Concentration
(µg/plate)

TA 100

TA1535

WP2uvrA

TA 98

TA 1537

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

112

-

12

-

43

-

24

 -

8

-

50

133

-

13

-

54

 -

28

 -

8

-

100

120

-

13

-

56

 -

26

 -

8

-

200

129

-

15

-

56

 -

25

 -

15

-

500

116

-

16

-

54

-

20

-

10

-

1000

129

-

14

-

49

-

26

-

14

-

2000

122

-

18

-

51

-

23

-

10

-

5000

118

-

13

-

59

-

30

-

14

-

Positive control**

477

-

333

-

591

-

463

-

1913

-

Table 7.6.1/4:Number of revertants per plate (mean of duplicates) in the presence of metabolic activation in finding test (preincubation method)

PFC-MS

Concentration
(µg/plate)

TA 100

TA1535

WP2uvrA

TA 98

TA 1537

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

124

-

10

-

61

-

33

 -

17

-

50

142

-

12

-

64

 -

36

 -

23

-

100

135

-

14

-

58

 -

36

 -

16

-

200

123

-

17

-

82

 -

36

 -

22

-

500

117

-

12

-

78

-

36

-

27

-

1000

123

-

11

-

66

-

32

-

23

-

2000

127

-

13

-

77

-

26

-

24

-

5000

149

-

15

-

55

-

31

-

22

-

Positive control**

501

-

160

-

425

-

188

-

93

-

Table 7.6.1/5:Number of revertants per plate (mean of duplicates) in the absence of metabolic activation in Main test (preincubation method)

PFC-MS

Concentration
(µg/plate)

TA 100

TA1535

WP2uvrA

TA 98

TA 1537

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

116

-

10

-

39

-

26

 -

13

-

313

123

-

13

-

46

 -

22

 -

22

-

625

134

-

12

-

39

 -

24

 -

19

-

1250

116

-

9

-

42

 -

14*

 -

23

-

2500

121

-

12

-

43

-

15*

-

16

-

5000

117

-

9*

-

38*

-

16*

-

19

-

Positive control**

282

-

225

-

439

-

490

-

1553

-

Table 7.6.1/6: Number of revertants per plate (mean of duplicates) in the presence of metabolic activation in Main test (preincubation method)

PFC-MS

Concentration
(µg/plate)

TA 100

TA1535

WP2uvrA

TA 98

TA 1537

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

119

-

13

-

55

-

23

 -

18

-

313

134

-

13

-

44

 -

26

 -

21

-

625

144

-

16

-

46

 -

26

 -

19

-

1250

142

-

8

-

41

 -

27

 -

16

-

2500

127

-

14

-

40

-

19

-

15

-

5000

139

-

7*

-

32*

-

20*

-

19

-

Positive control**

546

-

162

-

426

-

189

-

88

-

*Solvent control = negative control: water

**Mutagens positive controls (-S9 mix):

- AF-2 (0.01 µg/plate) in TA100 and WP2 uvrA and (0.1 µg/plate) in TA98

- NaN3 (0.5 µg/plate) in TA1535

- ICR-191(1µg/plate) in TA1537

**Mutagens positive controls (+S9 mix):

- 2AA: (2 µg/plate) in TA1535 and TA1537, (1µg/plate) in TA100, (0.5µg/plate) in TA98 and (10 µg/plate) in WP2 uvrA.

Conclusions:
Interpretation of results:
negative (with and without metabolic activation)

Under the test conditions, Trifluoromethanesulfonic acid did not show mutagenic activity in the bacterial mutation test with Salmonella typhimurium and E.Coli.
Executive summary:

In a reverse gene mutation assay in bacteria (YUKO, 1994), performed similarly to the OECD guideline No.471 and in compliance with Good Laboratory Practice,Salmonella typhimurium strain TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA were used in presence and in absence of metabolic activation system from liver fraction of phenobarbital/5,6 benzoflavone-induced rats (S9-mix). One preliminary study and one main test were performed using a preincubation method. The test article was dissolved in water and dose levels tested were included between 313 and 5000 µg/plate with or without S9‑mix. A slight growth inhibition was observed at 5000 µg/plate for TA1535 and WP2uvrA (+/-S9mix) and up to 1250 µg/plate in TA98 without S9 mix and at 5000 µg/plate with S9 mix. However, no statistically significant decreasing of revertant colonies was found.

The positive controls induced the appropriate responses in the corresponding strains. Under the experimental conditions of the study, Trifluoromethanesulfonic acid did not demonstrate any in vitro reverse mutagenic potential in either the presence or absence of metabolic activation.

This study is considered as acceptable as it satisfied the main criteria of the OECD guideline No.471.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Bacterial Reverse Mutation assay:

One reverse bacterial gene mutation test (YUKO, 1994) was selected as a key study, (OECD 471, Kr: 2). In this study, Trifluoromethanesulphonic acid 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 limit concentration.These results are confirmed by a supporting study (Cinelli, 2003), (OECD 471 screening, Kr: 2) providing negative results with or without metabolic activation inS. typhimurium strains TA98, TA100. Therefore, Trifluoromethanesulphonic acid showed no mutagenic action in Bacteria.

In vitro Mammalian chromosome aberration test:

In vitro, one cytogenetic study in CHL cells (OECD 473, Kr: 2) was available 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.

In vitro Mammalian Cell Gene Mutation test:

One study was available 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.

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, it is likely that trifluoromethanesulphonic acid doesn't present genotoxic activity potential, therefore no classification is required according to the CLP Regulation.