Trifluoromethanesulphonic anhydride

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Trifluoromethanesulphonic acid

EC Number:

216-087-5

EC Name:

Trifluoromethanesulphonic acid

Cas Number:

1493-13-6

Molecular formula:

CHF3O3S

IUPAC Name:

trifluoromethanesulfonic acid

Test material form:

liquid

Details on test material:

See Confidential details on test material

Method

Target gene:

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

Species / strain

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.

Controls

Controls 1

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

Controls 2

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.