Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Since all the available in vitro tests showed negative results, trifluoroacetic acid is not to be considered as mutagenic. Further testing of in vivo genetic toxicity is not considered necessary.


Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1986-03-04 to 1986-03-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed similarly to the OECD (No. 471) with acceptable restrictions and was in compliance to the GLP. Sodium Trifluoroacetate was tested instead of trifluoroacetic acid (TFA) according to a reliable analogue approach in order to be free of the cytotoxic effect on bacteria due to the extreme acid pH of TFA (pH=0.45).
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: only the 2-Anthramine is used as a positive control for the efficacy of S9 mix. Strain S. typhimurium TA102 or E.coli WP2 were not used.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Trifluoroacetic acid (TFA) is a strong acid. Testing with the neutral salt sodium trifluoroacetate was considered appropriate to avoid cytotoxic effects on bacteria due to the extreme acid pH of TFA (pH=0.45).
Target gene:
Each strain derived from S. thyphimurium LT2 contains one mutation in the histidine operon, resulting in a requirement for histidine. See details in Table 7.6.1/1.
Species / strain / cell type:
S. typhimurium, other: TA1535, TA1537, TA1538, TA98 and TA100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: additional mutation in 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:
S9 fraction was obtained from the liver of Sprague-Dawley male rats treated by the intraperitoneal route with Aroclor 1254 (500 mg/kg) dissolved in maize oil.
Test concentrations with justification for top dose:
Preliminary test: 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 mg/plate.
Mutagenicity experiments: 0.1, 0.5, 1, 5 and 10 mg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data

The susbtance is dissolved in DMSO at the highest concentration of 100 mg/mL. The succesive dilutions were performed from this initial solution.
Untreated negative controls:
yes
Remarks:
sterility controls for the substance's solution, the solvent and the S9 mix.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-Aminoacridine (TA1537), 2-Nitrofluorene (TA98 and TA1538), Methyl methanesulphonate (TA 100) and Ethyl methanesulphonate (TA1535) for without S9 mix efficacy control. 2-Anthramine for with S9 mix efficacy control.See details in Table 7.6.1/3.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
direct incorporation method: 100 µL of the DMSO diluted substance were incorporated in glass tubes. Then were added the bacteria (0.1 mL), the S9 fraction (0.5 mL) in the case of test with metabolic activation and the agar. After agitation the mix was plated on a Petri plate containing Vogel and Bonner medium.

DURATION
- Preincubation period: no data
- Exposure duration: 48 hours
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: 3 plates/dose/strain. Two independent experiments were performed.
NUMBER OF CELLS EVALUATED: no data
DETERMINATION OF CYTOTOXICITY
- Method: no data
OTHER EXAMINATIONS:
- Determination of polyploidy: not required
- Determination of endoreplication: not required
- Other:
OTHER:
Evaluation criteria:
A reproducible 2-fold increase in the number of revertants compared with the vehicle controls was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
no data
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA1538, TA98 and TA100
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: not applicable

RANGE-FINDING/SCREENING STUDIES: a preliminary study was performed on the TA100 strain without metabolic activation. The tested concentrations of the substance were 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 mg/plate. No cytotoxicity in terms of decrease of the number of revertants or effect on the bacterial lawn was observed.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of revertants for the vehicle and positive controls was similar to the historical data.
ADDITIONAL INFORMATION ON CYTOTOXICITY: no additional information
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 7.6.1/4:Number of revertants per plate (mean of triplicates) in the absence of metabolic activation in first test (direct plate incorporation method)

TFAS Concentration
(mg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 1538

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

12.7

5.5

10.7

2.9

28.0

3.6

86.0

7.8

18.3

5.7

0.1

12.7

4.9

16.0

2.6

20.3

4.0

84.0

7.5

20.0

7.0

0.5

14.3

7.6

10.7

4.1

17.0

1.7

83.0

3.6

15.3

1.1

1

15.3

3.5

10.3

1.1

17.7

5.5

86.0

6.9

17.0

2.6

5

14.0

2.6

11.7

4.0

16.3

1.5

100.3

10.7

23.0

10.5

10

18.7

4.0

15.3

4.1

25.7

2.3

102.3

4.9

17.0

1.7

Positive control**

5723.0

-

989.0

-

264.5

-

301.0

-

199.0

-

*Solvent control = negative control: 100 µL DMSO

**Mutagens positive controls:

- 2.N.F. (0.001 mg/plate) in TA98 and TA1538 strains

- M.M.S. (0.1 mg/plate) in TA100 strain

- E.M.S. (10 mg/plate) in TA1535 strain

- 9 A.A. ( 0.05 mg/plate) in TA 1537 strain

 

 

Table 7.6.1/5:Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (S9 mix) in first test (direct plate incorporation method)

TFAS Concentration
(mg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 1538

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

15.7

8.1

12.0

2.6

27.3

8.5

107.7

13.0

32.0

2.6

0.1

20.3

2.3

11.3

1.1

29.0

5.0

106.3

16.0

36.0

7.0

0.5

19.3

1.5

19.3

6.6

28.0

1.0

99.3

3.0

27.7

4.1

1

15.3

4.0

12.3

2.9

31.3

2.5

94.7

6.6

30.0

9.1

5

15.0

3.0

15.0

3.0

31.7

5.8

98.7

12.3

28.0

3.0

10

17.3

3.2

11.3

0.6

26.7

6.5

109.3

13.0

26.7

11.0

Positive control**

225.0

-

150.5

-

1003.0

-

1453.0

-

831.0

-

*Solvent control = negative control: 100 µL DMSO

**Mutagens positive controls:

- 2.A. (0.002 mg/plate)

 

 

 Table 7.6.1/6:Number of revertants per plate (mean of triplicates) in the absence of metabolic activation in second test (direct plate incorporation method)

TFAS Concentration
(mg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 1538

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

9.7

4.5

12.0

2.0

33.7

3.0

99.7

19.5

20.0

4.0

0.1

4.3

2.1

8.3

3.0

29.7

4.1

103.0

8.6

20.3

5.8

0.5

6.0

1.0

6.0

1.0

27.7

2.3

109.3

3.2

18.7

1.1

1

9.0

3.0

10.3

1.1

25.0

6.0

108.7

18.4

19.3

7.5

5

8.7

5.8

7.3

2.5

25.7

8.5

104.7

2.5

22.0

1.7

10

7.3

4.5

8.3

5.8

34.3

3.5

100.7

17.4

18.0

3.6

Positive control**

7301.5

-

445.0

-

263.0

-

450.5

-

254.5

-

*Solvent control = negative control: 100 µL DMSO

**Mutagens positive controls:

- 2.N.F. (0.001 mg/plate) in TA98 and TA1538 strains

- M.M.S. (0.1 mg/plate) in TA100 strain

- E.M.S. (10 mg/plate) in TA1535 strain

- 9 A.A. ( 0.05 mg/plate) in TA 1537 strain

 

Table 7.6.1/7:Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (S9 mix) in second test (pre-incubation method)

 

TFAS Concentration
(mg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 1538

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

7.7

2.0

10.3

3.5

44.0

3.6

117.0

8.6

29.3

5.7

0.1

9.7

3.2

8.0

2.6

38.0

6.5

136.7

1.1

29.0

4.6

0.5

10.0

4.0

8.7

2.9

43.0

9.8

125.3

13.0

29.7

5.7

1

11.3

1.1

9.7

2.5

41.3

2.9

125.7

11.0

25.3

1.5

5

10.0

2.6

8.3

3.2

44.3

5.8

120.0

9.8

22.3

3.0

10

7.7

3.0

7.7

1.1

46.3

5.1

111.3

17.4

22.0

4.6

Positive control**

151.5

-

188.0

-

1510.0

-

2241.5

-

601.0

-

*Solvent control = negative control: 100 µL DMSO

**Mutagens positive controls:

- 2.A. (0.002 mg/plate)

Conclusions:
Under the test conditions, the test item Sodium Trifluoroacetate did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

In a reverse gene mutation assay in bacteria, performed similarly to the OECD No.471 guideline, strains TA1535, TA1537, TA1538, TA98 and TA100 of S. typhimurium were exposed to Sodium Trifluoroacetate diluted in DMSO at concentrations of 0.1, 0.5, 1.0, 5.0 and 10 mg/plate in the presence and absence of mammalian metabolic activation (fraction of S9 from the liver of Sprague-Dawley male rats treated by intraperitoneal injection with Aroclor 1254 dissolved in maize oil). The method of direct incorporation was used in this study.

Sodium Trifluoroacetate was tested up to limit concentration recommended in the guideline (5 mg/plate).

The positive controls induced the appropriate responses in the corresponding strains.

Under the test conditions, the test item Sodium trifluoroacetate did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium as there was no evidence of induced mutant colonies over background.

In this study Sodium Trifluoroacetate was used instead Trifluoroacetic acid (TFA) according to a reliable analogue approach in order to be free of the cytotoxic effect on bacteria due to the extreme acid pH of the TFA.

Therefore, Trifluoroacetic acid is not considered as mutagenic in the bacterial reverse mutation test.

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

             

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study in accordance with international guidelines. Sodium Trifluoroacetate was tested instead of trifluoroacetic acid (TFA) according to a reliable analogue approach in order to be free of the cytotoxic effect on bacteria due to the extreme acid pH of TFA (pH=0.45).
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
Trifluoroacetic acid (TFA) is a strong acid. Testing with the neutral salt sodium trifluoroacetate was considered appropriate to avoid cytotoxic effects on bacteria due to the extreme acid pH of TFA (pH=0.45).
Target gene:
The hypoxanthine-guanine phosphoribosyl transferase (hprt) gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 10 medium prepared as follow:
Horse serum (heat inactivated): 10% v/v
Penicillin/Streptomycin: 100 units/mL / 100 µg/L
Amphotericin B: 2.5 µg/mL
Pluronic: 0.5 mg/L
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9: Aroclor 1254 induced rat liver post mitochondrial fraction
Test concentrations with justification for top dose:
Experiment 1 (+/- S9): 200, 400, 600, 800, 100, 1200 and 1360 µg/mL.
Experiment 2 (+/- S9): 150, 300, 500, 700, 900, 1100 and 1360 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline-1-oxide (NQO) 0.1 and 0.15 µg/mL; Benzo[a]pyrene (BP) 2 and 3 µg/mL
Remarks:
no remarks
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours incubation at 37±1°C
- Expression time (cells in growth medium): Cultures were maintained in flasks for a period of at least 7 days during which the hprt- mutation will be expressed.
- Selection time (if incubation with a selection agent): one to two weekss
- Fixation time (start of exposure up to fixation or harvest of cells): no data

SELECTION AGENT (mutation assays): thio-6-guanine (6TG)

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth
Evaluation criteria:
For valid data, the test article was considered to be mutagenic in this assay if:
1. The mutant frequency at one or more concentrations is significantly greater than that of the negative control (p<0.05)
2. There is a significant concentration relationship as indicated by the linear trend analysis (p<0.05)
3. The effects described above are reproducible.
Results which only partially satisfy the above criteria will be dealt with on a case by case basis. Positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Extreme caution will be exercised with positive results obtained at levels of RS lower than 10%.
Statistics:
Statistical significance of mutant frequencies will be carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) will be compared with the LMF from each test article treatment, and secondly the data will be checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
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:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: In the range-finder, there was no evidence of marked toxicity at concentrations up to and including 1360 µg/mL (equivalent to 10 mM) in the absence and presence of S-9. See table 7.6.1/3.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment 1 the highest concentration selected, 1360 µg/mL, gave 97% and 84% RS in the absence and presence of S 9, respectively.
In experiment 2 the highest concentration selected, 1360 µg/mL, gave 80% and 112% RS in the absence and presence of S 9, respectively.

DEVIATION FROM PROTOCOL:
In the presence of S-9 in Experiment 1, the positive controls did not meet the acceptance criteria stated in the protocol. However, both positive control concentrations showed clear increases in mutant frequency which were outside the historical negative control ranges generated by the last 20 studies, updated at the time of each experiment. The data were therefore considered acceptable and valid on this basis.

Table 7.6.1/1:Experiment 1 (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

 

- S-9

Treatment

(µg/mL)

 

+S-9

%RS

MF§

%RS

MF§

0

100

1.27!

 

0

100

3.29

 

200

129

4.02

NS

200

90

3.20

NS

400

144

3.66

NS

400

97

3.21

NS

600

107

2.15

NS

600

105

4.03

NS

800

117

2.17

NS

800

86

3.65

NS

1000

110

3.25

NS

1000

86

2.87

NS

1200

119

2.84

NS

1200

90

3.47

NS

1360

97

3.71

NS

1360

84

1.65

NS

Linear trend NS

Linear trend NS

NQO

B[a]P

0.1

90

24.82

 

2

74

14.42

 

0.15

69

46.58

 

3

34

23.58

 

§ = 6‑TG resistant mutants/106viable cells 7 days after treatment

%RS = Percent relative survival adjusted by post treatment cell counts

NS = Not significant

! = Based on one replicate only

Table 7.6.1/2:Experiment 2 (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

 

- S-9

Treatment

(µg/mL)

 

+S-9

%RS

MF§

%RS

MF§

0

100

0.37

 

0

100

2.94

 

150

98

0.75

NS

150

111

5.94

NS

300

87

0.86

NS

300

107

0.89

NS

500

82

0.20

NS

500

111

0.78

NS

700

99

1.15

NS

700

115

1.46

NS

900

91

0.71

NS

900

113

0.28

NS

1100

76

1.19

NS

1100

112

0.69

NS

1360

80

0.78

NS

1360

112

0.86

NS

Linear trendNS

Linear trendNS

NQO

B[a]P

0.1

71

5.94

 

2

80

21.34

 

0.15

36

16.29

 

3

62

6.38

 

§ =6‑TG resistant mutants/106viable cells 7 days after treatment

%RS =Percent relative survival adjusted by post treatment cell counts

NS =Not significant

! =Based on one replicate only

 

Conclusions:
Sodium trifluoroacetate (55.6% aq solution) was not mutagenic in the MLA when tested up to 10 mM in the absence and presence of S-9.
Executive summary:

In an in vitro mammalian cell gene mutation test performed according to the OECD test guideline No. 476 and in compliance with GLP, Sodium trifluoracetate as a 55.6% aqueous solution was assayed for its ability to induce mutation at the hprt locus (thio-6 -guanine resistance) in mouse lymphoma L5178Y cells.

The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post‑mitochondrial fraction (S‑9).

In the cytotoxicity range-finder experiment, 6 concentrations were tested in the absence and presence of S-9, ranging from 42.5 to 1360 µg/mL. There was no evidence of marked toxicity at concentrations up to and including 1360 µg/mL (equivalent to 10 mM) in the absence and presence of S-9.

In Experiment 1 seven concentrations, ranging from  200 to 1360 µg/mL, were tested in the absence and presence of S‑9.

In Experiment 2 seven concentrations, ranging from 150 to 1360 µg/mL were tested in the absence and presence of S-9.

 

When tested up to 1360 µg/mL (10 mM) in the absence and presence of S-9 in Experiments 1 and 2, no significant increases in mutant frequency were observed at any concentration analysed and there were no significant linear trends.

In the presence of S-9 in Experiment 1, the positive controls did not meet the acceptance criteria stated in the protocol. However, both positive control concentrations showed clear increases in mutant frequency which were outside the historical negative control ranges generated by the last 20 studies, updated at the time of each experiment. The data were therefore considered acceptable and valid on this basis.

Negative controls were valid for both experiments.

It is therefore conclude that sodium trifluoroacetate (55.6% aq solution) was not mutagenic when tested up to 10 mM in the absence and presence of S-9.

In this study Sodium Trifluoroacetate was used instead of Trifluoroacetic acid (TFA) according to a reliable analogue approach in order to be free of the cytotoxic effect on bacteria due to the extreme acid pH of the TFA (see §4.2).

Therefore, Trifluoroacetic acid is not considered as mutagenic in the mammalian cell gene mutation test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study in accordance with international guidelines. Sodium Trifluoroacetate was tested instead of trifluoroacetic acid (TFA) according to a reliable analogue approach in order to be free of the cytotoxic effect on bacteria due to the extreme acid pH of TFA (pH = 0.45).
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Trifluoroacetic acid (TFA) is a strong acid. Testing with the neutral salt sodium trifluoroacetate was considered appropriate to avoid cytotoxic effects on bacteria due to the extreme acid pH of TFA (pH=0.45).
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: human (female)
Details on mammalian cell type (if applicable):
- Type and identity of media: HEPES-buffered RPMI medium containing 20% (v/v) heat inactivated foetal calf serum and 50 µg/mL gentamycin. Phytohaemagglutinin is included in the medium at a concentration of approximately 2% of culture.
Blood from three healthy, non-smoking female volunteers was used for each experiment of this study.
No volunteer was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. The measured cell cycle time of the donors used at Covance falls within the range 13 +/- 1.5 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within two days of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from male Sprague-Dawley rats induced Arochlor 1254
Test concentrations with justification for top dose:
First test
Without S9 mix - 3 hours treatment, 17 hours recovery: 1000; 1200 and 1360 µg/mL
With S9 mix - 3 hours treatment, 17 hours recovery: 1000; 1200 and 1360 µg/mL

Second test
Without S9 mix - 20 hours continuous treatment: 750; 1200 and 1360 µg/mL
With S9 mix - 3 hours treatment, 17 hours recovery: 750; 1200 and 1360 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: 4-Nitroquinoline-1-oxide (2.5 µg/mL); with S9: cyclophosphamide (20 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration: First experiment 3 h
Second experiment 20 h (withour S9) / 3 h (with S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine (final concentration: 1 µg/mL)
STAIN (for cytogenetic assays): 4% (v/v) Giemsa

NUMBER OF REPLICATIONS: 2 (4 for vehicle)

NUMBER OF CELLS EVALUATED: at least 1000 cells counted where possible

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Determination of hyperdiploidy : yes
Evaluation criteria:
The test substance will be considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeds the historical negative control (normal) range is observed in both replicate cultures.
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) is observed (p < 0.05).
3. There is a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
The test article will be considered as positive in this assay if all of the above criteria are met.
The test article will be considered as negative in this assay if none of the above criteria are met.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Evidence of a concentration-related effect is considered useful but not essential in the evaluation of a positive result. Biological relevance will be taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments, or effects occurring only at high or very toxic concentrations, and the types and distribution of aberrations. Analysis of additional cells from vehicle or treated cultures or further experimental work may be deemed necessary to aid evaluation of the data.
Statistics:
The statistical method used will be Fisher's exact test. Probability values of p < 0.05 will be accepted as significant.
The proportions of aberrant cells in each replicate will be used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test. Probability values of p < 0.05 will be accepted as significant.
Species / strain:
lymphocytes: human
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:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: yes

Table 7.6.1/1 Results of chromosome analysis – Experiment 1 (3 +17) with metabolic activation

 

Vehicle

1000 µg/mL

1200 µg/mL

1360 µg/mL

NQC

Cytotoxicity

no

 

 

 

yes

Cells scored

201

202

202

200

100

Gaps

0

0

0

0

 

Chromosome aberrations

Deletions

1

0

0

1

 

Exchange

0

0

0

0

 

Chromatid aberrations

Deletions

1

1

1

1

 

Exchange

0

1

0

0

 

Aberrant cell

+ Gaps

2

2

1

2

 

- Gaps

2

2

1

2

 

Mitotic index(%)

8.7

9.2

8.75

8.55

 

Mitotic index ± SD

 

 

 

 

 

Numerical aberrations

Polyploid

0

1

1

0

0

Hyperdiploid

1

0

1

0

0

Endoreduplicated

0

1

0

0

0

 

Table 7.6.1/2 Results of chromosome analysis – Experiment 1 (3 +17) without metabolic activation

 

Vehicle

1000 µg/mL

1200 µg/mL

1360 µg/mL

NQC

Cytotoxicity

no

 

 

 

yes

Cells scored

202

201

200

201

182

Gaps

0

1

0

0

 

Chromosome aberrations

Deletions

2

1

1

1

 

Exchange

0

0

0

0

 

Chromatid aberrations

Deletions

3

3

2

1

 

Exchange

0

0

0

0

 

Aberrant cell

+ Gaps

5

5

3

2

 

- Gaps

5

4

3

2

 

Mitotic index(% of control)

8.6

10

9.95

10.3

 

Mitotic index ± SD

 

 

 

 

 

Numerical aberrations

Polyploid

0

0

0

1

0

Hyperdiploid

2

1

0

0

0

Endoreduplicated

0

0

0

0

0

Table 7.6.1/3 Results of chromosome analysis – Experiment 2 (3 +17) with metabolic activation

 

Vehicle

1050 µg/mL

1200 µg/mL

1360 µg/mL

CPA

Cytotoxicity

no

 

 

 

yes

Cells scored

201

202

202

201

67

Gaps

2

0

1

0

 

Chromosome aberrations

Deletions

0

0

0

1

 

Exchange

0

0

0

0

 

Chromatid aberrations

Deletions

3

0

1

2

 

Exchange

0

0

0

0

 

Aberrant cell

+ Gaps

5

0

2

3

 

- Gaps

3

0

1

3

 

Mitotic index(% of control)

8.275

7.9

7.2

6.45

 

Mitotic index ± SD

 

 

 

 

 

Numerical aberrations

Polyploid

1

0

1

1

0

Hyperdiploid

0

1

0

0

0

Endoreduplicated

0

1

1

0

0

 

Table 7.6.1/4 Results of chromosome analysis – Experiment 2 (20) without metabolic activation

 

Vehicle

750 µg/mL

1200 µg/mL

1360 µg/mL

CPA

Cytotoxicity

no

 

 

 

yes

Cells scored

200

201

203

201

75

Gaps

1

2

1

0

 

Chromosome aberrations

Deletions

2

0

0

1

 

Exchange

0

0

0

2

 

Chromatid aberrations

Deletions

3

0

1

0

 

Exchange

0

0

0

0

 

Aberrant cell

+ Gaps

6

2

2

3

 

- Gaps

5

0

1

3

 

Mitotic index(% of control)

8.25

7.15

5.35

5.4

 

Mitotic index ± SD

 

 

 

 

 

Numerical aberrations

Polyploid

0

0

1

0

0

Hyperdiploid

0

1

2

1

0

Endoreduplicated

0

0

0

0

0

Conclusions:
Under the test conditions, Sodium trifluoroacetate (56%) did not induce chromosome aberrations in cultured human peripheral blood lymphocytes in the presence and the absence of S-9 when tested up to a maximum concentration equivalent to 10 mM.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to Sodium trifluoroacetate 55.6 % aqueous solution. In the fist experiment cells were exposed both in the presence and absence of S-9 to concentrations of 0, 1000, 1250, 1360 µg/mL for 3 hours and sampled at 20 hours after the beginning of treatment. In the second experiment, cells were exposed continuously for 20 hours in the absence of S-9 (0, 750, 1200 and 1360 µg/mL) or 3 hours in the presence of S-9 (0, 1050, 1200 and 1360 µg/mL).

Positive controls induced the appropriate response. Chromosome aberrations were not induced over background at any tested concentrations in the absence and the presence of activation system in both experiments.

Under the test conditions, Sodium trifluoroacetate (56%) was not clastogenic in human lymphocytes exposed in vitro.

In this study Sodium Trifluoroacetate was used instead of Trifluoroacetic acid (TFA) according to a reliable analogue approach in order to be free of the cytotoxic effect on bacteria due to the extreme acid pH of the TFA

Therefore, Trifluoroacetic acid is not considered as clastogenic in humans cells exposed in vitro.

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

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro genotoxicity studies have been conducted with the sodium salt of Trifluoroacetate (TFA) instead of Trifluoroacetic acid (TFA) to avoid cytotoxic effect on bacteria due to the extreme acid pH of TFA (pH = 0.45 for 10% aqueous aolution, see § 4.20). Under physiological conditions the acid is promptly dissociated to trifluoroacetate and therefore, it is considered to be appropriate to assess the toxicological potential of the salt moiety. More information about the read-across justification is included in the Reporting format as attached to the respective IUCLID entry (section 13).

Bacterial Reverse Mutation assay:

In a reverse gene mutation assay in bacteria, performed similarly to the OECD guideline No.471, strains TA1535, TA1537, TA1538, TA98 and TA100 of S. typhimurium were exposed to Sodium Trifluoroacetate diluted in DMSO at concentrations of 0.1, 0.5, 1.0, 5.0 and 10 mg/plate in the presence and absence of mammalian metabolic activation (fraction of S9 from the liver of Sprague-Dawley male rats treated by intraperitoneal injection with Aroclor 1254 dissolved in maize oil). The method of direct incorporation was used in this study.

Sodium Trifluoroacetate was tested up to limit concentration recommended in the guideline (5 mg/plate). The positive controls induced the appropriate responses in the corresponding strains. Under the test conditions, the test item Sodium trifluoroacetate did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium as there was no evidence of induced mutant colonies over background. Therefore, Trifluoroacetic acid is not considered as mutagenic in the bacterial reverse mutation test. This study is considered as acceptable as it satisfied the main criteria of the OECD guideline No. 471.

In vitro Mammalian Chromosome Aberration test:

In an in vitro chromosome aberration test performed according to OECD guideline No 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to Sodium trifluoroacetate as 55.6 % aqueous solution. In the fist experiment cells were exposed both in the presence and absence of S-9 to concentrations of 0, 1000, 1250, 1360 µg/mL for 3 hours and sampled at 20 hours after the beginning of treatment. In the second experiment, cells were exposed continuously for 20 hours in the absence of S-9 (0, 750, 1200 and 1360 µg/mL) or 3 hours in the presence of S-9 (0, 1050, 1200 and 1360 µg/mL).

Positive controls induced the appropriate response.

Chromosome aberrations were not induced over background at any tested concentrations (up to limit concentration of 10mM) in the absence and the presence of activation system in both experiments. Under the test conditions, aqueous solution of Sodium trifluoroacetate (56% purity) was not clastogenic in human lymphocytes exposed in vitro. Therefore, Trifluoroacetic acid is not considered as clastogenic in humans cells exposed in vitro. This study is considered as acceptable and satisfies the requirement for the cytogenicity endpoint.

In vitro Mammalian Cell Gene Mutation test:

In an in vitro mammalian cell gene mutation test performed according to the OECD test guideline No. 476 and in compliance with GLP, Sodium trifluoracetate as a 55.6% aqueous solution was assayed for its ability to induce mutation at the hprt locus (thio-6 -guanine resistance) in mouse lymphoma L5178Y cells. In the cytotoxicity range-finder experiment, 6 concentrations were tested in the absence and presence of S-9, ranging from 42.5 to 1360 µg/mL. There was no evidence of marked toxicity at concentrations up to and including 1360 µg/mL (equivalent to 10 mM) in the absence and presence of S-9. In Experiment 1 seven concentrations, ranging from 200 to 1360 µg/mL, were tested in the absence and presence of S 9. In Experiment 2 seven concentrations, ranging from 150 to 1360 µg/mL were tested in the absence and presence of S-9.

When tested up to the limit concentration of 1360 µg/mL (10 mM) in the absence and presence of S-9 in Experiments 1 and 2, no significant increases in mutant frequency were observed at any concentration analysed and there were no significant linear trends.

In the presence of S-9 in Experiment 1, the positive controls did not meet the acceptance criteria stated in the protocol. However, both positive control concentrations showed clear increases in mutant frequency which were outside the historical negative control ranges generated by the last 20 studies, updated at the time of each experiment. The data were therefore considered acceptable and valid on this basis. Negative controls were valid for both experiments.

It is therefore concluded that sodium trifluoroacetate (55.6% aq solution) was not mutagenic when tested up to 10 mM in the absence and presence of S-9. Therefore, Trifluoroacetic acid is not considered as mutagenic in the mammalian cell gene mutation test. This study is considered as acceptable and satisfies the requirement for the mutagenicity endpoint in mammalian cells.

Regarding the overall negative results from the in vitro genotoxicity studies, it is likely that Trifluoroacetic acid doesn't induce any genotoxic activity potential.

Justification for classification or non-classification

Based on the available in vitro genotoxicity studies, the test substance does not need to be classified for genotoxicity according to Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008