Trifluoroacetic acid

Endpoint summary

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

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 Mar 2005 to 10 May 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254 and obtained from Molecular Toxicology Incorporated, USA. The activity of the S-9 mix used in each experiment was confirmed by AAN or B[a]P treatments (again in triplicate) of the strains in the presence of S-9.

Test concentrations with justification for top dose:

- Range-Finder Experiment: An initial toxicity Range-Finder Experiment was carried out in strain TA100 only, in the absence and presence of S-9, using final concentrations of Trifluoroacetate (TFA) at 1.6, 8, 40, 200, 1000 and 5000 µg/plate, plus negative (purified water) and positive controls. No evidence of toxicity was observed following any of these treatments. Therefore 5000 µg/plate was selected as the top dose for the experiment.
- Experiment 1: 1.6, 8, 40, 200, 1000 and 5000 µg/plate
- Experiment 2: 156.25, 312.5, 625 , 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: The test article was completely soluble in the aqueous assay system at all concentrations treated

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2 separate experiments

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation). Triplicate plates with or without S-9 mix were used. Negative (solvent) and positive controls were included in quintuplicate and triplicate respectively, without and with S-9 mix. These platings were achieved by the following sequence of additions to 2.5 mL molten agar at 46±1°C:
· 0.1 mL bacterial culture
· 0.1 mL test article solution or control
· 0.5 mL 10% S-9 mix or buffer solution

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 1 hour at 37±1°C in second experiment
- Exposure duration/duration of treatment: plates were incubated at 37±1°C in the dark for 3 days

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Following incubation, the plates were examined for evidence of toxicity to the background bacterial lawn, and where possible revertant colonies were counted. Colonies were counted electronically using a Seescan Colony Counter (Seescan Plc) or manually where confounding factors such as split agar affected the accuracy of the automated counter. The background bacterial lawn was inspected for signs of toxicity. Individual plate counts from both experiments were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined.

Rationale for test conditions:

Concentration selection: concentrations for the final experiment were selected based on the results of a Range-Finder Experiment in strain TA100 in absence and presence of S-9

Evaluation criteria:

The assay was considered valid if the following criteria were met:
1. the negative control counts fell within the historical control values,
2. the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation,
3. no more than 5% of the plates were lost through contamination or some other unforeseen event.

The test article was considered to be mutagenic if:
1. the assay was valid (see above)
2. Dunnett's test gave a significant response (p ≤ 0.01) and the data set(s) showed a significant dose correlation
3. the positive responses described above were reproducible

Statistics:

The m-statistic was calculated to check that the data were Poisson-distributed, and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis

Species / strain:

S. typhimurium TA 98

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

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

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

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

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

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

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

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

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

Positive controls validity:

valid

Additional information on results:

Results of the formulations analyses demonstrated achieved concentrations within 100±10% of the nominal test article concentrations for all treatment concentrations in each of the main mutation experiments

Conclusions:

It was concluded that Sodium Trifluoroacetate did not induce mutation in five histidine requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102) when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 mg/plate, in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

Sodium trifluoroacetate was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to the OECD 471 Guideline and under GLP. The experiments were carried out using five histidine-requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA102, TA 100 and TA 98) both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. Based on the results of a solubility test, the test item was formulated in purified water.

An initial toxicity Range-Finder Experiment was carried out in strain TA100 only, in the absence and presence of S-9, using final concentrations of Sodium trifluoroacetate at 1.6, 8, 40, 200, 1000 and 5000 µg/plate, plus negative (solvent) and positive controls. No evidence of toxicity was observed following any of these treatments. Experiment 1 treatments of all the test strains in the absence and presence of S-9 retained the same test doses as employed for the Range-Finder experiment. In Experiment 2 a narrowed dose range was employed (156.25-5000 µg/plate) in order to examine more closely those concentrations approaching the maximum test dose. No evidene of toxicity was observed following any of these treatments. 

Results of the formulations analyses demonstrated achieved concentrations within 100±10% of the nominal test article concentrations. Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. No dose-related and reproducible increases in revertant numbers were observed following any of the treatments of any of the tester strains in the absence or presence of S-9. Therefore, this study was considered to have provided no evidence of any mutagenic activity. 

Reference 2

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:

guideline study with acceptable restrictions

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.

Reference 3

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:

guideline study with acceptable restrictions

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:

Sodium trifluoroacetate was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to the OECD 471 Guideline and under GLP. The experiments were carried out using five histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA102, TA100 and TA98) both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. Based on the results of a solubility test, the test item was formulated in purified water. 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 the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008