Trifluoroacetic acid

Genetic toxicity: in vitro

Administrative data

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).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Sodium trifluoroacetate (NaTFA)

IUPAC Name:

Sodium trifluoroacetate (NaTFA)

Test material form:

solid

Method

Species / strain

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

Controls

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Applicant's summary and conclusion

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.