Ethyl trifluoroacetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 26 april 2007 to 30 May 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to the OECD (No. 471) and EC (B13/14) guidelines and is in compliance with the GLP.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Groupe Interministériel des produits chimiques, 8th March 2007

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl trifluoroacetate

EC Number:

206-851-6

EC Name:

Ethyl trifluoroacetate

Cas Number:

383-63-1

Molecular formula:

C4H5F3O2

IUPAC Name:

ethyl 2,2,2-trifluoroacetate

Details on test material:

- Name of test material (as cited in study report): study plan: Trifluoroacétate d'éthyle; Labeling: Ethyl Trifluoroacetate; Analytical certificate: Ethyl Trifluoroaxetate. All names corresponded to the same test item.
- Physical state: clear colorless liquid
- Stability under test conditions: no data, assume to be stable (sponsor responsibility)
- Storage condition of test material: at room temperature and protected from light and humidity

Method

Target gene:

Each strains derived from S. thyphimurium LT2 contains one mutation in the histine operon, resulting in a requirement for histidine.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: additional mutations in rfa and uvrB genes. For the strains TA98 and TA100 presence of an additional plasmid pKM101 in order to enhance their sensitivity of detection of some mutagens. See Table 7.6.1/1

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: additional mutation in rfa gene and presence of an additional plasmid pKM101 in order to enhance their sensitivity of detection of some mutagens. The histidine mutation is located on the multicopy plasmid pAQ1. See Table 7.6.1/1

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.

Test concentrations with justification for top dose:

Preliminary test: from 10 to 5000 µg/plate
Main test (2 independent tests): from 312.5 to 5000 µg/plate
See details in Table 7.6.1/2.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Dimethylsulfoxide), batch No. K35781950609, VWR (Fontenay-Sous-Bois, France)
- Justification for choice of solvent/vehicle: the test item is freely soluble in in this vehicle at a concentration of 100 mg/mL.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

dissolved in DMSO

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: TA 1535 and TA 100 (see details in Table 7.6.1/3)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

dissolved in DMSO

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: TA 1537 (see details in Table 7.6.1/3)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

dissolved in DMSO

Positive control substance:

2-nitrofluorene

Remarks:

Migrated to IUCLID6: TA 98 (see details in Table 7.6.1/3)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

dissolved in distilled water

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: TA 102 (see details in Table 7.6.1/3)

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

dissolved in DMSO

Positive control substance:

other: 2 Anthramine for TA 1535, TA 1537, TA 98 and TA 102 (see details in Table 7.6.1/3)

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

dissolved in DMSO

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: TA 100 (see details in Table 7.6.1/3)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (for both experiment without S9 mix and for the first experiment with S9 mix); preincubation (for the second experiment with S9 mix).

DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: 48 or 72h
- Expression time (cells in growth medium): revertants are scored immediately after the end of exposure.
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS:on eplate for the preliminary test, 3 replicate for both independent main tests

NUMBER OF CELLS EVALUATED: the number of all revertants per plate was scored

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies and/or thining of the bacterial lawn

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable

Evaluation criteria:

This study is considered valid if the number of revertants in the vehicle controls is consistent with the historical data of the testing facility and if the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.
A reproducible 2-fold increase (for the TA 98, Ta 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship 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.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

A slight increase in the number of revertants was noted at 5000 µg/plate in the TA 100 strain in the second experiment without S9 mix. Since this slight increase was neither dose-related nor reproducible, it was not considered as biologically relevant.

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

see details in Tables 7.6.1/4, 7.6.1/5, 7.6.1/6, 7.6.1/7

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see details in Tables 7.6.1/4, 7.6.1/5, 7.6.1/6, 7.6.1/7

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not tested
- Effects of osmolality: not tested
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no precipitate was observed in the Petri plates

RANGE-FINDING/SCREENING STUDIES: Since the test item was freely soluble and non-toxic in the preliminary test, the highest selected dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.

COMPARISON WITH HISTORICAL CONTROL DATA: the number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered as valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No noteworthy toxicity was observed in any of the five strains, in either experiment, with ot without S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

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

TFAE Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		TA 102
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	24	4	10	2	27	19	139	11	391	56
321.5	25	3	6	1	24	1	135	21	346	14
625	25	9	10	3	22	5	130	14	393	11
1250	21	2	8	5	29	5	140	9	445	70
2500	21	4	6	1	25	8	157	42	361	7
5000	16	6	6	2	23	5	156	52	371	33
Positive control**	535	47	554	187	268	40	638	58	2878	138

*Solvent control = negative control: 100 µL water

**Mutagens positive controls:

- NaN₃(1 µg/plate) in TA 1535 and TA 100 strains

- 9AA (50 µg/plate) in TA 1537 strain

- 2NF (0.5 µg/plate) in TA 98 strain

- MMC ( 0.5 µg/plate) in TA 102 strain

  

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

 

TFAE Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		TA 102
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	13	3	8	3	26	3	118	8	359	23
321.5	21	8	14	3	32	5	123	32	562	61
625	10	5	15	4	35	8	114	14	438	34
1250	19	6	13	3	39	8	131	10	512	90
2500	15	1	11	2	29	3	123	10	389	16
5000	8	2	7	3	44	17	113	16	367	36
Positive control**	195	10	145	22	1465	140	722	237	3911	899

*Solvent control = negative control: 100 µL water

**Mutagens positive controls:

- 2AM (2µg/plate) in TA1535, TA 1537, TA 98 strains

- 2AM (10µg/plate) in TA 102 strain

- BAP (5µg/plate) in TA 100 strain

 

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

TFAE Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		TA 102
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	11	3	9	4	26	7	116	21	447	54
321.5	20	8	9	3	28	8	115	5	378	38
625	18	5	6	2	25	7	156	61	447	34
1250	21	5	6	3	33	5	120	6	471	33
2500	23	5	2	2	27	6	120	19	503	66
5000	18	3	3	1	38	22	235	95	480	36
Positive control**	602	15	362	0	181	15	729	16	2274	50

*Solvent control = negative control: 100 µL water

**Mutagens positive controls:

- NaN₃(1 µg/plate) in TA 1535 and TA 100 strains

- 9AA (50 µg/plate) in TA1537 strain

- 2NF (0.5 µg/plate) in TA 98 strain

- MMC ( 0.5 µg/plate) in TA 102 strain

 

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

 

TFAE Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		TA 102
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	15	9	17	5	37	6	124	10	443	42
321.5	10	3	11	1	36	9	144	20	530	87
625	17	7	5	3	35	1	126	14	505	174
1250	16	7	10	4	45	19	134	6	520	109
2500	14	2	14	2	38	7	108	9	553	16
5000	16	2	13	1	42	7	119	8	682	174
Positive control**	146	13	130	18	1255	133	756	76	2213	169

*Solvent control = negative control: 100 µL water

**Mutagens positive controls:

- 2AM (2µg/plate) in TA 1535, TA 1537, TA 98 strains

- 2AM (10µg/plate) in TA 102 strain

- BAP (5µg/plate) in TA 100 strain

Applicant's summary and conclusion

Conclusions:

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

Under the test conditions, the test item Ethyl 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 according to the OECD No.471 and EC No.B13/14 guidelines, and in compliance with the GLP, strains TA1535, TA1537, TA100, TA98 and TA102 of S. typhimurium were exposed to ethyl trifluoroacetate (99.94%) diluted in DMSO in the presence and absence of mammalian metabolic activation (fraction of S9 from the liver of rats treated with Aroclor 1254). In the preliminary test, 6 concentrations of test substance were tested (10, 100, 500, 1000, 2500 and 5000 µg/plate). Since the test item was freely soluble and non-toxic in the preliminary test, the highest selected dose-level for the two independent main tests was 5000 µg/plate, according to the criteria specified in the international guidelines. Three replicates were realized per concentration of Ethyl trifluoroacetate in the main test. Furthermore, method of direct incorporation and protocol with a preincubation step were tested.

The positive controls induced the appropriate responses in the corresponding strains. Ethyl Trifluoroacetate was tested therefore up to limit concentration (5000 µg/plate) and no cytotoxicity was observed. No significant increase of the number of revertants was observed in any bacterial strains tested with Ethyl trifluoroacetate (at any concentrations).

Under the test conditions, Ethyl 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. 

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