Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

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).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Sodium Trifluoroacetate (Trifluoroacetate de sodium, TFAS)
- Physical state: white powder
- Storage condition of test material: no data
No other information

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).

Method

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
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.
Controls
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

Results and discussion

Test results
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'.

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 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)

Applicant's summary and conclusion

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.