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Toxicological information

Genetic toxicity: in vitro

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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:
2003-10-02 to 2003-12-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according 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.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
: only the 2-Anthramine is used as a positive control for the efficacy of S9 mix.
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
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): 2,2,2 trifluoroethanol (TFE)
- Physical state: colorless liquid
- Lot/batch No.: 02 092 01
- Expiration date of the lot/batch: no data
- Stability under test conditions: assumed to be stable during the test (sponsor responsibility)
- Storage condition of test material: at room temperature
- Other: the pH of the undiluted test item, measured at CIT, was approximately 4.

Method

Target gene:
Each strains derived from S. thyphimurium LT2 contains one mutation in the histidine operon, resulting in a requirement for histidine.
Species / strainopen allclose all
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 / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: mutated in rfa gene and presence of an additional plasmid pKM101 in order to enhance the sensitivity of detection of some mutagens. See Table 7.6.1/1
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was purchased from Moltox (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: 10, 100, 500, 1000, 2500, 5000 µg/plate.
Mutagenicity experiments: 312.5, 625, 1250, 2500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water (Batch No. EVE19D (Fresenius Kabi, Sèvres, France)
- Justification for choice of solvent/vehicle: the test item is freely soluble in the water at 50 mg/mL
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-Aminoacridine, 2-Nitrofluorene, Mitomycin C 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 plate incorporation: test item solution (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
or preincubation method: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate.
DURATION
- Preincubation period: 60 min at 37°C
- Exposure duration: 48 or 72 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. For the mutagenicity experiments, two independent experiments were performed.
NUMBER OF CELLS EVALUATED: no data
DETERMINATION OF CYTOTOXICITY
- Method: observation of the decrease in the number of revertant colonies and/or thinning of the bacterial lawn
OTHER EXAMINATIONS:
- Determination of polyploidy: not required
- Determination of endoreplication: not required
- Other:
OTHER:
Evaluation criteria:
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 resultsopen allclose all
Key result
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
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
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
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
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
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
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
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
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
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: not applicable

RANGE-FINDING/SCREENING STUDIES: No observation of cytotoxicity and mutagenicity in the 3 strains tested (TA98, TA100 and TA102) at the concentrations of 10, 100, 500, 1000, 2500, and 5000 µg/plate.

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

Any other information on results incl. tables

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

TFE 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

2

6

2

30

5

85

9

468

21

321.5

16

5

6

5

36

14

125

50

476

45

625

14

5

7

1

45

34

107

21

452

46

1250

13

5

5

1

38

10

118

23

491

12

2500

13

5

9

4

47

24

106

7

539

64

5000

11

2

6

2

24

7

150

56

495

19

Positive control**

517

27

380

67

201

19

544

35

1909

105

$: Mean of triplicate

*Solvent control = negative control: 100 µL water

**Mutagens positive controls:

- NaN3(1 µg/plate) in TA1535 and TA100 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 7.6.1/5:Number of revertants per plate in the presence of metabolic activation (S9 mix) in the first test (direct plate incorporation method) 

TFE 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*

17

5

12

2

34

9

113

10

628

30

321.5

12

2

9

3

35

7

120

25

651

30

625

17

6

10

2

31

1

133

12

645

55

1250

13

3

7

1

38

3

111

17

652

17

2500

18

2

11

5

33

14

123

16

643

33

5000

16

2

10

7

25

2

104

1

657

48

Positive control**

181

16

218

51

731

85

512

85

2004

331

$: Mean of triplicate

*Solvent control = negative control: 100 µL water

**Mutagens positive controls:

- 2AM (2µg/plate) in TA1535, TA1537, TA98, TA100 strains

- 2AM (10µg/plate) in TA102 strain

 

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

TFE 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

5

6

2

19

6

95

10

382

33

321.5

12

6

6

2

13

6

99

11

421

37

625

15

2

8

3

23

4

88

12

412

32

1250

10

3

11

5

19

1

109

3

413

28

2500

12

2

8

2

18

2

107

4

403

31

5000

10

6

8

2

19

4

113

14

436

5

Positive control**

490

30

261

20

158

25

599

15

2748

54

$: Mean of triplicate

*Solvent control = negative control: 100 µL water

**Mutagens positive controls:

- NaN3(1 µg/plate) in TA1535 and TA100 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 in the presence of metabolic activation (S9 mix) in the second test (pre-incubation method)

 

TFE 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*

19

5

7

4

28

3

116

4

619

25

321.5

15

3

7

4

33

6

131

13

612

1

625

13

2

7

3

22

4

134

10

612

27

1250

13

1

9

4

27

3

123

6

609

30

2500

13

1

9

2

26

6

118

9

624

25

5000

12

4

8

2

28

2

109

10

657

13

Positive control**

152

17

56

6

1384

55

660

28

3261

178

$: Mean of triplicate

*Solvent control = negative control: 100 µL water

**Mutagens positive controls:

- 2AM (2µg/plate) in TA1535, TA1537, TA98, TA100 strains

- 2AM (10µg/plate) in TA102 strain

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the test item 2,2,2-trifluoroethanol 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,  2,2,2-trifluoroethanol (99.95%) diluted in water at concentrations of 312.5, 625, 1250, 2500, and 5000 µg/plate was tested in S. typhimurium TA1535, TA1537, TA100, TA98 and TA102 in the presence and the absence of mammalian metabolic activation (S9) using the direct incorporation or the preincubation method.The positive controls induced the appropriate responses in the corresponding strains.

No induced revertants over background was observed in any strains of S. typhimurium up to the highest concentration of 2,2,2-trifluoroethanol.

Under the test conditions, 2,2,2-trifluoroethanol did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 471.