Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Reverse Mutation assay:

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 inS. typhimuriumTA1535, 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 in the presence and the absence of mammalian metabolic activation (S9 mix).

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.

Mammalian chromosome aberration assay:

In a chromosome aberration assay in mammalian cells, performed according to the OECD No.473,  2,2,2-trifluoroethanol (TFE) (99.9% purity) diluted in physiological saline solution was tested in CHL/IU: Chinese Hamster Lung cells in the presence and the absence of mammalian metabolic activation (S9) at concentrations of 250; 500 and 1000 µg/mL (2.5; 5 and 10 mM at final concentration).

TFE was incubated with the cells for 6, 24 or 48 hours and then the cells were analysed for the presence of chromosomal aberrations 18 hours after exposure (in the case of the 6 hrs exposure period) or immediately after the end of the exposure period (in the case of the 24 or 48 hrs exposure period).

Mitomycin C and Benzo(a)pyrene were used as positive controls and induced appropriate responses.

No increase in the occurence of chromatid or chromosome aberration was observed with and without metabolic activation and for all exposure tested period. No cytotoxicity was observed up to 10 mM, the maximum required concentration tested.

Under the test conditions, 2,2,2-trifluoroethanol did not show any cytogenic activity in the chromosome aberration test using hamster cells. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 473.

Mammalian cell gene mutation :

In an in vitro mammalian cell gene mutation test performed according to the OECD test guideline No. 476 and in compliance with GLP, Trifluoethanol 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 31.25 to 1000 µg/mL. There was no evidence of marked toxicity at concentrations up to and including 1000 µg/mL (equivalent to 10 mM) in the absence and presence of S-9.

In Experiment 1 seven concentrations, ranging from  100 to 1000 µg/mL, were tested in the absence and presence of S‑9.

In Experiment 2 seven concentrations, ranging from 300 to 1000 µg/mL in the absence of S-9 and from 150 to 1000 µg/mL in the presence of S-9.

When tested up to 1000 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.

Positive and negative controls were valid for both experiments.

It is therefore conclude that trifluoroethanol was not mutagenic when tested up to 10 mM in the absence and presence of S-9.

Link to relevant study records

Referenceopen allclose all

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

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

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-11-25 to 1998-03-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
: no detail on the susbtance
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
other: CHL/IU: Chinese Hamster Lung cells
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (Gibco Laboratories, Lot No. 37N7765)
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix. The metabolic activation was only used for the exposure period of 6 hours. No metabolic activation in the case of the 24 or 48 hrs exposure period.
Test concentrations with justification for top dose:
0, 5, 15, 50, 150, 500, 1500 and 5000 µg/mL for the cytotoxicity assay.
250, 500 and 1000 µg/mL for the analysis of the chromosomal aberration: 2.5; 5 and 10 mM at final concentrations
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: physiol. saline
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
Isotonic Sodium chloride solution
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: 20 µg/mL; with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
Isotonic Sodium chloride solution
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: 0.15 µg/mL (for the 6h exposure period), 0.05 µg/mL (for the 24 or 48h exposure period); without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 6, 24 or 48 hours (see details in Table 7.6.1/1).
- Expression time (cells in growth medium): 0 or 18 hours
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 or 48 hours

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): no data
STAIN (for cytogenetic assays): no data

NUMBER OF REPLICATIONS: no data

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no
Evaluation criteria:
The D20 value was used. It is an estimated dose of the required substance under test to produce abnormalities in 20% of the metaphase figures.
Statistics:
A binomial test was conducted between the negative control final test and the negative control background data on the occurrences of chromosomal aberration, and no significant differences were found. Then a binomial test was conducted between the negative control background data and substance processing group for each test or the positive control group.
Key result
Species / strain:
other: CHL/IU: Chinese Hamster Lung 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:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
no data

RANGE-FINDING/SCREENING STUDIES: 24 and 48 hours processing tests for continous processing methods and a 50 % cell growth inhibition concentration is estimated to be above 1000 µg/mL (final concentration: 10 mM). The maximum dose used for the chromosomal aberration was 1000 µg/mL for all test systems and was setup in 3 -dose stages (500 and 250 µg/mL for each stage with a common ratio of 2 or less).

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: see details in Table 7.6.1/2.

Table 7.6.1/2: Chromosomal aberration study results. The results are presented for the 2x100 evaluated cells.

Processing time

S9 mix

Doses (µg/mL)

Chromatid break

Chromatid exchange

Chromosome break

Chromosome exchange

Other

Total number of cell aberrations

Gap

Polyploid

Cell proliferation (mitotic index)

6-18

-

Negative control

2

0

0

0

0

2

0

2

9.8

-

250

1

0

1

0

0

2

1

1

10.7

-

500

1

0

1

0

0

2

1

0

7.4

-

1000

1

1

0

0

0

2

0

2

7.6

-

MMC (0.15)

20

48

1

0

0

58*

3

0

4.9

+

Negative control

0

0

0

0

0

0

0

2

10.9

+

250

1

0

0

0

0

1

0

1

11.1

+

500

0

0

0

0

0

0

1

3

10.6

+

1000

1

1

0

0

0

2

1

0

9.6

+

B[a]P (20)

18

50

0

2

0

63*

0

0

8.2

24-0

-

Negative control

1

1

0

0

0

2

0

2

9.7

-

250

0

0

0

0

0

0

1

1

10.1

-

500

1

0

0

0

0

1

0

1

9.3

-

1000

1

0

0

0

0

1

0

2

9.4

-

MMC (0.05)

18

46

0

0

0

56*

2

0

4.7

48-0

-

Negative control

0

0

0

0

0

0

1

2

7.7

-

250

0

0

0

0

0

0

0

1

6.6

-

500

1

0

0

0

0

1

0

10

8.5

-

1000

0

0

0

0

0

0

2

1

7.2

-

MMC (0.05)

21

60

0

0

3

73*

0

1

4.1

*: p<0.05

Conclusions:
Under the test conditions, the test item 2,2,2 trifluoroethanol did not show any cytogenic activity in the chromosomal aberration test in Hamster lung cells.
Executive summary:

In a chromosome aberration assay in mammalian cells, performed according to the OECD No.473,  2,2,2-trifluoroethanol (TFE) (99.9% purity) diluted in physiological saline solution was tested in CHL/IU: Chinese Hamster Lung cells in the presence and the absence of mammalian metabolic activation (S9) at concentrations of 250; 500 and 1000 µg/mL (2.5; 5 and 10 mM at final concentration).

TFE was incubated with the cells for 6, 24 or 48 hours and then the cells were analysed for the presence of chromosome aberration 18 hours (in the case of the 6 hrs exposure period) or immediately after the end of the exposure period (in the case of the 24 or 48 hrs exposure period).

Mitomycin C and Benzo(a)pyrene were used as positive controls and induced appropriate responses.

No increase in the occurence of chromatid or chromosome aberration was observed with and without metabolic activation and for all exposure tested period. No cytotoxicity was observed up to 10 mM, the maximum required concentration tested.

Under the test conditions, 2,2,2-trifluoroethanol did not show any cytogenic activity in the chromosome aberration test using hamster cells. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 473.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
In the presence of S-9 in Experiment 2, the positive controls did not meet the acceptance criteria stated in the protocol.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
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): 50, 100, 200, 400, 600, 800, 900 and 1000 µg/mL.
Experiment 2 (+/- S9): 150, 300, 450, 600, 700, 800, 900 and 1000 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: no data
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
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.
Key result
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: Following 3 hour treatment the highest concentration tested was 1000 µg/mL (equivalent to 10mM). No marked toxicity was observed at any concentration tested and relative survival was 74% and 89% in the absence and presence of S-9, respectively. See table 7.6.1/3.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment 1 eight concentrations, ranging from 50 to 1000 µg/mL, were tested in the absence and presence of S 9. Seven days after treatment the lowest concentration tested in the absence and presence of S-9 (50 g/mL) was not selected to determine viability and 6TG resistance as there were sufficient non-toxic concentrations. All other concentrations were selected. The highest concentration analysed was 1000 µg/mL in the absence and presence of S 9, which gave 158% and 101% RS, respectively (see Table 8). It may be noted that the relative survival values in the absence of S 9 appear erroneously high. This is due to slightly lower than normal, although still acceptable, cloning efficiency in the vehicle control and subsequently low survival counts. The cell count data in this experiment confirm the lack of toxicity and the data were therefore accepted as valid.
In Experiment 2 eight concentrations, ranging from 150 to 1000 µg/mL, were tested in the absence and presence of S 9. Seven days after treatment concentrations of 150 g/mL in the absence of S-9 and 300 g/mL in the presence of S 9 were not selected to determine viability and 6TG resistance as there were sufficient non-toxic concentrations. All other concentrations were selected. The highest concentration analysed was 1000 g/mL in the absence and presence of S 9, which gave 96% and 87% RS, respectively.

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 !

2.46

 

0

100

2.62

 

100

171

1.66

NS

100

105

3.23

NS

200

169

2.37

NS

200

114

1.35

NS

400

171

0.91

NS

400

99

1.02

NS

600

186

1.34

NS

600

97

2.35

NS

800

165

3.67

NS

800

102

3.23

NS

900

174

2.28

NS

900

101

3.43

NS

1000

158

2.42

NS

1000

101

1.78

NS

Linear trend NS

Linear trend NS

NQO

B[a]P

0.1

134

20.32

 

2

59

23.01

 

0.15

117

25.53

 

3

40

25.01

 

§ = 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

1.13

 

0

100

0.99

 

300

99

0.80

NS

150

90

1.60

NS

450

97

0.58

NS

450

92

1.46

NS

600

94

1.90

NS

600

95

1.11

NS

700

93

1.09

NS

700

95

0.79

NS

800

103

0.44

NS

800

103

0.46

NS

900

101

0.90

NS

900

77

0.58

NS

1000

96

2.67

NS

1000

87

0.71

NS

Linear trend NS

Linear trend NS

NQO

B[a]P

0.1

103

12.49

 

2

41

9.13

 

0.15

53

26.39

 

3

38

18.64

 

%RS =Percent relative survival adjusted by post treatment cell counts

NS =Not significant

! =Based on one replicate only

 

Conclusions:
Trifluoroethanol was not mutagenic 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, Trifluoethanol 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 31.25 to 1000 µg/mL. There was no evidence of marked toxicity at concentrations up to and including 1000 µg/mL (equivalent to 10 mM) in the absence and presence of S-9.

In Experiment 1 seven concentrations, ranging from  100 to 1000 µg/mL, were tested in the absence and presence of S‑9.

In Experiment 2 seven concentrations, ranging from 300 to 1000 µg/mL in the absence of S-9 and from 150 to 1000 µg/mL in the presence of S-9.

 

When tested up to 1000 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 2, 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 range 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 trifluoroethanol was not mutagenic when tested up to 10 mM in the absence and presence of S-9.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available according to the Regulation (EC) No 1272/2008.

Self classification:

2,2,2 Trifluoroethanol is not self-classified for genotoxicity according to the Regulation (EC) No 1272/2008 (CLP).