2,2,2-trifluoroethanol

Endpoint summary

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

Reference

Reference 1

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

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

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.

Reference 2

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

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.

Reference 3

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

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

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).