Registration Dossier
Registration Dossier
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EC number: 206-581-9 | CAS number: 355-37-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro Gene Mutation study in Bacteria: negative in the bacterial reverse mutation assay
In vitro cytogenicity / chromosome aberration study in mammalian cells: did not induce chromosome aberrations in the absence or presence of metabolic activation
In vitro gene mutation study in mammalian cells: not considered to be mutagenic in the TK mutation test system
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1994-04-06 t 1994-06-01
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- Standards for Toxicity Investigations (Japan's Ministry of Labor, No. 77, September 1, 1988) and Notification on Partial Revision of Testing Methods Relating to the New Chemical Susbtances (Notification No. 700 of the Planning and Coordination Bureau, EA, No. 1039 of the Pharmaceutical Affairs Bureau, MHW & No. 1014 (1986) of the Basic Industries Bureau, MITI, December 5, 1986).
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Asahi Glass Co., Ltd.; Batch no. 31001
- Expiration date of the lot/batch: Not specified
- Purity test date: Not specified
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in a cold and dark place
- Stability under test conditions: Not specified
FORM AS APPLIED IN THE TEST (if different from that of starting material): Colourless clear liquid
OTHER SPECIFICS:
Purity: 99.9%
Impurities: CClF2HCCl2F (0.05%)
Molecular Weight: 320.05
Boiling Point 71°C
Solubility: Oil soluble
Degree of Solublity Water (<10 mg/L)
DMSO (<5% w/v)
Acetone (≥ 10% w/v) - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat Liver S9
- Test concentrations with justification for top dose:
- Dose range-finding test: 0, 50, 100, 200, 500, 1000, 2000, or 5000 µg/plate
Main Test: 0, 313, 625, 1250, 2500, or 5000 µg/plate (Based on the results of the dose range-finding test, 5000 µg/plate was selected as the highest dose and the lower four doses diluted with a geometric progression of 2 were set). - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Not specified - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- not specified
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); 2-Aminoanthracene (2AA); 2-Methoxy-6-chloro--9-[3-(2-chloroethyl)-aminopropylamino]acridine.2HCl
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): Main Test - TA98: 2.0 x 10^9 viable cells/mL; TA100: 1.6 x 10^9 viable cells/mL; TA1535: 2.1 x 10^9 viable cells/mL; TA1537: 1.8 x 10^9 viable cells/mL; WP2 uvrA: 1.9 x 10^9 viable cells/mL
DURATION
- Preincubation period: 37 ± 0.5°C for 8-10 hours
- Expression time (cells in growth medium): 20 minutes
- Exposure duration: 48 hours
SELECTION AGENT (mutation assays): Not specified
NUMBER OF REPLICATIONS: 3 (negative control); 2 (Test substance and positive controls)
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The appearance of revertant colonies (size and number), deposition of the test substance and growth inhibition were examined with a stereo microscope.
NUMBER OF CELLS EVALUATED: The number of revertant colonies was counted with a manual counter or a colony analyzer (CA-7, Toyo-sokki Co., Ltd.). The count using the colony analyzer was finalized after correcting with count drop. Each plate was measured three times, and the average of these three individual measurements with rounding off the figures below decimal point was adopted as the number of revertant colonies. - Evaluation criteria:
- The test substance was judged to be positive, when the number of revertant colonies was increased more than twice that of the negative control in a dose-dependant manner, and the reproducibility of results was also obtained. It was judged to be negative in the other cases.
- Statistics:
- Statistical analysis was not applied.
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The sterility test confirmed the absence of micro-organisms.
- Conclusions:
- 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane was considered to have no reverse mutagenic potential under the conditions of the study.
- Executive summary:
In a key bacterial reverse mutation assay, the in vitro mutagenic potential of the test material, 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane, was evaluated in Salmonella typhimurium strains TA98, TA100, T1535, and TA 1537 and Escherischia coli strain WP2uvrA using pre-incubation method in the absence and presence of a metabolic activation system (±S9).
Based on a dose range-finding study, the concentrations of the test material used in the main study were 0, 313, 625, 1250, 2500, or 5000 µg/plate.
It was observed that the number of revertant colonies in all strains was less than twice that of the negative control at all dose levels tested in the absence and presence of metabolic activation (±S9). The positive controls exhibited a significant increase in the number of revertant colonies and the number of revertants for positive as well as negative controls was within the historical range.
Based on the results observed in the study, 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane was considered to be negative in this bacterial reverse mutation assay.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1994-04-05 to 1994-06-17
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- 'Notification on Partial Revision of Testing Methods Relating to the New Chemical Substances" (Notification No. 700 of the Planning and Coordination Bureau, EA, No. 1039 of the Pharmaceutical Affairs Bureau, MHW & No. 1014 (1986) of the Basic Industries Bureau, MITI, December 5, 1986).
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Asahi Glass Co., Ltd. (Japan); Batch no. 31001
- Expiration date of the lot/batch: Not specified
- Purity test date: Not specified
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in a cold and dark place
- Stability under test conditions: Not specified
FORM AS APPLIED IN THE TEST (if different from that of starting material): Colourless Clear Liquid
OTHER SPECIFICS:
Purity: 99.9%
Impurities: CClF2HCCl2F (0.05%)
Molecular weight: 320.05
Boiling Point: 71°C
Solubility: Oil soluble
Degree of Solubility: Water (Less than 10 mg/L) - Species / strain / cell type:
- other: Chinese Hamster Lung Fibroblast Cells (CHL Cells, clone No. 11)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: National Institute of Hygienic Sciences
- Suitability of cells: a) widely used in in vitro chromosome aberration tests; b) show quite high sensitivity to chemical mutagens; c) data available about their chromosomal aberrations
- Cell cycle length, doubling time or proliferation index: Modal number of chromosomes is 25 per cell; doubling time is about 15 hours
- Number of passages if applicable: Passage number of cells was 30 for 24, 48 hours treatment by the direct method; and 31 by the metabolic activation method
- Methods for maintenance in cell culture if applicable: Eagle's minimal essential medium supplemented with new born calf serum at a rate of 10% v/v (10% NCS/MEM) was used.
- Modal number of chromosomes: 25 per cell
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 10% NCS/MEM medium made from pure water. 5 mL of the 10% NCS/MEM medium containing 1.5 x 10^4 0.5 x 10^4 cells/mL was used for passage along with a culture flask with screw cap on the base area of 25 cm^2 and cultured for 2-3 days. Monolayer cells in logarithmic growth phase were then exposed to the test material at 2-3 days cultivation after seeding. Cell culture was carried out in a CO2 incubator under the following conditions:
Temperature: 37 ± 0.5°C
Humidity: approximately 100%
Atmosphere: Air containing 5% CO2
- Properly maintained: yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat Liver S9 prepared using intraperitoneal injections of phenobarbital (30 mg/Kg x 1 time; 60 mg/Kg x 3 times); 5,6-benzoflavone (80 mg/Kg x 1 time)
- Test concentrations with justification for top dose:
- Cell Growth Inhibition and Cell Division Inhibition Test:
24 hours treatment by Direct Method: 0, 10, 30, 50, 80, 100, and 300 µg/mL
48 hours treatment by Direct Method: 0, 10, 30, 50, 80, 100, 200, and 300 µg/mL
Metabolic Activation Method: 0, 10, 30, 50, 100, 300, 500, 1000, 3000, and 5000 µg/mL
Fifty percent growth inhibition concentrations of the test material were about 40 µg/mL for the 24 and 48 hour treatments by the direct method, and it could not be could not be calculated in the metabolic activation method, since cytotoxicity of the test material was weak. Mitotic metaphases of chromosomes sufficient for assessing chromosomal aberration were observed at 50 µg/mL for the 24 hours treatment and at 100 µg/mL for the 48 hours treatment by the direct method, and at 5000 µg/mL by the metabolic activation method. Therefore, the chromosomal aberration test was carried out at the dose levels mentioned below:
Chromosomal Aberration Test:
24 hours treatment by Direct Method: 0, 12.5, 25, and 50 µg/mL
48 hours treatment by Direct Method: 0, 25, 50, and 100 µg/mL
Metabolic Activation Method: 0, 1250, 2500, and 500 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The dose range-finding test carried out with 5 mL medium including the test material was prepared with a sodium carboxymethylcellulose (CMC) aqueous solution. No cytotoxicity was observed and hence, the solvent was changed to acetone. As a result, cytotoxicity was observed, and acetone selected as the vehicle. - Untreated negative controls:
- yes
- Remarks:
- Solvent treated group used as negative control
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1.5 x 10^4 or 0.5 x 10^4 cells/mL
DURATION
- Preincubation period: 2-3 days
- Exposure duration:
A] Cell Growth Inhibition Test: Direct Method: 24 - 48 hours; Metabolic Activation Method: 6 hours
B] Cell Division Inhibition Test: Direct Method: 24 - 48 hours; Metabolic Activation Method: 6 hours
C] Chromosome Aberration Test: Direct Method: 24 - 48 hours; Metabolic Activation Method: 6 hours
- Expression time (cells in growth medium): 18 hours
SPINDLE INHIBITOR (cytogenetic assays): Chromosome Aberration Test: Direct Method and Metabolic Activation Method: 2 hours prior to end of incubation, Colcemid was added to the medium to give a final concentration of 0.1 µg/mL.
STAIN (for cytogenetic assays): 2% Giesma solution
NUMBER OF REPLICATIONS: Two flasks per dose
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Two slides per flask were prepared as follows: Cells were detached from each flask with 0.25% trypsin and collected by centrifugation. The supernantant was discarded and 0.075 M KCl was added to the tubes and kept hypotonically at 37°C for 15 minutes. Methanol-acetic acid (3:1) solution was poured into the tube to fix the cells and the tube centrifuged. A small amount of methanol-acetic acid solution was added to pelleted cells to give a slightly cloudy suspension. The cell suspension was then dropped onto microscopic slides and spread. The slides were then dried and stained with a 2% Giesma solution.
NUMBER OF CELLS EVALUATED: The number of cell with chromatid type or chromosome type structural aberrations (gap, break, exchange, etc.) and numerical aberrations (polyploid, endoreduplication) were checked by observing 100 well spread metaphases for each flask.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): The incidence of each cell with structural and/or numerical aberrations were obtained by observing 200 metaphases for each dose.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes - Evaluation criteria:
- Results were evaulated as follows:
The incidence of cells (mean value for two flasks) with aberrations including gap was:
Less than 5%: Negative (-)
5% or more, less than 10%: Suspect positive (±)
10% or more, less than 20%: Positive (+)
20% or more, less than 50%: Positive (++)
50% or more: Positive (+++)
The test material was judged to be positive for induction of chromosomal aberration when the incidence of 10% or more was dose-related or reproducible. - Statistics:
- No statistical method used.
- Key result
- Species / strain:
- other: Chinese Hamster Lung Fibroblast Cells (CHL cells, Clone No. 11)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Values for the two flasks were not markedly different. The incidence of cells with any aberration did not exceed 5% in the negative control while the incidence of cells with structural aberrations excluding gap was 10% or more in the positive control.
There were no fluctuations in the test conditions and no contamination with micro-organisms which may have affected the results of the study. The test was therefore, considered to be valid.
Chromosome Aberration Test:
1) Direct Method:
A] 24 hours treatment: The incidence of cells with structural chromosomal aberrations including gap were 0.5% at 12.5 µg/mL, 1.5% at 25 µg/mL, and 1.0% at 50 µg/mL suggesting a negative effect. The incidence in the solvent treated group was 1.0% (within normal range). The positive control group treated with MMC clearly showed structural aberrations of 50.0%. The incidence of polyploid cells was less than 5% in each treatment group (negative).
B] 48 hours treatment: The incidence of cells with structural chromosomal aberrations including gap were 1.0% at 25 µg/mL, 0.5% at 50 µg/mL, and 0.5% at 100 µg/mL suggesting a negative effect. The incidence in the solvent treated group was 0.5% (within normal range). The positive control group treated with MMC clearly showed structural aberrations of 65.5%. The incidence of polyploid cells was less than 5% in the treatment group (negative).
2) Metabolic Activation Method:
A] With S9 Mix (+S9): The incidence of cells with structural aberrations including gap were 0.5% at 1250 µg/mL, 1.0% at 2500 µg/mL, and 2.0% at 5000 µg/mL suggesting a negative effect. The incidence in the solvent treated group was 1.0% (within normal range). The positive control group treated with CPA clearly showed chromosomal aberration of 80.5%. The incidence of polyploid cells was less than 5% in each treatment group (negative).
B] Without S9 Mix (-S9): The incidence of cells with structural chromosomal aberrations including gap were 0.5% at 1250 µg/mL, 0.5% at 2500 µg/mL, and 1.5% at 5000 µg/mL suggesting a negative effect. In the solvent treated group and positive control group, the incidences were 1.0 and 0.0% (within normal range). The incidence of polyploid cells was less than 5% in each treatment group (negative). - Conclusions:
- Based on the results observed in the study, it was concluded that the test material did not induce chromosome aberrations in the absence or presence of metabolic activation.
- Executive summary:
In a key in vitro cytogenicity study, the mutagenic potential of the test material (1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane) was evaluated using Chinese Hamster Lung Fibroblast (CHL) cells in the absence and presence of metabolic activation. Mitomycin C (MMC) and Cyclophosphamide monohydrate (CPA) were employed as positive controls in the absence of metabolic activation (Direct Method) and in the presence of metabolic activation (Metabolic Activation Method), respectively.
Inhibition tests on cell growth and cell division were carried out to determine the dose levels of the test material to be used in the study. Based on the results of these tests, the chromosome aberration tests were carried out at doses of 12.5, 25, and 50 µg/mL (Direct Method – 24 hours treatment); 25, 50, and 100 µg/mL (Direct Method – 48 hours treatment); and 1250, 2500, and 5000 µg/mL (Metabolic Activation Method). MMC (0.05 µg/mL) was used as the positive control for 24 hours and 48 hours treatment by the Direct Method, while CPA (10 µg/mL) was used as the positive control for the Metabolic Activation Method.
The test material induced no structural aberrations or numerical aberrations within the dose range which included doses showing cytotoxicity in the 24 and 48 hours treatments by the direct method and at less than 5000 µg/mL in the metabolic activation method. On the other hand, MMC and CPA induced evident chromosomal aberrations.
Based on the results observed in the study, it was concluded that the test material did not induce chromosome aberrations in the absence or presence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-03-29 to 2016-05-24
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: in vitro mammalian gene mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AGCChemicalsEurope Ltd. (UK); Batch no. 41251
- Expiration date of the lot/batch: 01 March 2017 (nominal expiry date) (taken from label)
- Purity test date: Not specified
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: until 01 March 2017 (nominal expiry date) (taken from label)
FORM AS APPLIED IN THE TEST (if different from that of starting material): Colourless liquid
OTHER SPECIFICS:
Purity: 100%
Molecular weight: 320
Volatile: 121 mmHg at 25°C - Target gene:
- thymidine kinase (TK)
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001)
- Suitability of cells: L5178Y mouse lymphoma cells are used because they are sensitive indicators of mutagenic activity of a broad range of chemical classes. The TK mutational system is able to detect base pair alterations, frame shift mutations and small deletions and clastogenic effect.
- Methods for maintenance in cell culture if applicable: Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1 x 10^6 cells/ml.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: See below
Horse serum: Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium: For 3 hour exposure: Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure: Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/ml trifluorothymidine (TFT) (Sigma).
Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
Environmental conditions
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 57 - 100%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.0 - 37.9°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes; Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10 medium containing 10^-4 M hypoxanthine (Sigma), 2 x 10^-7M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10^-5M thymidine (Merck) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver microsomal enzymes (S9 homogenate) were prepared from male Sprague Dawley dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
- Test concentrations with justification for top dose:
- Dose Range-finding Test (±S9): 5.4, 17, 52, 164, 512 µg/mL
Mouse Lymphoma Test (±S9): 0.05, 0.17, 0.54, 1.7, 5.4, 17, 52, 164 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Not specified - Untreated negative controls:
- yes
- Remarks:
- Ethanol
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Cell density was preferably kept below 1 x 10^6 cells/mL.
DURATION
- Exposure duration: A] Dose range finding test: 3 or 24 hours; B] Mutagenicity test: +S9: 3 hours; -S9: 3 and 24 hours
- Expression time (cells in growth medium): 2 days post treatment
- Selection time (if incubation with a selection agent): 11-12 days
SELECTION AGENT (mutation assays): trifluorothymidine (TFT)
STAIN (for cytogenetic assays): 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
NUMBER OF REPLICATIONS: Solvent controls and treatment groups five plates with 2000 cells/well and the positive controls ten plates with 1000 cells/well
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFTselection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Rationale for test conditions:
- L5178Y mouse lymphoma cells are used because they are sensitive indicators of mutagenic activity of a broad range of chemical classes. The TK mutational system is able to detect base pair alterations, frame shift mutations and small deletions and clastogenic effect. Cells deficient in thymidine kinase (TK), due to the forward mutation (TK+/- to TK-/-) are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). TK deficient cells cannot incorporate the analogue into its phosphorylated derivative (nucleotide); the nucleotides needed for cellular metabolism are obtained solely from de novo synthesis. In the presence of TK, TFT is converted into nucleotides, which is lethal to the cells. Thus, cells that are able to proliferate in culture medium containing TFT are mutated, either spontaneously or by the action of the test item, to a TK deficient phenotype. Furthermore, by applying the TFT-selection procedure it is possible to discriminate between two different classes of TFT-resistant mutants (small and large colonies). The large colonies are believed to be the result of mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. The small colonies are believed to be the result of chromosomal damage to the TK and adjacent genes.A test article, which induces a positive response in this assay, is presumed to be a potential mammalian cell mutagen.
- Evaluation criteria:
- In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Solubility
The test matrial precipitated in the exposure medium at concentrations of 164 μg/mL and above. The test material was tested beyond the limit of solubility to obtain adequate toxicity data, the concentration used as the highest test item concentration for the dose range finding test was 512 μg/mL.
Dose range finding test
3 hour treatment: Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test material concentration of 512 μg/mL compared to the suspension growth of the solvent control.
24 hour treatment: No toxicity in the relative suspension growth was observed up to test material concentrations of 512 μg/mL compared to the solvent control.
Mutation experiment
Evaluation of toxicity: No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix in both experiments.
Evaluation of the mutagenicity: No significant increase in the mutation frequency at the TK locus was observed after treatment with the test material either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls.
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Except in the second experiment, in which the mutation frequency of one of the solvent control cultures was just above upper control limit. This limit is a 95% control limit and a slightly higher response is within the expected response range.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
The growth rate over the two-day expression period for cultures treated with ethanol was between 19 and 23 (3 hours treatment) and 111 and 120 (24 hours treatment). - Conclusions:
- In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.
In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency.
Based on the results observed, 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane (AsahiklinTM AC-2000) was not considered to be mutagenic in the TK mutation test system. - Executive summary:
In a key guideline (OECD 490) in vitro gene mutation study in mammalian cells, the mutagenic potential of the test material ((1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane (AsahiklinTM AC-2000)) was evaluated using L5178Y mouse lymphoma cells in the absence and presence of metabolic activation (±S9).
Two experiments were carried out. In the first experiment, the test material was tested up to concentrations of 164 µg/mL in the absence and presence of S9-mix with the incubation time being 3 hours. In the second experiment, the test material was tested up to concentrations of 164 µg/mL in the absence of S9-mix with the incubation time being 24 hours.
No toxicity was observed in the absence and presence of metabolic activation. The test material precipitated in the culture medium at 164 µg/mL, the highest concentration tested in the mutagenicity test.
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. Additionally, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
The growth rate over the two-day expression period for cultures treated with ethanol was between 19 and 23 (3 hours treatment) and 111 and 120 (24 hours treatment).
In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.
In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency.
Based on the results observed, 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane (AsahiklinTM AC-2000) was not considered to be mutagenic in the TK mutation test system.
Referenceopen allclose all
Table 1. Results of the Main Test |
||||||
With (+) or Without (-S9) Mix |
Test Substance Dose (µg/plate) |
Number of Revertants per Plate |
||||
Base-pair Substitution Type |
Frameshift Type |
|||||
TA 100 |
TA 1535 |
WP2uvrA |
TA 98 |
TA 1537 |
||
-S9 Mix |
Negative Control |
131 153 (134) 117 |
12 8 (10) 9 |
44 47 (43) 37 |
22 20 (19) 14 |
10 12 (10) 8 |
313 |
174 152 (163) |
17 13 (15) |
61 46 (54) |
26 34 (30) |
15 4 (10) |
|
625 |
119 113 (116) |
9 18 (14) |
52 53 (53) |
17 24 (21) |
22 5 (14) |
|
1250 |
162 158 (160) |
14 15 (15) |
51 43 (47) |
24 28 (26) |
5 11 (8) |
|
2500 |
147 147 (147) |
10 17 (14) |
48 42 (45) |
19 29 (24) |
11 12 (12) |
|
5000 |
147 147 (147) |
11 15 (13) |
41 43 (42) |
26 16 (21) |
8 7 (8) |
|
|
||||||
+S9 Mix |
Negative Control |
148 148 (148) 148 |
21 28 (22) 16 |
51 47 (49) 49 |
34 29 (34) 39 |
24 19 (17) 8 |
313 |
149 168 (159) |
13 18 (16) |
57 52 (55) |
41 37 (39) |
14 22 (18) |
|
625 |
133 143 (138) |
11 14 (13) |
50 61 (56) |
43 48 (46) |
22 13 (18) |
|
1250 |
147 134 (141) |
14 12 (13) |
53 52 (53) |
29 30 (30) |
11 25 (18) |
|
2500 |
112 147 (130) |
12 14 (13) |
55 41 (48) |
38 30 (34) |
17 19 (18) |
|
5000 |
158 138 (148) |
15 22 (19) |
54 56 (55) |
36 35 (36) |
15 20 (18) |
|
|
||||||
Positive Control (-S9 Mix) |
Chemical |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
ICR-191 |
Dose (µg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
1 |
|
Number of Revertants /plate |
424 398 (411) |
295 297 (296) |
444 412 (428) |
395 445 (420) |
1925 1948 (1937) |
|
|
||||||
Positive Control (+S9 Mix) |
Chemical |
2AA |
2AA |
2AA |
2AA |
2AA |
Dose (µg/plate) |
1 |
2 |
10 |
0.5 |
2 |
|
Number of Revertants /plate |
647 632 (640) |
151 130 (141) |
447 450 (449) |
140 149 (145) |
122 123 (123) |
|
1) values in parenthesis show the mean of each plate
2) AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)
3) 2AA: 2-Aminoanthracene
4) ICR-191: 2-Methoxy-6-chloro--9-[3-(2-chloroethyl)-aminopropylamino]acridine.2HCl
5) NaN3: Sodium Azide
Table 1. Results of the Cell Growth Inhibition Test and Cell Division Inhibition Test |
||||||||||||
Direct Method |
24 hours Treatment |
Concentration of the test material (µg/mL) |
0 |
10 |
30** |
50** |
80** |
100** |
300** |
|
|
|
Cell Growth Rate (%)* |
100 |
89.2 |
59.3 |
45.1 |
38.4 |
36.2 |
30.2 |
|
|
|
||
Mitotic Metaphase Index*** |
+++ |
+++ |
++ |
+ |
± |
± |
± |
|
|
|
||
48 hours Treatment |
Concentration of the test material (µg/mL) |
0 |
10 |
30** |
50** |
80** |
100** |
200** |
300** |
|
|
|
Cell Growth Rate (%)* |
100 |
85.6 |
66.7 |
39.1 |
29.9 |
18.8 |
14.5 |
13.0 |
|
|
||
Mitotic Metaphase Index*** |
+++ |
+++ |
+++ |
+++ |
+++ |
++ |
± |
± |
|
|
||
|
||||||||||||
Metabolic Activation Method |
Concentration of the test material (µg/mL) |
0 |
10 |
30 |
50 |
100** |
300** |
500** |
1000** |
3000** |
5000** |
|
Cell Growth Rate (%)* |
100 |
101.4 |
95.9 |
98.2 |
73.6 |
78.6 |
81.8 |
57.7 |
75.5 |
55.5 |
||
Mitotic Metaphase Index*** |
+++ |
+++ |
+++ |
+++ |
+++ |
++ |
+++ |
+++ |
+++ |
++ |
||
* Mean value of 2 flasks
** Precipitation of test material was observed
*** Judgement
+++: Sufficient; ++: Abundant; + a few; ±: Rare; -: None
Table 2. Results of Chromosome Aberration Test (without metabolic activation -S9) |
||||||||||||||
Treatment |
Treatment Time (h) |
Concn µg/mL |
Observed |
Number of Cells* |
Number and Percentage (%) of Cells Showing Structural Chromosome Aberrations* |
|||||||||
No. of Polyploids |
Judgement |
Gap g |
Chromatid Type |
Chromosome Type |
Others |
Total |
Judgement |
|||||||
ctb |
cte |
csb |
cse |
-g |
+g |
|||||||||
Solvent (Acetone) |
24 |
0 |
100 |
0 |
|
0 |
2 |
0 |
0 |
0 |
0 |
2 |
2 |
|
100 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
||||
200 |
1 (0.5) |
0 (0.0) |
2 (1.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
2 (1.0) |
2 (1.0) |
|
||||
48 |
0 |
100 |
0 |
|
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
|
100 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
|
||||
200 |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
|
||||
|
||||||||||||||
Test Material |
24 |
12.5 |
100 |
1 |
-
|
1 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
- |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||
200 |
1 (0.5) |
1 0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
|||||
25** |
100 |
0 |
-
|
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
- |
||
100 |
1 |
0 |
1 |
0 |
0 |
1 |
0 |
2 |
2 |
|||||
200 |
1 (0.5) |
0 (0.0) |
2 (1.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
3 (1.5) |
3 (1.5) |
|||||
50** |
100 |
1 |
- |
1 |
1 |
0 |
0 |
0 |
0 |
1 |
2 |
- |
||
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||
200 |
1 (0.5) |
1 (0.5) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
2 (1.0) |
|||||
48 |
25** |
100 |
0 |
- |
1 |
0 |
1 |
0 |
0 |
0 |
1 |
2 |
- |
|
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||
200 |
0 (0.0) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
2 (1.0) |
|||||
50** |
100 |
1 |
- |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
||
100 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
|||||
200 |
1 (0.5) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
1 (0.5) |
|||||
100** |
100 |
1 |
- |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
1 |
- |
||
100 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||
200 |
2 (1.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
1 (0.5) |
|||||
|
||||||||||||||
Positive Control (MMC) |
24 |
0.05 |
100 |
0 |
- |
4 |
19 |
35 |
1 |
1 |
0 |
50 |
52 |
+++ |
100 |
0 |
9 |
12 |
39 |
0 |
1 |
0 |
46 |
48 |
|||||
200 |
0 (0.0) |
13 (6.5) |
31 (15.5) |
74 (37.0) |
1 (0.5) |
2 (1.0) |
0 (0.0) |
96 (48.0) |
100 (50.0) |
|||||
48 |
0.05 |
100 |
0 |
- |
4 |
14 |
64 |
0 |
2 |
0 |
70 |
71 |
+++ |
|
100 |
0 |
3 |
13 |
55 |
1 |
1 |
0 |
59 |
60 |
|||||
200 |
0 (0.0) |
7 (3.5) |
27 (13.5) |
119 (59.5) |
1 (0.5) |
3 (1.5) |
0 (0.0) |
129 (64.5) |
131 (65.5) |
|||||
* number of cells observed, polyploids and cells showing structural chromosomal aberrations per each plate in line 1 & 2, and the total in line 3, and percentage (%) of cells showing aberrations in parenthesis with every concentration of treatment.
Gap (g): total value of chromatic-type and chromosome type
Sum total (-g): total number and percentage of cells except those which have only gaps. If there were many aberrations in once cell, that cell was calculated as once cell showing aberrations. For e.g. if there are 2 breaks and two exchanges in one cell, that cell was calculated as one cell showing aberrations, once cell showing breaks, and one cell showing exchanges.
ctb: Chromatid break
cte: Chromatid exchange
csb: Chromosome break
cse: Chromosome exchange
Others: Fragmentation etc. (except pulverization)
** Precipitate of the Test material observed
MMC: Mitomycin C
Table 3. Results of Chromosome Aberration Test (with metabolic activation +S9) |
||||||||||||||
Treatment |
S9 mix |
Concn µg/mL |
Observed |
Number of Cells* |
Number and Percentage (%) of Cells Showing Structural Chromosome Aberrations* |
|||||||||
No. of Polyploids |
Judgement |
Gap g |
Chromatid Type |
Chromosome Type |
Others |
Total |
Judgement |
|||||||
ctb |
cte |
csb |
cse |
-g |
+g |
|||||||||
Solvent (Acetone) |
- |
0 |
100 |
0 |
|
0 |
0 |
1 |
0 |
0 |
0 |
1 |
1 |
|
100 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
|||||
200 |
0 (0.0) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
2 (1.0) |
|||||
+ |
0 |
100 |
0 |
|
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
|
100 |
1 |
1 |
0 |
1 |
0 |
0 |
0 |
1 |
2 |
|||||
200 |
1 (0.5) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
2 (1.0) |
|||||
|
||||||||||||||
Test Material |
-
|
1250** |
100 |
0 |
-
|
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
100 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
1 |
|||||
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
1 (0.5) |
|||||
2500** |
100 |
0 |
-
|
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
||
100 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
|||||
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
1 (0.5) |
|||||
5000** |
100 |
0 |
- |
0 |
0 |
1 |
0 |
1 |
0 |
2 |
2 |
- |
||
100 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
1 |
|||||
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
2 (1.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
3 (1.5) |
3 (1.5) |
|||||
+ |
1250** |
100 |
0 |
- |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
1 |
- |
|
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
1 (0.5) |
|||||
2500** |
100 |
0 |
- |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
- |
||
100 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
1 |
|||||
200 |
0 (0.0) |
0 (0.0) |
1 (0.5) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
2 |
2 (1.0) |
|||||
5000** |
100 |
0 |
- |
1 |
0 |
0 |
0 |
1 |
0 |
1 |
2 |
- |
||
100 |
0 |
0 |
1 |
0 |
0 |
1 |
0 |
2 |
2 |
|||||
200 |
0 (0.0) |
1 (0.5) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
2 (1.0) |
0 (0.0) |
3 (1.5) |
4 |
|||||
|
||||||||||||||
Positive Control (CPA) |
- |
10 |
100 |
0 |
|
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|||||
+ |
10 |
100 |
0 |
|
3 |
16 |
75 |
0 |
0 |
0 |
81 |
82 |
+++ |
|
100 |
0 |
5 |
19 |
73 |
1 |
1 |
0 |
78 |
79 |
|||||
200 |
0 (0.0) |
8 (4.0) |
35 (17.5) |
148 (74.0) |
1 (0.5) |
1 (0.5) |
0 (0.0) |
159 (79.5) |
161 (80.5) |
|||||
Amount of S9 (1%), Treatment time of the test material (6h), Recovery time of cells after treatment of the test material (18h)
* number of cells observed, polyploids and cells showing structural chromosomal aberrations per each plate in line 1 & 2, and the total in line 3, and percentage (%) of cells showing aberrations in parenthesis with every concentration of treatment.
Gap (g): total value of chromatic-type and chromosome type
Sum total (-g): total number and percentage of cells except those which have only gaps. If there were many aberrations in once cell, that cell was calculated as once cell showing aberrations. For e.g. if there are 2 breaks and two exchanges in one cell, that cell was calculated as one cell showing aberrations, once cell showing breaks, and one cell showing exchanges.
ctb: Chromatid break
cte: Chromatid exchange
csb: Chromosome break
cse: Chromosome exchange
Others: Fragmentation etc. (except pulverization)
** Precipitate of the Test material observed
CPA: Cyclophosphamide monohydrate
Table 1. Experiment 1: Cytotoxic and mutagenic response of AsahiklinTMAC-2000 in the mouse lymphoma L5178Y test system |
|||||||
Dose (µg/mL) |
RSG (%) |
CEday2 (%) |
RCE (%) |
RTG (%) |
Mutation frequency per 106 survivors |
||
Total |
Small |
Large |
|||||
Without Metabolic Activation (3 hours treatment) |
|||||||
SC1 |
100 |
101 |
100 |
100 |
72 |
27 |
43 |
SC2 |
97 |
102 |
44 |
53 |
|||
0.05 |
89 |
102 |
104 |
92 |
50 |
16 |
34 |
0.17 |
88 |
116 |
118 |
103 |
65 |
8 |
55 |
0.54 |
109 |
98 |
99 |
108 |
68 |
33 |
33 |
1.7 |
96 |
104 |
105 |
101 |
72 |
34 |
35 |
5.4 |
91 |
89 |
90 |
81 |
67 |
33 |
33 |
17 |
106 |
91 |
92 |
98 |
89 |
39 |
46 |
52 |
93 |
68 |
69 |
64 |
59 |
19 |
39 |
1641 |
99 |
93 |
94 |
93 |
96 |
38 |
53 |
MMS |
67 |
56 |
56 |
38 |
841 |
475 |
274 |
With Metabolic Activation (3 hours treatment) |
|||||||
SC1 |
100 |
75 |
100 |
100 |
112 |
52 |
55 |
SC2 |
95 |
114 |
53 |
55 |
|||
0.05 |
110 |
108 |
127 |
140 |
82 |
40 |
38 |
0.17 |
102 |
88 |
103 |
105 |
101 |
39 |
58 |
0.54 |
112 |
93 |
109 |
122 |
76 |
37 |
36 |
1.7 |
115 |
70 |
83 |
95 |
112 |
52 |
55 |
5.4 |
105 |
84 |
99 |
104 |
84 |
33 |
48 |
17 |
106 |
111 |
131 |
139 |
64 |
26 |
36 |
52 |
112 |
113 |
133 |
149 |
62 |
21 |
39 |
1641 |
109 |
108 |
127 |
139 |
76 |
21 |
53 |
CP |
94 |
65 |
77 |
72 |
794 |
393 |
301 |
Note: all calculations were made without rounding off
RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growth;
SC = Solvent control = Ethanol; MMS = Methylmethanesulfonate; CP = Cyclophosphamide
1= test material precipitated in the exposure medium
Table 2. Experiment 2: Cytotoxic and mutagenic response of AsahiklinTM AC-2000 in the mouse lymphoma L5178Y test system |
|||||||
Dose (µg/mL) |
RSG (%) |
RCE (%) |
RCE |
RTG (%) |
Mutation frequency per 106 survivors |
||
Total |
Small |
Large |
|||||
Without Metabolic Activation (24 hours treatment) |
|||||||
SC1 |
100 |
79 |
100 |
100 |
125 |
112 |
11 |
SC2 |
69 |
121 |
101 |
17 |
|||
0.05 |
102 |
81 |
110 |
113 |
143 |
126 |
14 |
0.17 |
96 |
81 |
110 |
105 |
148 |
123 |
21 |
0.54 |
101 |
93 |
125 |
126 |
100 |
85 |
13 |
1.7 |
109 |
89 |
120 |
131 |
115 |
94 |
18 |
5.4 |
108 |
86 |
116 |
126 |
122 |
93 |
25 |
17 |
99 |
91 |
123 |
122 |
101 |
82 |
16 |
52 |
92 |
101 |
136 |
125 |
109 |
85 |
20 |
1641 |
97 |
83 |
111 |
108 |
133 |
109 |
21 |
MMS |
81 |
72 |
98 |
79 |
744 |
566 |
115 |
Note: all calculations w ere made without rounding off
RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growth;
SC = Solvent control = ethanol; MMS = Methylmethanesulfonate
1 = test material precipitated in the exposure medium
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro Gene Mutation study in Bacteria:
In a key bacterial reverse mutation assay (Hita Research Laboratories, 1994b), the in vitro mutagenic potential of the test material, 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane, was evaluated in Salmonella typhimurium strains TA98, TA100, T1535, and TA 1537 and Escherischia coli strain WP2uvrA using pre-incubation method in the absence and presence of a metabolic activation system (±S9).
Based on a dose range-finding study, the concentrations of the test material used in the main study were 0, 313, 625, 1250, 2500, or 5000 µg/plate.
It was observed that the number of revertant colonies in all strains was less than twice that of the negative control at all dose levels tested in the absence and presence of metabolic activation (±S9). The positive controls exhibited a significant increase in the number of revertant colonies and the number of revertants for positive as well as negative controls was within the historical range.
Based on the results observed in the study, 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane was considered to be negative in this bacterial reverse mutation assay.
In Vitro Cytogenicity / Chromosome Aberration study in Mammalian Cells:
In a key in vitro cytogenicity study (Hita Research Laboratories, 1994c), the mutagenic potential of the test material (1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane) was evaluated using Chinese Hamster Lung Fibroblast (CHL) cells in the absence and presence of metabolic activation. Mitomycin C (MMC) and Cyclophosphamide monohydrate (CPA) were employed as positive controls in the absence of metabolic activation (Direct Method) and in the presence of metabolic activation (Metabolic Activation Method), respectively.
The chromosome aberration tests were carried out at doses of 12.5, 25, and 50 µg/mL (Direct Method – 24 hours treatment); 25, 50, and 100 µg/mL (Direct Method – 48 hours treatment); and 1250, 2500, and 5000 µg/mL (Metabolic Activation Method). MMC (0.05 µg/mL) was used as the positive control for 24 hours and 48 hours treatment by the Direct Method, while CPA (10 µg/mL) was used as the positive control for the Metabolic Activation Method.
The test material induced no structural aberrations or numerical aberrations within the dose range which included doses showing cytotoxicity in the 24 and 48 hours treatments by the direct method and at less than 5000 µg/mL in the metabolic activation method. On the other hand, MMC and CPA induced evident chromosomal aberrations.
Based on the results observed in the study, it was concluded that the test material did not induce chromosome aberrations in the absence or presence of metabolic activation.
In vitro Gene Mutation study in Mammalian Cells:
In a key Guideline (OECD 490) in vitro gene mutation study in mammalian cells (Charles River Laboratories Den Bosch BV., 2016b), the mutagenic potential of the test material ((1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane) was evaluated using L5178Y mouse lymphoma cells in the absence and presence of metabolic activation (±S9).
Two experiments were carried out. In the first experiment, the test material was tested up to concentrations of 164 µg/mL in the absence and presence of S9-mix with the incubation time being 3 hours. In the second experiment, the test material was tested up to concentrations of 164 µg/mL in the absence of S9-mix with the incubation time being 24 hours.
No toxicity was observed in the absence and presence of metabolic activation. The test material precipitated in the culture medium at 164 µg/mL, the highest concentration tested in the mutagenicity test.
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. Additionally, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
The growth rate over the two-day expression period for cultures treated with ethanol was between 19 and 23 (3 hours treatment) and 111 and 120 (24 hours treatment).
In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.
In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency.
Based on the results observed, 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane was not considered to be mutagenic in the TK mutation test system.
Justification for classification or non-classification
Not classified for mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 or UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
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