Registration Dossier
Registration Dossier
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EC number: 246-791-8 | CAS number: 25291-17-2
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test (OECD 471): negative with S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA with and without metabolic activation
HPRT test (OECD 476): negative in Chinese hamster ovary cells with and without metabolic activation
Chromosome Aberration test (OECD 473): negative in Chinese lung fibroblasts with and without metabolic activation
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 Dec 2006 - 28 Feb 2007
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- only 200 metaphases per dose were scored
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- only 200 metaphases per dose were scored
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- other: Chinese hamster lung fibroblasts (CHL/IU)
- Details on mammalian cell type (if applicable):
- - Cell proliferation: doubling time 15 h
- Type and identity of media: L-glutamine (final concentration: 0.292 g/L) and sodium hydrogen carbonate (final concentration: approx. 1.85 g/L) were added to Eagle's minimum essential medium and basel medium (MEM) was prepared. This medium was then supplemented with 10% heat-inactivated NBCS.
- Properly maintained: yes
- Checked for Mycoplasma contamination: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated i.p. with phenobarbital and 5,6-benzoflavone
- Test concentrations with justification for top dose:
- Pre-experiment:
6 h treatment: 13.5, 27, 54.1, 108, 216*, 433*, 865*, 1730* and 3460* µg/mL without S9 mix and 13.5, 27, 54.1, 108, 216, 433*, 865*, 1730* and 3460* µg/mL with S9 mix
24 h treatment: 13.5, 27, 54.1, 108*, 216*, 433*, 865*, 1730* and 3460* µg/mL without S9 mix
*selected for scoring of chromosome aberrations
Main experiment:
6 h treatment: 108, 216, 433, 865*, 1730* and 3460* µg/mL with and without S9 mix
24 h treatment: 108, 216, 433, 865*, 1730* and 3460* µg/mL without S9 mix
*selected for scoring of chromosome aberrations - Vehicle / solvent:
- - Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: The test substance was not soluble in water or DMSO. The test substance was soluble in acetone at 346 mg/mL and was not indicated any change in colour nor exothermic at room temperature within 2 h after preparation. Therefore, acetone was selected as a solvent. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Remarks:
- mitomycin C (MMC): 0.1 µg/mL (6 h, -S9), 0.05 µg/mL (24 h, -S9); cyclophosphamide (CPA): 6 µg/mL (6 h, +S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 6 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 6 h treatment: 24 h; 24 h treatment: 24 h
SPINDLE INHIBITOR (cytogenetic assays): demecolcine, 50 µL of 10 µg/mL solution
STAIN (for cytogenetic assays): 2 vol% Giemsa solution in 1/15 mol/L PBS (pH 6.8)
NUMBER OF REPLICATIONS: each dose was set in duplicate
NUMBER OF CELLS EVALUATED: 200 metaphases were evaluated per dose
DETERMINATION OF CYTOTOXICITY
- Method: cell growth rate and IC50 value
OTHER EXAMINATIONS:
- Determination of polyploidy: yes - Evaluation criteria:
- The findings were judged to be positive when the frequencies of cells with structural aberrations or numerical aberrations were 10% or more with a dose-related increase, or the frequencies of aberrant cells were 5% or more both in the chromosomal aberration test and the confirmation test. The other cases were judged to be negative.
- Key result
- Species / strain:
- other: Chinese hamster lung fibroblasts (CHL/IU)
- 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 examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at 216 µg/mL and above at the start and end of treatment and at the end of the culture.
- Other confounding effects: Colour change of medium and corrosion of the culture dish were not observed at any doses.
RANGE-FINDING/SCREENING STUDIES: The results of the cell growth inhibition test revealed no cytotoxic properties of the test substance in the short-term treatment with and without metabolic activation and the 24 h treatment, therefore the highest dose for the main study was selected at 3460 µg/mL.
ADDITIONAL INFORMATION ON CYTOTOXICITY: In the short-term treatments with and without metabolic activation and the 24 h treatment, cytotoxic properties of the test substance were not observed. The IC50 value in all experiments was calculated to be > 3460 µg/mL. - Conclusions:
- Interpretation of results:
negative - Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 12 - 31 Jan 2007
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (for S. typhimurium strains)
trp operon (for E. coli strain) - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone
- Test concentrations with justification for top dose:
- Pre-experiment: 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate with and without metabolic activation
Main experiment: 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: The test substance was insoluble at 50 mg/mL in distilled water or DMSO, respectively, and soluble in acetone at 100 mg/mL. The test substance solution of 100 mg/mL prepared with acetone was considered to be stable from the facts that there was no change in colour nor heat generation at room temperature within 2 h after preparation. Therefore, acetone was preferably selected as a solvent. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium azide (NaN3), 2-aminoanthracene (2-AA), 2-(2-furyl]-3-(5-nitro-furyl)acrylamide (AF-2), 2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine (ICR-191)
- Remarks:
- +S9: 2-AA (0.5 µg/plate, TA98; 1 µg/plate, TA100; 2 µg/plate TA1535 and TA1537; 10 µg/plate WP2uvrA); -S9: AF-2 (0.01 µg/plate, TA100 and WP2uvrA; 0.1 µg/plate TA98); NaN3 (0.5 µg/plate, TA1535); ICR-191 (0.5 µg/plate, TA1537)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
NUMBER OF REPLICATIONS: duplicate plates for the test substance and positive control group, triplicate plates for the the negative control group
DETERMINATION OF CYTOTOXICITY
- Method: inhibition of bacterial growth - Evaluation criteria:
- The test substance was judged to be positive when the number of revertant colonies increased to twice or more that in the negative (solvent) control in a concentration-dependent manner and also the reproducibility of the rest results was obtained. In all other cases, it was judged to be negative.
- Key result
- Species / strain:
- other: S. typhimurium TA1535, TA1537, TA98 and TA100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- 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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed at any of the doses tested with and without metabolic activation.
RANGE-FINDING/SCREENING STUDIES: A total of 6 doses consisting of 5000 µg/plate as the highest dose and 5 lower doses diluted with a geometric progression of 4 were employed. The number of revertant colonies in the test substance treatment groups with and without metabolic activation was less than twice that in the solvent control. The bacterial growth inhibition was not observed at any doses in all test strains with and without metabolic activation. Based on the results of the preliminary experiment, the highest dose was selected at 5000 µg/plate. - Conclusions:
- Interpretation of results:
negative - Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 Oct - 01 Dec 2017
- 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)
- Version / remarks:
- adopted Jul 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2008
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- National Institute of Pharmacy and Nutrition
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: American Type Culture Collection (Manassas,Virginia, United States)
- Lot No.: 58244452
- Methods for maintenance in cell culture: For each experiment, one or more vials were thawed rapidly, the cells were diluted in F12-10 medium and incubated at 37oC (± 0.5 C) in a humidified atmosphere (5± 0.3% CO2 in air). When cells were growing well, subcultures were established in an appropriate number of flasks. Trypsin-EDTA (0.25% Trypsin, 1 mM EDTA) solution was used for cell detachment to subculture.
MEDIA USED
- Type and identity of media: All used F12 media were supplemented with L-glutamine (0.01 mL/mL) and antibiotic-antimycotic solution (0.01 mL/mL consisting of penicillin, streptomycin and amphotericin-B). The culture medium was additionally supplemented with 10% (v/v) fetal bovine serum, and the treatment media with 1% (v/v) fetal bovine serum. Hypoxanthine-free Ham’s F-12 medium supplemented with 10% (v/v) fetal bovine serum was used for preparation of the selection culture medium.
- Periodically checked for Mycoplasma contamination: yes, for each batch of frozen stock
- Periodically 'cleansed' against high spontaneous background: yes, prior to use in this test, the culture was cleansed of pre-existing mutant cells by culturing in HAT medium. - Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and β-naphthoflavone.
- Test concentrations with justification for top dose:
- Based on the results of a preliminary toxicity test, the dose levels for the mutagenicity test were selected as follows:
Experiment I
Without S9 mix: 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL (5 h)
With S9 mix: 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL (5 h)
Experiment II
Without S9 mix: 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL (24 h)
With S9 mix: 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL (5 h) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone for the test item and DMSO for the positive controls
- Justification for choice of solvent/vehicle: Based on the results of a short solubility test, Distilled water and Dimethyl sulfoxide (DMSO) were not suitable solvents for the test, however the test item was soluble at 400 mg/mL concentrations in Acetone which was suitable for the test. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 2 x 10E06 cells/100 mm cell culture dish
DURATION
- Exposure duration: 5 and 24 h, respectively
- Expression time: 7 days
- Selection time: 7 days
- Fixation time: 5 min
SELECTION AGENT: 6-thioguanine (10 µg/mL)
NUMBER OF REPLICATIONS: Duplicate cultures were used for each treatment.
METHODS OF STAINING TECHNIQUE USED: After fixation, colonies were stained using 10%Giemsa solution for 30 minutes, dried and manually counted.
DETERMINATION OF CYTOTOXICITY
- Method: Relative survival - Evaluation criteria:
- The test item was considered to be mutagenic in this assay if the following criteria are met:
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship.
Results which only partially met the criteria were dealt with on a case-by-case basis (historical control data of untreated control samples was taken into consideration if necessary). Similarly, positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological significance. In cases with survival lower than 10%, extreme caution is taken in the interpretation.
According to the relevant OECD guideline, the biological relevance of the results was considered first, statistical significance was not the only determination factor for a positive response. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There were no large changes in pH after treatment in any cases.
- Effects of osmolality: There were no large changes in osmolality after treatment in any cases.
- Precipitation: Precipitation of the test substance was observed at 250 µg/mL and higher for the 5-h treatment in the presence of metabolic activation and at 500 µg/mL for the 5-h and 24-h treatment in the absence of metabolic activation. The precipitation did not interfere with the reading of the results.
RANGE-FINDING/SCREENING STUDIES: In the preliminary experiment, a 5-hour treatment in the presence and absence of S9-mix and a 24-hour treatment in the absence of S9-mix were performed with a range of test concentrations to determine toxicity immediately after the treatments. The highest test concentration in the preliminary test was 2000 μg/mL (the recommended maximum concentration). Based on the results of the preliminary toxicity test, cytotoxicicity was not observed up to a concentration of 2000 µg/mL with and without metabolic activation for the 5-h and the 24-h treatment period, respectively.
HISTORICAL CONTROL DATA
- Positive historical control data: The positive controls induced a clear increase in mutant frequency and were within the range of the historical control data, and were therefore considered as valid.
- Negative (solvent/vehicle) historical control data: The mutant frequency in the negative (vehicle/solvent) control cultures was in accordance with the historical control data, or slightly below the historical control values for the untreated control, which was not considered to affect the validity of the test, and were therefore considered as valid. - Conclusions:
- Interpretation of results:
negative
Referenceopen allclose all
Table 1: Results of the main experiment.
Test item |
Concentration in µg/mL |
Cell growth rate (%) |
Frequency of cells with aberrations (%)* |
|
Structural aberration |
Numerical aberration |
|||
Exposure period 6 h, preparation interval 24 h, without S9 mix |
||||
Acetone |
0 |
100 |
0.5 |
0.0 |
MMC |
0.1 |
ND |
64.5 |
0.0 |
Test substance |
865P |
89.7 |
3.5 |
2.5 |
1730P |
94.0 |
0.0 |
0.5 |
|
3460P |
90.4 |
1.5 |
1.0 |
|
Exposure period 6 h, preparation interval 24 h, with S9 mix |
||||
Acetone |
0 |
100 |
0.0 |
0.5 |
CPA |
6 |
ND |
41.0 |
0.5 |
Test substance |
865P |
74.9 |
2.0 |
1.0 |
1730P |
90.1 |
1.5 |
0.5 |
|
3460P |
85.3 |
1.0 |
0.5 |
|
Exposure period 24 h, preparation interval 24 h, without S9 mix** |
||||
Acetone |
0 |
100 |
2.0 |
3.0 |
MMC |
0.05 |
ND |
76.0 |
0.5 |
Test substance |
865P |
85.9 |
1.0 |
0.5 |
1730P |
87.5 |
2.5 |
1.0 |
|
3460P |
91.0 |
2.5 |
2.0 |
|
*: the frequency of cells with chromosomal aberrations was calculated by observing 200 metaphases per dose
**: the medium was exchanged before the demecolcine solution was added 2 h prior to cell harvest to remove the precipitation of the test substance
CPA: cyclophosphamide
MMC: mitomycin C
ND: not detected
P: precipitation of the test substance occurred
Table 2: Results of cell growth inhibition
| Frequency of cells with aberrations (%) * | ||||||
| Substance | Concentration in µg/mL | Treatment - recovery time (h) | S9 mix | Cell Growth rate (%) | Strucutral aberration | Numerical aberration |
| Acetone | 0 | 6-18 | - | 100 | 0.0 | 0.0 |
| Test item | 13.5 | 6-18 | - | 98.8 | n.o. | n.o. |
| 27.0 | 6-18 | - | 95.6 | n.o. | n.o. | |
| 54.1 | 6-18 | - | 91.4 | n.o. | n.o. | |
| 108 | 6-18 | - | 88.1 | n.o. | n.o. | |
| 216 | 6-18 | - | 78.9 | 0.0 | 0.0 | |
| 433 | 6-18 | - | 75.4 | 2.0 | 2.0 | |
| 865 | 6-18 | - | 68.0 | 0.0 | 0.0 | |
| 1730 | 6-18 | - | 75.6 | 4.0 | 2.0 | |
| 3460 | 6-18 | - | 84.1 | 2.0 | 2.0 | |
| Acetone | 0 | 6-18 | + | 100 | 0.0 | 0.0 |
| Test item | 13.5 | 6-18 | + | 91.1 | n.o. | n.o. |
| 27.0 | 6-18 | + | 89.7 | n.o. | n.o. | |
| 54.1 | 6-18 | + | 99.6 | n.o. | n.o. | |
| 108 | 6-18 | + | 93.5 | n.o. | n.o. | |
| 216 | 6-18 | + | 88.9 | n.o. | n.o. | |
| 433 | 6-18 | + | 80.7 | 0.0 | 0.0 | |
| 865 | 6-18 | + | 84.0 | 4.0 | 2.0 | |
| 1730 | 6-18 | + | 87.5 | 0.0 | 2.0 | |
| 3460 | 6-18 | + | 74.7 | 0.0 | 0.0 | |
| Acetone | 0 | 24-0 | - | 100 | 0.0 | 0.0 |
| Test item | 13.5 | 24-0 | - | 95.1 | n.o. | n.o. |
| 27.0 | 24-0 | - | 92.0 | n.o. | n.o. | |
| 54.1 | 24-0 | - | 92.1 | n.o. | n.o. | |
| 108 | 24-0 | - | 84.9 | 0.0 | 0.0 | |
| 216 | 24-0 | - | 74.2 | 0.0 | 0.0 | |
| 433 | 24-0 | - | 82.1 | 0.0 | 0.0 | |
| 865 | 24-0 | - | 84.0 | few meta | ||
| 1730 | 24-0 | - | 71.0 | few meta | ||
| 3460 | 24-0 | - | 78.6 | few meta | ||
n.o.: not observed
few meta: the frequency of metaphases was extremely few
* The frequency of cells with chromosomal aberrations was calculated by observing 50 metaphases per dose. The highest dose was set at 3460 µg/mL equivalent to 10 mmol/L, as the maximum dose in case of no cytotoxicity on the guidelines, the dose levels based on a geometric progression of 2 were selected.
Table 1. Test results of the range-finding study (preincubation).
With or without S9-Mix |
Test substance concentration [μg/plate] |
Mean number of revertant colonies per plate (average of 2 plates) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
WP2 uvrA |
TA98 |
TA1537 |
||
– |
0 (acetone) |
129 |
12 |
42 |
22 |
16 |
– |
4.88 |
125 |
14 |
38 |
22 |
19 |
– |
19.5 |
112 |
13 |
39 |
24 |
18 |
– |
78.1 |
109 |
11 |
38 |
22 |
19 |
– |
313 |
113 |
16 |
40 |
28 |
21 |
– |
1250 |
101 |
11 |
45 |
19 |
22 |
– |
5000 |
102 |
7 |
36 |
21 |
17 |
Positive controls, –S9 |
Name |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
ICR-191 |
Concentrations [μg/plate] |
0.01 |
0.5 |
0.01 |
0.1 |
0.5 |
|
Mean No. of colonies/plate (average of 2 plates) |
690 |
454 |
407 |
593 |
1265 |
|
+ |
0 (acetone) |
136 |
11 |
41 |
34 |
41 |
+ |
4.88 |
114 |
13 |
49 |
39 |
32 |
+ |
19.5 |
124 |
12 |
47 |
37 |
36 |
+ |
78.1 |
140 |
13 |
46 |
38 |
42 |
+ |
313 |
132 |
10 |
44 |
36 |
41 |
+ |
1250 |
124 |
11 |
44 |
32 |
35 |
+ |
5000 |
116 |
11 |
51 |
35 |
36 |
Positive controls, +S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations [μg/plate] |
1 |
2 |
10 |
0.5 |
2 |
|
Mean No. of colonies/plate (average of 2 plates) |
1250 |
252 |
450 |
427 |
283 |
|
NaN3: sodium azide
2-AA: 2-aminoanthracene
AF-2: 2-(2-furyl]-3-(5-nitro-furyl)acrylamide
ICR-191: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine
Table 2. Test results of the main experiment (preincubation).
With or without S9-Mix |
Test substance concentration [μg/plate] |
Mean number of revertant colonies per plate (average of 2 plates) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
WP2 uvrA |
TA98 |
TA1537 |
||
– |
0 (acetone) |
140 |
15 |
25 |
27 |
9 |
– |
313 |
146 |
10 |
33 |
27 |
11 |
– |
625 |
135 |
10 |
32 |
27 |
6 |
– |
1250 |
148 |
7 |
20 |
32 |
5 |
– |
2500 |
128 |
14 |
26 |
31 |
7 |
– |
5000 |
139 |
13 |
31 |
33 |
9 |
Positive controls, –S9 |
Name |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
ICR-191 |
Concentrations [μg/plate] |
0.01 |
0.5 |
0.01 |
0.1 |
0.5 |
|
Mean No. of colonies/plate (average of 2 plates) |
792 |
459 |
387 |
534 |
2099 |
|
+ |
0 (acetone) |
168 |
15 |
25 |
41 |
17 |
+ |
313 |
181 |
13 |
28 |
40 |
24 |
+ |
625 |
170 |
6 |
35 |
36 |
19 |
+ |
1250 |
167 |
7 |
32 |
40 |
20 |
+ |
2500 |
173 |
13 |
25 |
39 |
15 |
+ |
5000 |
158 |
8 |
23 |
41 |
21 |
Positive controls, +S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations [μg/plate] |
1 |
2 |
10 |
0.5 |
2 |
|
Mean No. of colonies/plate (average of 2 plates) |
1185 |
241 |
305 |
394 |
277 |
|
NaN3: sodium azide
2-AA: 2-aminoanthracene
AF-2: 2-(2-furyl]-3-(5-nitro-furyl)acrylamide
ICR-191: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine
Table 2: Summary of Results (Experiment I)
| S9 mix | Treatment period (hours) | Test item or control concentration | Total number of colonies | Cloning Efficiency (CE) | Relative Survival (%) on plates | Total number of colonies | Mutant frequency |
| + | 5 | 2000 µg/mL | 809 | 0.674 | 76 | 37 | 9.7 |
| 1000 µg/mL | 840 | 0.700 | 79 | 26 | 6.9 | ||
| 500 µg/mL | 828 | 0.690 | 78 | 18 | 5.1 | ||
| 250 µg/mL | 871 | 0.726 | 82 | 19 | 5.0 | ||
| 125 µg/mL | 892 | 0.743 | 84 | 15 | 4.1 | ||
| 62.5 µg/mL | 864 | 0.720 | 81 | 20 | 5.3 | ||
| 31.25 µg/mL | 884 | 0.737 | 83 | 16 | 4.4 | ||
| Negative control | 1061 | 0.884 | 100 | 19 | 4.6 | ||
| Negative control for DMBA (DMSO) | 929 | 0.774 | 88 | 21 | 5.6 | ||
| Untreated control | 1087 | 0.906 | 102 | 20 | 5.0 | ||
| Positive control (DMBA) | 41 | 0.034 | 4 | 1332 | 358.4** | ||
| - | 5 | 2000 µg/mL | 1008 | 0.840 | 92 | 19 | 4.4 |
| 1000 µg/mL | 1081 | 0.901 | 99 | 22 | 6.2 | ||
| 500 µg/mL | 1001 | 0.834 | 91 | 25 | 6.6 | ||
| 250 µg/mL | 1019 | 0.849 | 93 | 17 | 4.4 | ||
| 125 µg/mL | 1064 | 0.887 | 97 | 17 | 4.5 | ||
| 62.5 µg/mL | 1100 | 0.917 | 100 | 13 | 3.2 | ||
| 31.25 µg/mL | 1040 | 0.867 | 95 | 26 | 5.5 | ||
| Negative control (0.5% Acetone) | 1096 | 0.913 | 100 | 19 | 4.6 | ||
| Negative control for EMS (DMSO) | 1112 | 0.927 | 101 | 22 | 5.6 | ||
| Untreated control | 1052 | 0.877 | 96 | 22 | 5.7 | ||
| Positive control (EMS) | 821 | 0.684 | 75 | 1041 | 303.7** |
DMBA= 7,12-Dimethylbenz[a]anthracene, 15µg/mL
EMS = Ethyl methanesulfonate, 0.4 µL/mL
** = Statistically significant increase (at p< 0.01) compared to the relevant vehicle control
Table 3: Summary of results (Experiment II)
| S9 mix | Treatment period (hours) | Test item or control concentration | Total number of colonies | Cloning Efficiency (CE) | Relative Survival (%) on plates | Total number of colonies | Mutant frequency |
| + | 5 | 2000 µg/mL | 939 | 0.783 | 88 | 14 | 4.5 |
| 1000 µg/mL | 957 | 0.798 | 90 | 15 | 4.3 | ||
| 500 µg/mL | 980 | 0.817 | 92 | 16 | 4.1 | ||
| 250 µg/mL | 1046 | 0.872 | 98 | 14 | 4.0 | ||
| 125 µg/mL | 1001 | 0.834 | 94 | 17 | 5.3 | ||
| 62.5 µg/mL | 998 | 0.832 | 94 | 20 | 5.5 | ||
| 31.25 µg/mL | 1022 | 0.852 | 96 | 21 | 5.7 | ||
| Negative control | 1067 | 0.889 | 100 | 30 | 8.3 | ||
| Negative control for DMBA (DMSO) | 1058 | 0.882 | 99 | 17 | 5.1 | ||
| Untreated control | 1045 | 0.871 | 98 | 17 | 4.6 | ||
| Positive control (DMBA) | 31 | 0.026 | 3 | 1378 | 362.0** | ||
| - | 24 | 2000 µg/mL | 1060 | 0.883 | 99 | 16 | 4.6 |
| 1000 µg/mL | 1106 | 0.992 | 103 | 19 | 4.7 | ||
| 500 µg/mL | 1112 | 0.927 | 103 | 31 | 9.4 | ||
| 250 µg/mL | 1018 | 0.848 | 95 | 28 | 8.6 | ||
| 125 µg/mL | 1033 | 0.861 | 96 | 18 | 4.6 | ||
| 62.5 µg/mL | 1080 | 0.900 | 100 | 16 | 4.7 | ||
| 31.25 µg/mL | 1099 | 0.916 | 102 | 16 | 4.7 | ||
| Negative control (0.5% Acetone) | 1076 | 0.897 | 100 | 29 | 7.2 | ||
| Negative control for EMS (DMSO) | 1074 | 0.895 | 100 | 19 | 5.4 | ||
| Untreated control | 1041 | 0.868 | 97 | 18 | 4.9 | ||
| Positive control (EMS) | 517 | 0.431 | 48 | 1659 | 796.4** |
DMBA= 7,12-Dimethylbenz[a]anthracene, 15µg/mL
EMS = Ethyl methanesulfonate, 0.4 µL/mL
** = Statistically significant increase (at p< 0.01) compared to the relevant vehicle control
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Gene mutation in bacteria
A bacterial gene mutation assay with the registered substance was performed similar to OECD guideline 471 and in compliance with GLP (Key, 2007). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA were exposed to the test substance using the preincubation method. Test substance concentrations of 4.88 to 5000 µg/plate and 313 to 5000 µg/plate were selected for two independent experiments with and without metabolic activation, respectively. No substantial increase in the mean number of revertants per plate was observed in any of the test strains compared to the control, neither in the presence nor absence of metabolic activation. The test substance did not show cytotoxic properties and did not precipitate on the plates up to 5000 µg/plate. All positive and the vehicle control values were found to be within the respective historical control ranges. Under the conditions of this experiment, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-ene did not show mutagenicity in the selected S. typhimurium and E. coli strains in the presence and absence of metabolic activation.
In vitro Cytogenicity in mammalian cells
The clastogenic activity of the registered substance was investigated in an in vitro mammalian chromosome aberration test in Chinese lung fibroblasts performed similar to OECD guideline 473 and GLP (Key, 2007). The test substance was dissolved in acetone and two independent experiments were performed. In the first experiment, the test substance was tested up to 3460 μg/mL for a 6-h exposure time with a 24 -h fixation time in the absence and presence of metabolic activation (S9 mix). In the second experiment, the test substance was tested up to 3460 μg/mL for a 24-h continuous exposure time with a 24-h fixation time in the absence of S9 mix. Precipitation was observed at 216 µg/mL and above at the start and end of treatment and at the end of the culture. In both experiments, cytotoxic properties of the test substance were not observed. The IC50 value in all experiments was calculated to be > 3460 µg/mL. The number of cells with chromosome aberrations found in the solvent control cultures was below 5% and thus considered to be valid. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9 mix) functioned properly. The test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9 mix, in either of the two independently repeated experiments. No effects of the test substance on the number of polyploid cells were observed both in the absence and presence of S9 mix. Therefore it can be concluded that 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-ene does not have the potential to inhibit mitotic processes and to induce numerical chromosome aberrations under the experimental conditions described in this report.
In vitro Gene mutations in mammalian cells
An in vitro mammalian cell gene mutation assay was performed according to OECD guideline 476 and under GLP conditions with the registered substance, in Chinese hamster ovary cells (CHO) (Key, 2017). The cells were treated for 5 h with and without metabolic activation (S9 mix: cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with phenobarbital and β-naphthoflavone), and for 24 h without metabolic activation. Based on the results of a preliminary toxicity test the test substance was tested up to the highest recommended concentration: 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL with and without metabolic activation. Precipitation of the test substance was observed at 250 µg/mL and higher for the 5-h treatment in the presence of metabolic activation and at 500 µg/mL for the 5-h and 24-h treatment in the absence of metabolic activation. The precipitation did not interfere with the reading of the results. 7,12-Dimethylbenz[a]anthracene and ethylmethanesulphonate were used as positive controls with and without S9 mix, respectively. Vehicle controls and a negative control (no treatment) were included. A reduced cloning efficiency was not observed with and without metabolic activation. The positive and negative controls were valid and within the range of the historical control data. No biological significant increase in the mutation frequency at the HPRT locus was observed after treatment either in the absence or in the presence of S9-mix. Therefore, it was concluded that 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-ene was not mutagenic in Chinese hamster ovary cells (CHO) under the experimental conditions described.
Conclusion for genetic toxicity
The available data show that 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-ene is not mutagenic in bacteria and mammalian cells (Chinese hamster ovary cells) in vitro, and not clastogenic in Chinese hamster lung fibroblasts.
Justification for classification or non-classification
The available data on genetic toxicity with 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-ene do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.
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