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EC number: 919-489-5 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
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- Nanomaterial pour density
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- Nanomaterial catalytic activity
- Endpoint summary
- Stability
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015-05-27 to 2015-07-28
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 015
- Report date:
- 2015
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: in vitro mammalian cell gene mutation
Test material
- Reference substance name:
- Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile
- EC Number:
- 919-489-5
- Molecular formula:
- C13H23N
- IUPAC Name:
- Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile
1
Method
- Target gene:
- HPRT locus
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- mammalian (rat) microsomal fraction S9 mix, Phenobarbital/β-naphthoflavone induced
- Test concentrations with justification for top dose:
- Experiment 1:
Exposure period 4 hours, with S9-mix: 1.9, 3.8, 7.5, 15, 30, 60, 120, 240 µg/mL (phase separation was observed from 30 µg/mL onwards)
Exposure period 4 hours, without S9-mix: 0.94, 1.9, 3.8, 7.5, 15, 30, 45, 60 µg/mL
Experiment 2:
Exposure period 4 hours, with S9-mix: 0.94, 1.9, 3.8, 7.5, 15, 30, 45, 60 µg/mL (phase separation was observed from 45 µg/mL onwards)
Exposure period 24 hours, without S9-mix: 0.94, 1.9, 3.8, 7.5, 15, 30, 45, 60 µg/mL - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- 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
DURATION
- Exposure duration: 4 hours or 24 hours
- Expression time (cells in growth medium): 7 days
- Selection time: 8 days
SELECTION AGENT: 6-TG
STAIN: 10 % methylene blue in 0.01 % KOH solution
NUMBER OF REPLICATIONS: 5 flasks were seeded with the cells after the expression time, two additional flasks were seeded in non-selective medium for determination of the viability
NUMBER OF CELLS EVALUATED: 500 cells were seeded per flask
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration. - Statistics:
- A linear regression was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment I: at 3.8 µg/mL and above without S9-mix, Experiment II: at 15 µg/mL and above without S9-mix, at 45.0 μg/mL with metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
Phase separation was noted at the highest evaluated concentration of experiment I and II in the presence of metabolic activation.
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both cultures occurred in the first experiment at 3.8 μg/mL and above in the absence of metabolic activation. In the second experiment exceedingly severe cytotoxic effects were noted at 15.0 μg/mL and above without metabolic activation, and at 45.0 μg/mL with metabolic activation.
COMPARISON WITH HISTORICAL CONTROL DATA:
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The mutant frequency did not exceed the historical range of solvent controls. Only in the second culture of experiment I without metabolic activation the range of the historical solvent control data (1.6 - 45.7 mutant colonies/10^6 cells) was slightly exceeded at 7.5 μg/mL (49.6 mutant colonies per 10^6 cells).
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered without metabolic activation. In the presence of metabolic activation an extremely steep cytotoxic gradient at the onset of phase separation did not permit any coverage of this range.
Any other information on results incl. tables
|
concentration [µg/mL] |
S9 mix |
relative cloning efficiency I [%] |
relative cell density [%] |
relative cloning efficiency II [%] |
mutant colonies/10exp6 cells |
induction factor |
relative cloning efficiency I [%] |
relative cell density [%] |
relative cloning efficiency II [%] |
mutant colonies/10exp6 cells |
induction factor |
Experiment 1 (4 h treatment) |
|
|
culture I |
culture II |
||||||||
Solvent control (DMSO) |
|
- |
100 |
100 |
100 |
22.9 |
1 |
100 |
100 |
100 |
22.1 |
1 |
Positive control (EMS) |
150 |
- |
80.8 |
125 |
93.3 |
205.6 |
9 |
75.7 |
127.4 |
10..1 |
231.6 |
10.5 |
Test item |
0.94 |
- |
93.5 |
112.6 |
56.1 |
24.7 |
1.1 |
82.3 |
121.8 |
66.1 |
16 |
0.7 |
1.9 |
- |
94.4 |
98.2 |
54.6 |
32.6 |
1.4 |
95.4 |
107.4 |
61.8 |
19 |
0.9 |
|
3.8 |
- |
84.1 |
18.9 |
50.7 |
8.8 |
0.4 |
80.7 |
23.7 |
27.2 |
18.6 |
0.8 |
|
7.5 |
- |
28.2 |
15.3 |
42.5 |
9.8 |
0.4 |
25.8 |
17.6 |
31.1 |
49.6 |
2.2 |
|
15 |
- |
6.8 |
8.3 |
culture was not continued 1) |
2.22 |
7.7 |
culture was not continued 1) |
|||||
30 |
- |
0 |
8.3 |
culture was not continued 1) |
0 |
0 |
culture was not continued 1) |
|||||
45 |
- |
0 |
7.4 |
culture was not continued 1) |
0 |
0 |
culture was not continued 1) |
|||||
60 |
- |
0 |
7.2 |
culture was not continued 1) |
0 |
0 |
culture was not continued 1) |
|||||
Solvent control (DMSO) |
|
+ |
100 |
100 |
100 |
20.9 |
1 |
100 |
100 |
100 |
7.5 |
1 |
Positive control (DMBA) |
1.1 |
+ |
84.2 |
71.9 |
91.6 |
176.5 |
8.4 |
74.4 |
79.7 |
116.5 |
186.6 |
24.7 |
Test item |
1.9 |
+ |
100.3 |
60.5 |
103.2 |
12.4 |
0.6 |
102.7 |
78.2 |
75.6 |
21.3 |
2.8 |
3.8 |
+ |
95.5 |
63.6 |
99.1 |
19.4 |
0.9 |
96 |
77.9 |
81.3 |
38.3 |
5.1 |
|
7.5 |
+ |
102.2 |
55.3 |
109.7 |
24.1 |
1 |
85.3 |
70.6 |
81.3 |
21.6 |
2.9 |
|
15 |
+ |
101.5 |
72.8 |
103.9 |
14.4 |
0.7 |
84.2 |
81.1 |
63.4 |
30.4 |
4 |
|
30 |
+ |
95.2 |
64 |
96.1 |
13.5 |
0.6 |
82.4 |
73.6 |
82.2 |
22.2 |
2.9 |
|
60 |
+ |
0 |
9 |
culture was not continued 1) |
0 |
0 |
culture was not continued 1) |
|||||
120 |
+ |
0 |
0 |
culture was not continued 1) |
0 |
0 |
culture was not continued 1) |
|||||
240 |
+ |
0 |
0 |
culture was not continued 1) |
0 |
0 |
culture was not continued 1) |
|||||
Experiment 2 (24 h treatment) |
|
|
culture I |
culture II |
||||||||
Solvent control (DMSO) |
|
- |
100 |
100 |
100 |
8.1 |
1 |
100 |
100 |
100 |
20.7 |
1 |
Positive control (EMS) |
150 |
- |
85.5 |
87.1 |
102.9 |
227.2 |
28 |
95.1 |
107.3 |
96.6 |
183.3 |
8.8 |
Test item |
0.94 |
- |
86.7 |
94.7 |
99.6 |
21.5 |
2.6 |
86.7 |
116.2 |
101.7 |
12.3 |
0.6 |
1.9 |
- |
86.2 |
73.2 |
102.5 |
15.5 |
1.9 |
88.3 |
83.2 |
99.7 |
22.1 |
1.1 |
|
3.8 |
- |
81.8 |
75.2 |
98.8 |
9.6 |
1.2 |
81.7 |
96.6 |
99.6 |
22 |
1.1 |
|
7.5 |
- |
86.1 |
culture was not continued 2) |
85.2 |
culture was not continued 2) |
|||||||
15 |
- |
7.1 |
109.7 |
99.5 |
17.1 |
2.1 |
4.3 |
97.1 |
102.6 |
19.6 |
0.9 |
|
30 |
- |
0 |
9.7 |
101.1 |
15.8 |
1.9 |
0 |
14.3 |
102.9 |
9.3 |
0.4 |
|
45 |
- |
0 |
0 |
culture was not continued 1) |
0 |
0 |
culture was not continued 1) |
|||||
60 |
- |
0 |
0 |
culture was not continued 1) |
0 |
0 |
culture was not continued 1) |
|||||
Experiment 2 (4 h treatment) |
|
|
culture I |
culture II |
||||||||
Solvent control (DMSO) |
|
+ |
100 |
100 |
100 |
17 |
1 |
100 |
100 |
100 |
19.7 |
1 |
Positive control (DMBA) |
2.2 |
+ |
79.1 |
98.4 |
100 |
329.3 |
19.4 |
71.7 |
104.3 |
102.3 |
311.6 |
15.8 |
Test item |
0.94 |
+ |
87.4 |
culture was not continued 3) |
93.5 |
culture was not continued 3) |
||||||
1.9 |
+ |
84.7 |
culture was not continued 3) |
85.6 |
culture was not continued 3) |
|||||||
3.8 |
+ |
79.9 |
102.8 |
100 |
12.8 |
0.8 |
88.8 |
105.1 |
102.8 |
20.5 |
1 |
|
7.5 |
+ |
91.3 |
103.1 |
102.5 |
12.8 |
0.7 |
80.1 |
109.2 |
101.7 |
32.4 |
1.6 |
|
15 |
+ |
86.2 |
103.6 |
98.2 |
19.8 |
1.2 |
79.7 |
104.5 |
101.3 |
24.1 |
1.2 |
|
30 |
+ |
67.9 |
119.6 |
99.5 |
21.7 |
1.3 |
72 |
119.4 |
103.3 |
20.6 |
1 |
|
45 |
+ |
0 |
70.6 |
99.1 |
38.5 |
2.3 |
0 |
69.5 |
103.6 |
22.4 |
1.1 |
|
60 |
+ |
0 |
1.2 |
culture was not continued 1) |
0 |
1.3 |
culture was not continued 1) |
1) culture was not continued due to exceedingly severe cytotoxic effects
2) culture was not continued due to a technical error (no cell growth at all in both cultures)
3) culture was not continued since a minimum of only four analysable concentrations is required
Applicant's summary and conclusion
- Conclusions:
- The test item did not induce gene mutations at the HPRT locus in V79 cells.
- Executive summary:
The study according to OECD 476 was conducted to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The maximum test item concentration of the pre-experiment (1960 μg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. The tested concentrations in experiment 1 for an exposure period 4 hours, with S9-mix were 1.9, 3.8, 7.5, 15, 30, 60, 120, 240 µg/mL (phase separation was observed from 30 µg/mL onwards) and for an exposure period 4 hours, without S9-mixwere 0.94, 1.9, 3.8, 7.5, 15, 30, 45, 60 µg/mL. In experiment 2 the test concentrations with an exposure period of 4 hours were with S9-mix 0.94, 1.9, 3.8, 7.5, 15, 30, 45, 60 µg/mL (phase separation was observed from 45 µg/mL onwards) and with an exposure period of 24 hours without S9-mix 0.94, 1.9, 3.8, 7.5, 15, 30, 45, 60 µg/mL were used. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.
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