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EC number: 915-926-9 | 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
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- 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
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- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
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- Endpoint summary
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- Environmental data
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- 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
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- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
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- Genetic toxicity
- Carcinogenicity
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- Specific investigations
- Exposure related observations in humans
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- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012-10-11 to 2013-01-08
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 013
- Report date:
- 2013
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- first experiment 4 hours treatment with and without metabolic activation second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Test material
- Reference substance name:
- N,N-dibutyloleamide and oleic acid, sulphated, sodium salts
- EC Number:
- 915-926-9
- Molecular formula:
- C18H34Na2O6S + C26H52NNaO5S
- IUPAC Name:
- N,N-dibutyloleamide and oleic acid, sulphated, sodium salts
- Test material form:
- liquid
- Details on test material:
- The water content of the actual test item was 57.6% (w/w); the water content of the REACH registration substance was analytically determined to be 3.8% (w/w).
Constituent 1
Method
- Target gene:
- HPRT
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital / Beta-Naphtoflavone induced Rat liver S9
- Test concentrations with justification for top dose:
- Experiment I:
without metabolic activation: 0.7; 1.4; 2.8; 5.5; 11.0; 16.5; 22.0 µg/mL
with metabolic activation: 5.5; 11.0; 22.0; 44.0; 88.0; 132.0; 176.0(p) µg/mL
Experiment II:
without metabolic activation: 5.5; 11.0; 22.0; 44.0; 88.0; 132.0; 176.0(p) µg/mL
with metabolic activation. 5.5; 11.0; 22.0; 44.0; 88.0; 132.0; 176.0(p) µg/mL
p = precipitation
In experiment I the cultures at the two highest concentrations without metabolic activation and the highest concentration with metabolic activation were not continued due to exceedingly severe cytotoxicity. In experiment II the cultures at the highest concentration with and without metabolic activation were not continued for the same reason. The culture at the lowest concentration of experiment I with metabolic activation and of experiment II with and without metabolic activation were not continued as a minimum of only four analysable concentrations is required by the guidelines. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water (10 % v/v)
- Justification for choice of solvent/vehicle: suitability properties
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION:
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days
SELECTION AGENT (mutation assays): 6-thioguanine
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: > 1.5 x 10 e6
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 (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using. The number of mutant colonies obtained for the groups treated with the test item was 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 relevance and statistical significance were 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (measured in two pre-experiments: pH 7.38 / 7.31 in the solvent control versus pH 7.33 at 11800 µg test item/mL and pH 7.25 at 180 µg test item/mL)
- Effects of osmolality: No relevant increase (283 mOsm in the solvent control and 296 mOsm at 11800 µg test item/mL and 286 mOsm measured at 180 µg test item/mL)
- Evaporation from medium: Not examined
- Precipitation: Pre-experiment: The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal of the test item. Following 4 hours treatment precipitation occurred at 180 µg/mL and above without metabolic activation and at 184.4 µg/mL and above with metabolic activation. Following 24 hours treatment without metabolic activation precipitation was observed at the maximum concentration of 11800 µg/mL.
Main experiments: Precipitation at the end of treatment, visible to the unaided eye, was not observed at evaluated concentrations.
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20 % relative survival or cell density at sub-cultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up or beyond their limit of solubility. Precipitation should be evaluated at the end of treatment by the unaided eye.
Relevant cytotoxic effects indicated by a relative suspension growth below 50 % were observed at 22.5 µg/mL and above without metabolic activation and 184.4 µg/mL and above with metabolic activation following 4 hours treatment. After 24 hours treatment without metabolic activation strong toxic effects were observed at 184.4 µg/mL and above.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal of the test item. Following 4 hours treatment precipitation occurred at 180 µg/mL and above without metabolic activation and at 184.4 µg/mL and above with metabolic activation. Following 24 hours treatment without metabolic activation precipitation was observed at the maximum concentration of 11800 µg/mL.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
Based on the results of the pre-experiments, the individual concentrations of the main experiments were selected. A series of concentrations spaced by a factor of 2 was placed into the lower range. Narrower spacing was used at high concentrations to cover the cytotoxic range more closely.
COMPARISON WITH HISTORICAL CONTROL DATA:
The range of the historical solvent control data (3.4 - 36.6 mutant colonies / 106 cells) was exceeded in the second culture of the first experiment with metabolic activation at 11.0 µg/mL (49.3 mutant colonies / 106 cells) and at 132.0 µg/mL (45.7 mutant colonies/106 cells). However, the induction factor did not reach the threshold of three times the corresponding solvent control and statistical analysis showed that there was no dose dependent increase. Therefore, the increases were judged as biologically irrelevant.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effects were noted in the first and the second experiment without metabolic activation up to the maximum analyzable concentration of 11.0 µg/mL and 132.0 µg/mL respectively. Based on the very steep gradient of cytotoxicity exceedingly severe toxic effects occurred at the next higher concentrations of 16.5 µg/mL or 176.0 µg/mL even though the concentration spacing was smaller than factor 2 requested by the current OECD guideline. In both main experiments with metabolic activation, relevant cytotoxic effects occurred at 132.0 µg/mL in one culture (experiment I) or both cultures (experiment II). The later onset of cytotoxicity in the experimental parts with metabolic activation and following 24 hour treatment without metabolic activation indicates possible protein binding of the test item as the protein concentration during treatment is higher based on the S9-mix or serum present during long term treatment.
Any other information on results incl. tables
Table 1: Summary of results
conc. µg/mL |
P |
S9 mix |
relative cloning efficiency I % |
relative cell density % |
relative cloning efficiency II % |
mutant colonies/ 106cells |
induction factor |
relative cloning efficiency I % |
relative cell density % |
relative cloning efficiency II % |
mutant colonies/ 106cells |
induction factor |
|
Column |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
Experiment I / 4 h treatment |
culture I |
culture II |
|||||||||||
Solvent control with water |
- |
100.0 |
100.0 |
100.0 |
19.2 |
1.0 |
100.0 |
100.0 |
100.0 |
17.8 |
1.0 |
||
Positive control (EMS) |
150.0 |
- |
81.7 |
106.4 |
91.2 |
136.4 |
7.1 |
78.1 |
109.7 |
96.1 |
110.9 |
6.2 |
|
Test item |
0.7 |
- |
89.4 |
95.6 |
99.9 |
21.6 |
1.1 |
86.6 |
87.0 |
74.6 |
15.0 |
0.8 |
|
Test item |
1.4 |
- |
89.2 |
105.8 |
100.4 |
29.7 |
1.5 |
92.6 |
126.9 |
120.7 |
14.6 |
0.8 |
|
Test item |
2.8 |
- |
89.9 |
99.9 |
111.8 |
24.8 |
1.3 |
96.2 |
102.3 |
96.5 |
22.4 |
1.3 |
|
Test item |
5.5 |
- |
88.6 |
121.5 |
90.4 |
24.9 |
1.3 |
95.6 |
98.3 |
84.4 |
35.5 |
2.0 |
|
Test item |
11.0 |
- |
87.4 |
105.6 |
77.5 |
18.1 |
0.9 |
91.5 |
85.6 |
66.8 |
27.5 |
1.5 |
|
Test item |
16.5 |
- |
0.0 |
culture was not continued# |
0.0 |
culture was not continued# |
|||||||
Test item |
22.0 |
- |
0.0 |
culture was not continued# |
0.0 |
culture was not continued# |
|||||||
Solvent control with water |
+ |
100.0 |
100.0 |
100.0 |
11.2 |
1.0 |
100.0 |
100.0 |
100.0 |
27.2 |
1.0 |
||
Positive control (DMBA) |
1.1 |
+ |
80.9 |
69.8 |
96.4 |
358.8 |
31.9 |
80.4 |
95.6 |
82.2 |
571.3 |
21.0 |
|
Test item |
5.5 |
+ |
102.2 |
culture was not continued## |
107.9 |
culture was not continued## |
|||||||
Test item |
11.0 |
+ |
95.5 |
65.9 |
95.8 |
8.1 |
0.7 |
102.6 |
85.3 |
83.3 |
49.3 |
1.8 |
|
Test item |
22.0 |
+ |
96.8 |
71.5 |
93.5 |
12.1 |
1.1 |
101.8 |
85.5 |
92.7 |
30.1 |
1.1 |
|
Test item |
44.0 |
+ |
93.8 |
84.2 |
100.5 |
15.4 |
1.4 |
109.5 |
92.4 |
89.9 |
28.3 |
1.0 |
|
Test item |
88.0 |
+ |
90.0 |
63.8 |
88.9 |
20.8 |
1.8 |
105.6 |
89.0 |
95.8 |
21.7 |
0.8 |
|
Test item |
132.0 |
+ |
48.3 |
76.4 |
106.6 |
10.8 |
1.0 |
100.1 |
108.2 |
87.0 |
45.7 |
1.7 |
|
Test item |
176.0 |
P |
+ |
0.0 |
culture was not continued# |
0.0 |
culture was not continued# |
||||||
Experiment II / 24 h treatment |
culture I |
culture II |
|||||||||||
Solvent control with water |
- |
100.0 |
100.0 |
100.0 |
22.3 |
1.0 |
100.0 |
100.0 |
100.0 |
16.4 |
1.0 |
||
Positive control (EMS) |
150.0 |
- |
87.5 |
81.9 |
96.0 |
506.2 |
22.7 |
92.5 |
82.4 |
92.6 |
434.4 |
26.4 |
|
Test item |
5.5 |
- |
101.3 |
culture was not continued## |
102.6 |
culture was not continued## |
|||||||
Test item |
11.0 |
- |
100.7 |
95.3 |
94.4 |
12.4 |
0.6 |
99.8 |
110.5 |
94.5 |
19.4 |
1.2 |
|
Test item |
22.0 |
- |
95.8 |
92.7 |
94.0 |
14.3 |
0.6 |
100.6 |
117.0 |
96.2 |
19.2 |
1.2 |
|
Test item |
44.0 |
- |
94.5 |
105.0 |
96.4 |
11.9 |
0.5 |
105.2 |
113.3 |
96.6 |
8.5 |
0.5 |
|
Test item |
88.0 |
- |
96.3 |
111.2 |
97.3 |
14.2 |
0.6 |
101.5 |
101.5 |
96.5 |
20.3 |
1.2 |
|
Test item |
132.0 |
- |
93.7 |
74.8 |
92.4 |
9.9 |
0.4 |
91.2 |
97.5 |
97.3 |
26.2 |
1.6 |
|
Test item |
176.0 |
P |
- |
7.0 |
culture was not continued# |
3.4 |
culture was not continued# |
||||||
Experiment II / 4 h treatment |
|||||||||||||
Solvent control with water |
+ |
100.0 |
100.0 |
100.0 |
14.7 |
1.0 |
100.0 |
100.0 |
100.0 |
17.3 |
1.0 |
||
Positive control (DMBA) |
1.1 |
+ |
92.2 |
77.3 |
98.0 |
314.7 |
21.5 |
87.6 |
112.1 |
97.8 |
331.0 |
19.2 |
|
Test item |
5.5 |
+ |
97.9 |
culture was not continued## |
97.1 |
culture was not continued## |
|||||||
Test item |
11.0 |
+ |
99.3 |
106.7 |
102.4 |
26.8 |
1.8 |
98.0 |
142.9 |
97.6 |
20.4 |
1.2 |
|
Test item |
22.0 |
+ |
94.2 |
88.1 |
97.7 |
17.9 |
1.2 |
94.6 |
126.9 |
100.2 |
17.2 |
1.0 |
|
Test item |
44.0 |
+ |
94.7 |
88.5 |
98.4 |
13.7 |
0.9 |
93.2 |
131.9 |
104.6 |
14.1 |
0.8 |
|
Test item |
88.0 |
+ |
78.4 |
95.6 |
98.9 |
7.9 |
0.5 |
78.1 |
123.7 |
103.0 |
9.7 |
0.6 |
|
Test item |
132.0 |
+ |
37.5 |
81.3 |
102.5 |
17.8 |
1.2 |
39.9 |
133.1 |
103.3 |
16.4 |
1.0 |
|
Test item |
176.0 |
P |
+ |
0.0 |
culture was not continued# |
0.0 |
culture was not continued# |
P – Precipitation
#culture was not continued due to exceedingly severe cytotoxic effects
##culture was not continued as a minimum of only four analysable concentrations is required
Applicant's summary and conclusion
- Conclusions:
- Under the experimental conditions according to OECD 476 the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.
- Executive summary:
The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in a test according to OECD guideline 476. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. 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 11800 µg/mL used in the pre-experiments was equal to approximately 5452 µg/mL of the REACH registration substance. The test item was dissolved in deionised water. The dose 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. 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 item is considered to be non-mutagenic in this HPRT assay.
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