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EC number: - | 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
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- 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
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 10 February 2022 to 24 March 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 022
- Report date:
- 2022
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
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- Paraffin waxes (Fischer-Tropsch), isomerization
- Cas Number:
- 2658498-20-3
- Molecular formula:
- C25H52 - C150H302
- IUPAC Name:
- Paraffin waxes (Fischer-Tropsch), isomerization
- Details on test material:
- - Purity: 100%
- Molecular weight: Not applicable (UVCB)
- Physical state / Appearance: Solid / white
- Expiry Date / Retest Date: Not indicated
- Storage Conditions: Room temperature
- Stability in Solvent: Not indicated
Constituent 1
- Specific details on test material used for the study:
- TREATMENT OF TEST MATERIAL PRIOR TO TESTING
On the day of the experiment (immediately before treatment), the test item was suspended in THF by using a ultrasonic water bath and stirring by vortex. The final concentration of THF in the culture medium was 0.5% (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures (1).
All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable for this period.
The osmolarity and the pH-value were determined in culture medium of the solvent control and of the maximum concentration in the pre-experiment without metabolic activation (2):
Solvent control test item
100 µg/mL
Osmolarity [mOsm] 381 410
pH-value 7.37 7.38
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:
- Table 1 Doses applied in the gene mutation assay with the test item
(concentrations given in bold letters were chosen for the mutation rate analysis)
exposure period S9mix concentrations in µg/mL
Main experiment
4 hours - 0.09 0.27 0.8 1.6P 3.1 P 6.3 P
4 hours + 0.09 0.27 0.8 1.6P 3.1 P 6.3 P
P = Precipitation visible at the end of treatment
The cultures at the two highest concentration with and without metabolic activation were not continued to avoid analysis of too many concentrations showing precipitation. - Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: solubility 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:
DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 10 days
SELECTION AGENT (mutation assays): 6-Thioguanine
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: >1,5x10exp. 6
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- The gene mutation assay is considered acceptable if it meets the following criteria:
a) The mean values of the numbers of mutant colonies per 106 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistical significant increase compared with the concurrent solvent control.
c) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
d) An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90% cytotoxicity during treatment.
e) The criteria for the selection of the top concentration are fulfilled . - Statistics:
- The statistical analysis was performed on the mean values of culture I and II for the main experiment.
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated 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.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate a significant increase of the mutation frequency at test points exceeding the 95% confidence interval. A t-test is judged as significant if the p-value (probability value) is below 0.05.
A t-test was performed only for the positive controls since all mean mutant frequencies of the groups treated with the test item were well within the 95% confidence interval of our laboratory’s historical negative control data
However, both, biological and statistical significance will be considered together.
Results and discussion
Test results
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
There was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test item measured in the pre-experiment (solvent control: 381 mOsm, pH 7.37 versus 410 mOsm and pH 7.38 at 100 µg/mL).
- Evaporation from medium: Not examined
- Precipitation: Precipitation occurred at the concentration of 1.6 µg/mL and above after four hours treatment with and without S9 mix in the main experiment
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
Pre-Experiment
Test item concentrations between 0.8 µg/mL and 100 µg/mL were used in the pre-experiment with and without metabolic activation following 4 hours treatment. The highest concentration was limited by the solubility properties of the test item.
The test medium was checked for phase separation and precipitation at the end of the treatment period (4 hours) before the test item was removed. Precipitation occurred at 12.5 µg/mL and above with and without metabolic activation.
There was no relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below, neither with nor without metabolic activation
Based on the results of the pre-experiment the following concentrations were applied in the main experiment:
0.09; 0.27; 0.8; 1.6; 3.1; and 6.3 µg/mL
COMPARISON WITH HISTORICAL CONTROL DATA: Complies
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) were noted up to the highest concentration evaluated, which showed precipitation. - Remarks on result:
- other: strain/cell type: Chinese hamster lung fibroblasts (V79)
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Summary Table
|
|
|
| relative | relative | rel. adjusted | (MF) | 95% | statistical analysis | |
| conc. | P | S9 | cloning | cell | cloning | mutant/ | confidence | ||
| µg/mL | mix | efficiency I | density | efficiency I | colonies/ | interval | t-test | linear | |
|
|
|
| % | % | % | 106 cells |
|
| regression |
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
Experiment I / 4 h treatment | mean values of culture I and II |
|
| |||||||
Solvent control with THF |
|
| - | 100.0 | 100.0 | 100.0 | 12.0 | 3.3 - 21.3 |
| |
Positive control (EMS) | 300.0 | - | 99.5 | 94.3 | 93.7 | 183.0 | ꟷ | 0.000S |
| |
Test item | 0.09 | - | - | 110.9 | 92.0 | 100.9 | 8.2 | 3.3 - 21.3 | n.c. | 0.156 |
Test item | 0.27 | - | - | 106.5 | 88.0 | 94.0 | 11.5 | 3.3 - 21.3 | n.c. | |
Test item | 0.8 | - | - | 103.8 | 87.2 | 90.6 | 5.5 | 3.3 - 21.3 | n.c. | |
Test item | 1.6 | P | - | 97.0 | 91.4 | 89.0 | 6.1 | 3.3 - 21.3 | n.c. | |
Test item | 3.1 | P | - | culture not continued# |
| |||||
Test item | 6.3 | P | - | culture not continued# |
|
| ||||
Solvent control with THF | + | 100.0 | 100.0 | 100.0 | 11.2 | 3.4 - 22.5 |
| |||
Positive control (DMBA) | 2.3 | + | 96.2 | 118.5 | 114.7 | 86.4 | ꟷ | 0.000S |
| |
Test item | 0.09 | - | + | 91.3 | 110.1 | 100.5 | 13.1 | 3.4 - 22.5 | n.c. | 0.027 I |
Test item | 0.27 | - | + | 92.1 | 97.6 | 89.6 | 10.6 | 3.4 - 22.5 | n.c. | |
Test item | 0.8 | - | + | 94.0 | 117.3 | 108.6 | 9.8 | 3.4 - 22.5 | n.c. | |
Test item | 1.6 | P | + | 95.5 | 98.4 | 93.7 | 7.3 | 3.4 - 22.5 | n.c. | |
Test item | 3.1 | P | + | culture not continued# |
| |||||
Test item | 6.3 | P | + | culture not continued# |
|
|
MF Mutant frequency
P Precipitation at the end of treatment
S Significant trend (p < 0.05)
n.c. not calculated (mean MF below the upper limit of the 95% control limit)
I Inverse trend without biological relevance
# cultures not continued to avoid to many concentrations showing precipitation
Applicant's summary and conclusion
- Conclusions:
- - test item did not induce gene mutations at the HPRT locus in V79 cells;
- test item is considered to be non-mutagenic in this HPRT assay - Executive summary:
A study was performed to investigate the potential of the substance Paraffin waxes (Fischer-Tropsch), isomerization, suspended in THF, to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (OECD 476; GLP).
The treatment period was 4 hours with and without metabolic activation.
The maximum test item concentration of the pre-experiment (100 µg/mL) was limited by the solubility of the test item. The maximum test item concentration of the main experiment was 6.3 µg/mL, limited by precipitation of the test item as well.
Based on the results of the pre-experiment the following concentrations were applied in the main experiment:
0.09; 0.27; 0.8; 1.6; 3.1; and 6.3 µg/mL where precipitation of the test material occurred at the concentration of 1.6 µg/mL and above after four hours treatment.
In the main experiment in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration (1.6 µg/mL), which showed precipitation.
Consequently, the concentrations of 0.09 to 1.6 µg/mL were evaluated for mutagenicity in the presence and absence of metabolic activation.
No substantial and dose dependent increase of the mutation frequency was observed in the main experiment.
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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