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EC number: 239-816-9 | CAS number: 15721-78-5
- 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
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- The study was conducted in accordance with the Good Laboratory Practice Principles as Published by OECD in 1998, No 1 ENV/MC/CHEM(98)17
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 015
- Report date:
- 2015
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Principles of method if other than guideline:
- In vitro mammalian cell gene mutation assa ywas performed to determine the mutagenic nature of the test chemical
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)amine
- EC Number:
- 239-816-9
- EC Name:
- Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)amine
- Cas Number:
- 15721-78-5
- Molecular formula:
- C28-H43-N
- IUPAC Name:
- 4-(2,4,4-trimethylpentan-2-yl)-N-[4-(2,4,4-trimethylpentan-2-yl)phenyl]aniline
- Test material form:
- solid: particulate/powder
- Remarks:
- migrated information: powder
- Details on test material:
- CAS Number: 15721-78-5
Chemical Name: Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)amine
Molecular Formula: C28H43N
Molecular Weight: 393.65 g/mol
Nature of chemical: Organic
Consistency: Light apricot-colored powder
Activity (Clinical Indication): Industrial Chemical
Constituent 1
Method
- Target gene:
- Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.
This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.
Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
Species / strain
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable - Additional strain / cell type characteristics:
- other: Hypodiploid, modal No. 20
- Cytokinesis block (if used):
- No data
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats
- Test concentrations with justification for top dose:
- 0, 0.025, 0.05, 0.1 or 0.5 mM
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test chemical was dissolved in DMSO
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: In medium with pre-incubation
DURATION
-Pre-incubation: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration: 3 hours
-Expression time: 7 days
- Selection time: 14 days
- Fixation time: 7 days (harvest of cells)
SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet
NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.
NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated
DETERMINATION OF CYTOTOXICITY
- Cytotoxicity test: After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.
OTHER EXAMINATIONS: Not applicable
- Determination of polyploidy:
- Determination of endoreplication:
- Other: - Rationale for test conditions:
- No data
- Evaluation criteria:
- The cell line was observed for gene mutation at the HGPRT locus
- Statistics:
- No data
Results and discussion
Test results
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: Completed without S9 metabolic activation. A range of test concentrations (0, 0.01, 0,05, 0.1, 0.5, 1, 5, 10 or 50mM) was applied 24 hours after seeding to single cultures in fresh medium in 96-well plates. The cell population (control and treated cells) were assessed 24 and 48 hours after treatment using the colorimetric assayMTTand the BCA assay to assess cell viability and total protein concentration, respectively. From the basis of these results, the test concentrations of the chemical was chosen to be included in the gene toxicity test. Since no cytotoxicity was evident at the tested concentrations in this preliminary dose-finding test further testing concentrations were adapted to have a maximum test concentration of 0.5 mM. Since the test chemical was dissolved in DMSO, higher concentrations of the test chemical than the concentration mentioned above would result in a toxic effect of DMSO. The test chemical could only be dissolved in 100% DMSO.
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate: No data
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data - Remarks on result:
- other: No mutagenic potential
Applicant's summary and conclusion
- Conclusions:
- When CHO cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM the results did not show any evidence of gene toxicity without metabolic activation. CHO cells when exposed to the test chemical in the presence of metabolic activation at the concentration of 0, 0.025, 0.05 or 0.1 mM did not show any evidence of gene toxicity. However, treatment with 0.5 mM showed an indication of genetic toxicity.
- Executive summary:
With S9 metabolic activation system:
In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.1 or 0.5 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. The results showed an indication of gene toxicity when treated with 0.5 mM when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 0.025, 0.05 or 0.1 mM does not cause genetic mutation(s) in the presence of metabolic activation, whereas treatment with 0.5 mM may, when CHO cells are exposed to the test chemical in the presence of metabolic activation.
Without S9 metabolic activation system:
In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM and without S9-induced metabolic activation. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. Independently of treatment concentration, the results showed no evidence of gene toxicity when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM does not cause genetic mutation(s) in the absence of metabolic activation.
Overall conclusion
At time of exposure, the test chemical was added in the absence or presence of S9 liver microsomal fraction. The test chemical was added to each applicable well to give a final concentration of 0, 0.025, 0.05, 0.1 or 0.5 mM. Negative controls, solvent/vehicle controls and positive control substance(s) were also included in each experiment. pH and osmolality was not determined in the gene mutation test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity except when treated with 0.5 mM in the presence of 4% S9 liver microsomal fraction. Thus, the results show evidence of genotoxicity when CHO cells are exposed to the test chemical in the highest tested concentrations, i.e. at 0.5 mM. It was concluded that the test chemical does not give rise to gene mutations when used at concentrations of ≤ 0.1 mM in organisms with a fully functioning metabolic activation, whereas concentrations > 0.1 mM may induce gene mutations. However, when treated with test chemical in the absence of S9 liver microsomal fraction, no evidence of occurring gene mutations were detected. Hence, test chemical does not give rise to gene mutations in organisms who has no or a non-functional metabolic activation at the above mentioned concentrations. When the mutation frequency was determined, a frequency of 3.67 x 10-4 was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction, and a frequency of -1.92 x 10-4 was observed for 0.5 mM in the presence of S9 liver microsomal fraction. Since no other tested concentration of the test chemical and in the absence or presence of S9 liver microsomal fraction resulted in colonies, it was concluded that the test chemical does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at ≤ 2.5 mM for 3 hrs.
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