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EC number: 217-420-7 | CAS number: 1843-03-4
- 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
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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:
- 01 September 1992 to 10 November 1992
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to OECD test guidance in compliance with GLP.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 992
- Report date:
- 1992
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- 4,4',4''-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]
- EC Number:
- 217-420-7
- EC Name:
- 4,4',4''-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]
- Cas Number:
- 1843-03-4
- Molecular formula:
- C37H52O3
- IUPAC Name:
- 4-[4,4-bis(5-tert-butyl-4-hydroxy-2-methylphenyl)butan-2-yl]-2-tert-butyl-5-methylphenol
Constituent 1
Method
- Target gene:
- L5178Y TK+/- mouse lymphoma cells
Species / strain
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- A bank of L5178Y (-3.7.2c [TK+/-]) cells was stored in a liquid nitrogen freezer. The cell stocks have been shown to be free of mycoplasma by enzyme linked immunosorbent assay (ELISA).
The cultures were kept at 37°C under an atmosphere of 5% CO2 in air, either in a gassing incubator or in a hot room in roller bottles rotated on a roller apparatus. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- 0 - 125μg/ml
- Vehicle / solvent:
- Dimethylsulphoxide (DMSO), CTL reference number Y00876/001 (BDH Chemicals Ltd, England), was used as the vehicle for the positive controls.
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Migrated to IUCLID6: N-nitrosodimethylamine (NDMA)
- Details on test system and experimental conditions:
- Control Chemicals
Negative Control: Dimethylsulphoxide (DMSO), CTL reference number Y00876/001 (BDH Chemicals Ltd, England), was used as the vehicle for the positive controls. It was also tested independently as the solvent control (SC) at an equivalent volume (i.e .200μl added to 20ml cell cultures) both in the absence and presence of S9.
Positive Controls: The positive controls were ethyl methanesulphonate (EMS), CTL reference number Y01958/005 (Sigma, England); and N-nitrosodimethylamine (NDMA), CTL reference Y01468/005 (Sigma, England). EMS is a direct acting mutagen and was tested in the absence of S9 at a final dose level of 750μg/ml. NDMA, which requires metabolic activation to its reactive species, was tested in the presence of S9 at a final concentration of 600μg/ml. The shelf lives of these chemicals have not yet been defined as no loss in activity has been detected to date.
Test Methods
Preparation of Solutions of Test Sample and Positive Controls:
All stock solutions were prepared by dissolving the chemical in DMSO to produce a final concentration of 100 times that of the ultimate maximum dose level. Further appropriate serial dilutions of this main stock solution were made to provide the 100 times stock solution for subsequent dose levels.
Cell Maintenance: A bank of L5178Y (-3.7.2c [TK+/-]) cells was stored in a liquid nitrogen freezer. The cell stocks have been shown to be free of mycoplasma by enzyme linked immunosorbent assay (ELISA).
The cultures were kept at 37°C under an atmosphere of 5% CO2 in air, either in a gassing incubator or in a hot room in roller bottles rotated on a roller apparatus.
Summary of Methodology: A dose-ranging study was performed to determine concentrations of 1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane to be used in the main mutation assay. Subsequently, two series of exponentially growing suspension cultures of L5178Y cells (one with, and one without S9), were treated in duplicate with concentrations of 1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane or control chemical (as appropriate) for 4 hours. 1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane was tested both in the presence and absence of S9 up to a maximum dose level of 125μg/ml. After removal of the treatment medium, the cells were washed and a sample diluted to determine the survival immediately after treatment. The remaining cells were then cultured to allow any induced mutants to be detected. During this expression time the growth rate was monitored and the cells subcultured daily. At the end of the 72 hour expression time, samples were grown in both selective medium (supplemented with trifluorothymidine (TFT)) and non-selective medium, and the results obtained used to determine the mutant frequency per viable cell. - Evaluation criteria:
- Test data from individual experiments were considered valid if:
(a) the spontaneous mutant frequencies in the presence and absence of S9 fell within an acceptable range.
(b) the positive controls induced unequivocal positive responses. A positive response in a valid individual experiment is recorded when statistically significant, dose related increases in mutant frequency are observed across a range of toxicity. Such increases in mutant frequency are not considered indicative of a positive response if the increases occur only at doses eliciting excessive toxicity, i.e. <10% survival. These increases must also be associated with increases in the numbers of mutants over those observed with the concurrent negative control.
A negative result in a valid experiment is obtained when there is no significant dose related increase in mutant frequency.
A positive response in an individual experiment must be reproducible for the test chemical to be considered positive (i.e. mutagenic) in this assay. - Statistics:
- A statistical analysis is applied at the discretion of the Study Director in consultation with the Study Statistician. The data was examined by the Study Statistician who considered that a statistical analysis was not necessary.
Results and discussion
Test results
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- not specified
- Additional information on results:
- From a preliminary dose-ranging experiment, the maximum dose level of 1,1,3-Tris(2-methyl-4-4hydroxy-5-tertbutylphenyl)butane that was considered appropriate for testing was determined as 125μg/ml both with and without S9 since this dose produced significant toxicity and the next dose tested (250μg/ml) gave a visible precipitate at the end of the treatment time. In the subsequent main mutation experiments, this top dose level proved too toxic in the absence of S9 and lower dose level were used to provide an assessment over an adequate toxicity range.
Survival Data
In the first experiment, 1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane was examined in the presence and absence of S9 at dose levels up to 125μg/ml, which in the presence of S9 reduced survival to 9% of the negative control value. The highest surviving dose level in the absence of S9 in this experiment was 31.3μg/ml which gave 35% survival. In the second experiment, the highest surviving dose in the absence of S9 was 22μg/ml which gave 8% survival. In the presence of S9, 1% survival was observed at the top dose of 125μg/ml with 17% survival at a dose of 70μg/ml. The third experiment was conducted in the absence of S9 only, the highest surviving dose being 38μg/ml which gave 8% survival. Dose related toxicity was observed in all experiments and an evaluation of mutant frequency was made over a toxicity range extending to approximately 10% survival both in the presence and absence of S9.
Mutation Data
1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane have no reproducible dose related increases in mutant frequency and is therefore considered to have given a negative response in the assay.
The positive controls (EMS and NDMA) induced substantial increases in mutant frequency in all experiments, demonstrating that the assay was performing satisfactorily. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
SUMMARY OF DATA FOR EXPERIMENT 1
WITHOUT S9 |
|
WITH S9 |
||||
Dose (μg/ml) |
% Survival |
Mutant Frequency (x10-4) |
Dose (μg/ml) |
% Survival |
Mutant Frequency (x10-4) |
|
1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane |
1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane |
|||||
125 |
0 |
- |
125 |
9 |
2.6 |
|
62.5 |
0 |
- |
62.5 |
84 |
1.0 |
|
31.3 |
35 |
1.1 |
31.3 |
81 |
1.0 |
|
15.6 |
63 |
0.8 |
15.6 |
96 |
1.1 |
|
Negative Control |
Negative Control |
|||||
DMSO (10μl/ml) |
100 |
1.7 |
DMSO (10μl/ml) |
100 |
1.2 |
|
Positive Control |
Positive Control |
|||||
EMS 750 |
23 |
22.6 |
NDMA 600 |
82 |
18.2 |
SUMMARY OF DATA FOR EXPERIMENT 2
WITHOUT S9 |
|
WITH S9 |
||||
Dose (μg/ml) |
% Survival |
Mutant Frequency (x10-4) |
Dose (μg/ml) |
% Survival |
Mutant Frequency (x10-4) |
|
1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane |
1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane |
|||||
53 |
0 |
- |
125 |
1 |
- |
|
40 |
0 |
- |
94 |
0 |
- |
|
30 |
0 |
- |
70 |
17 |
0.7 |
|
22 |
8 |
1.6 |
53 |
49 |
1.2 |
|
117 |
39 |
0.9 |
40 |
71 |
1.6 |
|
Negative Control |
Negative Control |
|||||
DMSO (10μl/ml) |
100 |
1.0 |
DMSO (10μl/ml) |
100 |
1.1 |
|
Positive Control |
Positive Control |
|||||
EMS 750 |
47 |
13.7 |
NDMA 600 |
77 |
15.3 |
SUMMARY OF DATA FOR EXPERIMENT 3
WITHOUT S9 |
||
Dose (μg/ml) |
% Survival |
Mutant Frequency (x10-4) |
1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane |
||
50 |
0 |
- |
38 |
8 |
3.1 |
28 |
51 |
2.9 |
21 |
83 |
2.5 |
16 |
99 |
4.0 |
12 |
145 |
4.0 |
9 |
107 |
1.9 |
Negative Control |
||
DMSO (10μl/ml) |
100 |
3.3 |
Positive Control |
||
EMS 750 |
34 |
25.9 |
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative with & without metabolic activation
Under the conditions of this assay, 1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane is non-mutagenic to L5178Y TK+/- cells as determined by selection in trifluorothymidine in both the presence and absence of an auxiliary metabolising system (S9), when tested to doses limited by the toxicity of the test material. - Executive summary:
Study conducted to OCED Guidelines for Testing of Chemicals, Second addendum August 1984. Section 4, Genetic Toxicity, 476, In Vitro Mammalian Cell Gene Mutation Tests and in compliance with the Principles of Good Laboratory Practice (GLP).
To assess the mutagenic potential of 1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane to mammalian cells, L5178Y TK+/- mouse lymphoma cells were treated in vitro with various concentrations of the test sample, both in the presence and absence of an auxiliary metabolising system (S9). Mutant frequencies were assessed by cell growth in the presence of trifluorothymidine after a 72 hours expression time.
1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane was tested both in the presence and absence of S9 in two independent experiments and in the absence of S9 only in a third experiment. All experiments gave negative results in the presence and absence of S9 when tested to a maximum dose of 125μg/ml.
Under the conditions of this assay, 1,1,3-Tris(2-methyl-4-hydroxy-5-tertbutylphenyl)butane is non-mutagenic to L5178Y TK+/- cells in both the presence and absence of an auxiliary metabolising system (S9) when tested to doses limited by the toxicity of the test material.
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