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EC number: 940-393-4 | CAS number: 1702355-94-9
- 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
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016 / 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: HPRT Locus assay
Test material
- Reference substance name:
- N,N'-dibutyl-N,N'-bis(1,2,2,6,6-pentamethylpiperidin-4-yl)-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamine
- EC Number:
- 940-393-4
- Cas Number:
- 1702355-94-9
- Molecular formula:
- C35 H66 N8
- IUPAC Name:
- N,N'-dibutyl-N,N'-bis(1,2,2,6,6-pentamethylpiperidin-4-yl)-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamine
- Test material form:
- solid: bulk
- Details on test material:
- - State of aggregation: solid
Constituent 1
Method
- Target gene:
- HPRT locus
Species / strain
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: derived from the Chinese hamster
- Modal number of chromosomes: 20
- Normal (negative control) cell cycle time: 12-16h
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbital and β-naphthoflavone induced rat liver S9 mix
- Test concentrations with justification for top dose:
- Based on the data and the observations from the pretest and taking into account the current guidelines, the following doses were selected in this study.
First Experiment:
Without S9 mix, 4-hour exposure: 0.78 - 50 ug/ml
With S9 mix, 4-hour exposure: 0.78 - 50 ug/ml
Second Experiment:
Without S9 mix, 4-hour exposure: 2.50 - 40 ug/ml
With S9 mix, 4-hour exposure: 2.50 - 40 ug/ml
Third Experiment:
With S9 mix, 4-hour exposure: 2.5 - 40 ug /ml - Vehicle / solvent:
- In comparison to other commonly used vehicles (e.g. DMSO, acetone etc.) tetrahydrofurane (THF) is the most suitable. Therefore, THF was selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay. The final concentration of the vehicle THF in culture medium was 0.5% (v/v).
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- 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
- Cell density at seeding (if applicable): in 300 cm² flasks, 20x106 cells in 40 mL)
DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 3d incl. 2 passages
- Fixation time (start of exposure up to fixation or harvest of cells): days 7-9 Drying, fixation, staining and counting of the cloning efficiency 1 3rd passage of the treated cells; addition of selection medium ("TG" medium); and seeding of the cloning efficiency 2 (viability), from day 16 Drying, fixation, staining and counting of the selected colonies and cloning efficiency 2
NUMBER OF REPLICATIONS: each dose in triplicate, three independent experiments
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
OTHERS:
Changes in pH were recorded by a change in the indicator color of the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured for the top concentrations and for the vehicle controls with and without S9 mix.
Osmolality was measured in the top concentrations and the vehicle controls with and without S9 mix.
Test substance precipitation was assessed immediately after dosing the test cultures and at the end of treatment.
Cell morphology: The test cultures of all test substance concentrations were examined microscopically for cell morphology and cellular attachment at the end of the exposure period, which is a further indication for cytotoxicity. - Evaluation criteria:
- A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit). - Statistics:
- An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report (6). The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction (7, 8). The calculation was performed using R (9).
If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- CYTOTOXICITY
Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were observed in the 2nd Experiment in the absence of S9 mix and in the 1st and 2nd Experiment in the presence of S9 mix at the highest applied concentrations. Clearly reduced cell densities in the 2nd passage were obtained from about 40.00 μg/mL onward. Those test groups were discontinued. In addition, a clearly reduced cloning efficiency of below 20% of the respective vehicle control value was observed in the 1st Experiment in the presence of S9 mix at 50.00 μg/mL (CE1 relative: 8.1%).
CELL MORPHOLOGY
After 4 hours treatment, the cell morphology and attachment of the cells was not adversely influenced (grade > 2) at the highest applied concentrations in the 1st and 2nd Experiment in the absence of S9 mix in the 2nd Experiment in the presence of S9 mix and.
TREATMENT CONDITIONS
Osmolality and pH values were not influenced by test substance treatment. In this study, test substance precipitation was observed macroscopically in culture medium at the end of treatment from about 15.00 μg/mL onward under all experimental conditions
Any other information on results incl. tables
Summary tables for the three experiments are attached
Applicant's summary and conclusion
- Conclusions:
- In this study, in the 1st Experiment in the absence of metabolic activation and in the 1st and 2nd Experiment in the presence of metabolic activation the highest concentrations tested were clearly cytotoxic. In all experimental parts, the highest concentrations evaluated for gene mutations showed strong precipitation in culture medium. Two single statistically significant increased mutant frequencies as well as a positive trend in mutant colonies in the 1st Experiment in the presence of S9 mix were considered as not biological relevant.
Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in three experiments performed independently of each other.
Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
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