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EC number: 203-613-3 | CAS number: 108-75-8
- 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
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- 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
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2020-01-21 to 2020-02-06
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 020
- Report date:
- 2020
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)
- Version / remarks:
- June 2018
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- ARE-Nrf2 luciferase LuSens test method
Test material
- Reference substance name:
- 2,4,6-trimethylpyridine
- EC Number:
- 203-613-3
- EC Name:
- 2,4,6-trimethylpyridine
- Cas Number:
- 108-75-8
- Molecular formula:
- C8H11N
- IUPAC Name:
- 2,4,6-trimethylpyridine
- Test material form:
- liquid
Constituent 1
In vitro test system
- Details of test system:
- Lusens transgenic cell line [442D]
- Details on the study design:
- 442D
PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution/dilutions
On the day of the experiment (prior to start) 2,4,6-Collidine was dissolved or stably dispersed/suspended in DMSO to the final desired concentration.
The maximum final concentration of test item was 2000 μM.
For the MTT test (dose finding assay) twelve concentrations of the test item were analysed, if possible. Therefore, dilutions were prepared by 1:2 serial dilutions from the highest soluble concentration (200 mM).
- Preparation of the solvent and negative controls
Solvent Control:
DMSO (final concentration 1% (v/v) in Treatment Medium, purity: ≥ 99%)
Negative Control:
Name: Lactic acid (final concentration 5000 μM), CAS 50-21-5, purity: ~ 90% Solvent: Treatment Medium including 1% (v/v) DMSO
DOSE RANGE FINDING ASSAY:
The doses investigated in the main experiments (LuSens) were determined with a MTT test. The MTT test is based on the cleavage of the yellow tetrazolium salt MTT [=3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid] to form a blue-violet formazan dye by MTT reduction. One cytotoxicity experiment (dose finding assay) was performed to obtain a CV75. The test item was dissolved in the solvent and 1:2 serially diluted in the solvent to obtain the desired test item concentrations (twelve concentrations).
A final concentration of 120 μM EGDMA and 5000 μM lactic acid were used as positive and negative control, respectively.
The solvent (twelve replicates), the positive (two replicates) and the negative (three replicates) controls as well as the test item concentrations (each three replicates) were subsequently diluted 1:25 in Treatment Medium.
24 hours ± 30 minutes after seeding of the cells, the Seeding Medium were removed from the wells. Thereafter, 150 μL Treatment Medium were added per well and 50 μL of the test item dilutions, the solvent, negative and positive controls (1:25 dilution in Treatment Medium) and the medium control were added to the wells, respectively. At the end of the incubation period of 48 ± 1 hours under standard cell culture conditions, the cell cultures were microscopically evaluated for morphological alterations, precipitation or phase separation.
At the end of the incubation period, Treatment Medium was removed from the wells and the cells were washed at least twice with 200 μL DPBS including Ca2+/Mg2+. Thereafter, 200 μL of the MTT working solution were added to each treatment well and the cells were incubated for 3 hours ± 30 minutes under standard cell culture conditions. After rinsing the MTT working solution, the cells of each well were treated with 100 μL MTT lysis agent (Isopropanol with 0.04 N HCl) for at least 30 minutes, while gently shaking. Thereafter the microplate was transferred to a microplate reader equipped with a 570 nm filter to measure the absorbance (reference wavelength 690 nm).
- Final concentration range selected on basis of: The quantity of formazan is presumably directly proportional to the number of viable cells, as monitored by the absorbance. The CV75 value, a concentration showing 75% of LuSens cell survival (25% cytotoxicity), is calculated. Six test item concentrations were tested in the main experiment (LuSens). The highest concentration used was CV75 × 1.2. Five further test item dilutions were prepared by serial dilution with a dilution factor of 1.2.
APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
The following concentrations of the test item were tested in the main experiments: 394.1; 472.9; 567.5; 681.0; 817.2; 980.6 μM
Solvent control: twenty-four replicates, positive control: five replicates; negative control: six replicates, test item: three replicates for each concentration
- Application procedure
After incubation of the LuSens cells, Seeding Medium was removed and 150 μL of Treatment Medium was distributed in each well. Thereafter, 50 μL of the test item and control dilutions and the medium control (twelve replicates) were added into the corresponding wells. At the end of the incubation period of 48 ± 1 hours under standard cell culture conditions, the cell cultures were microscopically evaluated for morphological alterations, precipitation or phase separation.
Acceptance Criteria
• The average luciferase activity induction obtained with the positive control, 120 μM EGDMA should be ≥ 2.5, and the positive control should have a relative cell viability ≥ 70% as compared to the solvent control.
• The average luciferase activity induction obtained with the negative control, i.e., 5000 μM Lactic acid, as well as the basal expression of untreated cells should be < 1.5 fold as compared to the average solvent control.
• The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) should be below 20% in each main experiment.
• At least three test item concentrations should have cell viability of at least 70% relative to the solvent controls. Moreover, in case a result is to be considered negative, at least one concentration should be cytotoxic, i.e. have a cell viability < 70%, or the maximum concentration of 2000 μM (or 2000 μg/ mL for substances with no defined MW) should have been tested.
LUCIFERASE ACTIVITY MEASUREMENTS
Measurement of the Luciferase activity
The Steady-Glo® Mix was be prepared by adding Steady-Glo® buffer to one bottle of Steady-Glo® Substrate and mixing by inversion until the substrate was dissolved. The Steady-Glo® working solution was prepared by mixing one part of DPBS (without Ca2+/Mg2+) with one part of Steady-Glo®-Mix.
At the end of the incubation period the Treatment Medium was removed from the wells and the cells were washed at least twice with 200 μL DPBS including Ca2+/Mg2+. Thereafter, 200 μL of the Steady-Glo® working solution was added in each well. After slowly shaking of the microtiter plate for at least 10 min in the dark, the plate was transferred to a microplate reader and the luminescence was measured for 2 seconds per well. - Vehicle / solvent control:
- other: DMSO (final concentration 1% (v/v) in Treatment Medium, purity: ≥ 99%)
- Negative control:
- other: Lactic acid (final concentration 5000 μM), CAS 50-21-5, purity: ~ 90% Solvent: Treatment Medium including 1% (v/v) DMSO
- Positive control:
- other: EGDMA (final concentration 120 μM), CAS 97-90-5, purity: ≥ 97.5% Solvent: Treatment Medium including 1% (v/v) DMSO
Results and discussion
- Positive control results:
- The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (ME 1: 6.056; ME 2: 3.070) and statistically significant. The positive control had a relative cell viability ≥ 70% as compared to the solvent control (ME 1: 106.63%; ME 2: 113.20%).
In vitro / in chemico
Resultsopen allclose all
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 2
- Parameter:
- other: induction compared to solvent control
- Value:
- 64.8 %
- At concentration:
- 394.1 other: µM
- Cell viability:
- mean of 3 replicates: 84.59 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 2
- Parameter:
- other: induction compared to solvent control
- Value:
- 63.9 %
- At concentration:
- 472.9 other: µM
- Cell viability:
- mean of 3 replicates: 90.58 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 2
- Parameter:
- other: induction compared to solvent control
- Value:
- 68.5 %
- At concentration:
- 567.5 other: µM
- Cell viability:
- mean of 3 replicates: 94.46 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 2
- Parameter:
- other: induction compared to solvent control
- Value:
- 70.2 %
- At concentration:
- 681 other: µM
- Cell viability:
- mean of 3 replicates: 94.46 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 2
- Parameter:
- other: induction compared to solvent control
- Value:
- 66.6 %
- At concentration:
- 817.2 other: µM
- Cell viability:
- mean of 3 replicates: 87.35 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 2
- Parameter:
- other: induction compared to solvent control
- Value:
- 70.2 %
- At concentration:
- 980.6 other: µM
- Cell viability:
- mean of 3 replicates: 71.36 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 1
- Parameter:
- other: induction compared to solvent control
- Value:
- 155.5 %
- At concentration:
- 980.6 other: µM
- Cell viability:
- mean of 3 replicates: 71.67 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 1
- Parameter:
- other: induction compared to solvent control
- Value:
- 131.1 %
- At concentration:
- 817.2 other: µM
- Cell viability:
- mean of 3 replicates: 81.64 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 1
- Parameter:
- other: induction compared to solvent control
- Value:
- 113.4 %
- At concentration:
- 681 other: µM
- Cell viability:
- mean of 3 replicates: 82.95 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 1
- Parameter:
- other: induction compared to solvent control
- Value:
- 102.3 %
- At concentration:
- 567.5 other: µM
- Cell viability:
- mean of 3 replicates: 96.24 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 1
- Parameter:
- other: induction compared to solvent control
- Value:
- 102 %
- At concentration:
- 472.9 other: µM
- Cell viability:
- mean of 3 replicates: 83.80 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 1
- Parameter:
- other: induction compared to solvent control
- Value:
- 117.6 %
- At concentration:
- 394.1 other: µM
- Cell viability:
- mean of 3 replicates: 79.4%
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Outcome of the prediction model:
- negative [in vitro/in chemico]
Applicant's summary and conclusion
- Interpretation of results:
- other: The study results alone are not sufficient to decide on the classification for skin sensitisation.
- Remarks:
- The ARE-Nrf2 luciferase test (LuSens) can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), human cell line activation test method (h-CLAT)) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals. The technical proficiency
- Conclusions:
- The test item 2,4,6-Collidine did not activate the LuSens cells up to a concentration of 980.6 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).
- Executive summary:
The in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens) was performed to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of a skin sensitization AOP) of 2,4,6-Collidine. In the cytotoxicity test, cytotoxic effects were observed following incubation with the test item starting with the concentration of 0.98 μM up to the highest tested concentration of 2000 μM (threshold of cytotoxicity: < 75%). The CV75 value of the cytotoxicity test was calculated as 817.2 μM. The test item was tested in 2 independent main experiments. The following concentrations of the test item were tested in the main experiments (LuSens):
394.1; 472.9; 567.5; 681.0; 817.2; 980.6 μM
After treatment with the test item for 48 ± 1 hours the luciferase induction was not above or equal to (≥) 1.5 fold compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations. Therefore, the LuSens prediction is considered negative. The acceptance criteria were met. In conclusion, the test item 2,4,6-Collidine did not activate the LuSens cells up to a concentration of 980.6 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).
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