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Diss Factsheets

Toxicological information

Skin sensitisation

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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:
2020
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

Constituent 1
Chemical structure
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

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).