2,4,6-trimethylpyridine

Endpoint summary

Description of key information

The test item was negative in the DPRA (OECD 442C) and in the ARE-Nrf2 Luciferase Test (LuSens, OECD 442D). Thus, the test item was judged to be not skin sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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

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

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%).

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]

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).

 

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-01-27 to 2020-01-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

18 June 2019

Deviations:

yes

Remarks:

please refer to "Principles of method if other than guideline"

Principles of method if other than guideline:

Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate peptide buffer and acetonitrile (parallel dilution) instead of conducting a serial dilution as stated in the OECD 442C Guideline. This procedure was selected, since this preparation is similar to the preparation of the test item samples and controls. Furthermore, the DPRA proficiency study (study 1841800 at ICCR-Roßdorf GmbH Germany) was conducted under these conditions.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in approximately 20 mL aliquots of the appropriate buffer solution (cysteine in 100 mM phosphate buffer pH 7.5, lysine in 100 mM ammonium acetate buffer pH 10.2).
- Preparation of the test chemical solutions
The test item was weighed into volumetric flask and dissolved immediately before testing in acetonitrile to prepare a 100 mM stock solution.
- Preparation of the positive controls, reference controls and co-elution controls
The positive control chemical (Cinnamaldehyde) was prepared at a concentration of 100 mM in acetonitrile.
Reference Control A: For the verification of the HPLC system suitability (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=1 with 3 fold injections.
Reference Control B: For the stability of the reference controls over time (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=6
Reference Control C1: Peptide stability control for the solvent used to dissolve the test item and the positive control (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=3
Co-elution Control: Sample prepared of the respective peptide buffer and the test item or the positive control without peptide. n=1, each
The reference control A, B and C1 samples of both peptides were prepared at a concentration of 500 μM in acetonitrile.

INCUBATION
500 μM cysteine and lysine peptide solutions were incubated in glass autosampler vials with 5 mM or 25 mM of the test item, respectively. The reaction solutions were incubated in the dark at 22.5 - 30ºC for 24 ± 2 hours prior to initiation of the analysis run. The test item and the positive control were analysed in triplicate for both peptides. The appearance of the test item and positive control samples in the HPLC vials was visually inspected and documented after preparation and prior to initiation of the HPLC run.

DATA EVALUATION
The concentration of cysteine or lysine peptide was photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks and by calculating the concentration of peptide using the linear calibration curve derived from the standards.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

The positive control depletion values for cysteine and lysineldepletion are within the ranges of the acceptance criteria.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

2.3 %

At concentration:

25 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

1.56 %

At concentration:

5 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
Applying the depletion model for cysteine and lysine, reactivity is classed as “no or minimal reactivity” and the DPRA prediction is therefore negative and 2,4,6-Collidine is therefore considered negative. There were no co-elution peaks in either the cysteine or lysine assays.
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for reference controls A to C: yes
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Overall achieved depletion values

Test item	Cysteine peptide depletion (%)	Lysine peptide depletion (%)	Overall mean depletion (%)	Reactivity class	DPRA prediction
2,4,6-Collidine	1.56	2.30	1.93	Minimal	Negative

 

Table 2A Cysteine Peptide Depletion

Sample	Peak area	Peptide concentration1 (μM)	Peptide Depletion (%)	Mean Depletion (%)	SD (percent points)
Positive control	735217	145	69.72	69.8	0.207
	733761	145	69.72
	725870	143	70.12
2,4,6-Collidine	2387426	479	1.542	1.56	0.127
	2383671	479	1.702
	2389802	480	1.442

SD Standard Deviation

1 Samples prepared at a nominal concentration of 500 μM (376 μg/mL)

2 Calculated against a mean Reference Control (C1) area of 2424779 (n=3)

Table 2B Lysine Peptide Depletion

Sample	Peak area	Peptide concentration1 (μM)	Peptide Depletion (%)	Mean Depletion (%)	SD (percent points)
Positive control	1321166	274	45.92	44.2	1.58
	1397829	290	42.72
	1368105	284	44.02
2,4,6-Collidine	2364674	491	3.132	2.30	1.51
	2427437	504	0.5562
	2362398	491	3.222

SD Standard Deviation

1 Samples prepared at a nominal concentration of 500 μM (388 μg/mL)

2 Calculated against a mean Reference Control (C1) area of 2441011 (n=3)

Interpretation of results:

other: This study alone is not sufficient to decide on the classification of a substance as skin sensitiser.

Conclusions:

The negative Direct Peptide Reactivity Assay (DPRA)-result can be used as part of a testing battery (including e.g. h-CLAT (human cell line activation test), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway (AOP) for the assessment of the skin sensitisation potential of chemicals.

Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of 2,4,6-Collidine. This direct peptide reactivity assay can be used as part of a testing battery (including e.g. h-CLAT (human Cell Line Activation Test), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals. The test item was dissolved in acetonitrile when incubated for 24 ± 2 hours in the range between 22.5 and 30 °C. There were no co-elution peaks in either the cysteine or lysine assays. The test item 2,4,6-Collidine was analyzed by the DPRA method in both the cysteine and lysine containing synthetic peptides. With an overall depletion value of 1.93%, this places 2,4,6-Collidine in the reactivity class of “no to minimal” and hence it is predicted by DPRA not to be a skin sensitizer. In conclusion, solutions of 2,4,6-Collidine were successfully analyzed by the DPRA method in both the Cysteine and Lysine containing synthetic peptides. With no to minimal mean depletion of both peptides (1.93%) in the presence of the test item, 2,4,6-Collidine is therefore predicted by DPRA as negative and not to be a potential skin sensitizer based on this assay. The OECD 442C Test Guideline cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by UN GHS (1), for authorities implementing these two optional subcategories, nor to predict potency for safety assessment decisions. Data generated with the OECD 442C Test Guideline should be considered in the context of integrated approaches such as IATA (Integrated Approaches to Testing and Assessment). The Direct Peptide Reactivity Assay (DPRA) can be used as part of a testing battery (including e.g. h-CLAT (human cell line activation test), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway (AOP) for the assessment of the skin sensitisation potential of chemicals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

The chemical and biological mechanisms associated with skin sensitisation are summarised in the form of an Adverse Outcome Pathway (AOP). This AOP includes four key events:

1) The molecular initiating event is the covalent binding of electrophilic substances to nucleophilic centres in skin proteins.

2) The inflammatory responses and gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways in the keratinocytes.

3) The activation of dendritic cells, typically assessed by expression of specific cell surface markers, chemokines and cytokines.

4) The T-cell proliferation.

Studies addressing one of the key events can be used as part of a testing battery based on the OECD adverse outcome pathway (AOP) for the assessment of the skin sensitisation potential of chemicals. For 2,4,6-Collidine, Key event 1 is addressed by the DPRA assay. The second key event is addressed by the LuSens test. As the results of both tests were negative, no further testing is required.

DPRA Assay

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of 2,4,6-Collidine. This direct peptide reactivity assay can be used as part of a testing battery (including e.g. h-CLAT (human Cell Line Activation Test), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals. The test item was dissolved in acetonitrile when incubated for 24 ± 2 hours in the range between 22.5 and 30 °C. There were no co-elution peaks in either the cysteine or lysine assays. The test item 2,4,6-Collidine was analyzed by the DPRA method in both the cysteine and lysine containing synthetic peptides. With an overall depletion value of 1.93%, this places 2,4,6-Collidine in the reactivity class of “no to minimal” and hence it is predicted by DPRA not to be a skin sensitizer. In conclusion, solutions of 2,4,6-Collidine were successfully analyzed by the DPRA method in both the Cysteine and Lysine containing synthetic peptides. With no to minimal mean depletion of both peptides (1.93%) in the presence of the test item, 2,4,6-Collidine is therefore predicted by DPRA as negative and not to be a potential skin sensitizer based on this assay.

The OECD 442C Test Guideline cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by UN GHS (1), for authorities implementing these two optional subcategories, nor to predict potency for safety assessment decisions. Data generated with the OECD 442C Test Guideline should be considered in the context of integrated approaches such as IATA (Integrated Approaches to Testing and Assessment). The Direct Peptide Reactivity Assay (DPRA) can be used as part of a testing battery (including e.g. h-CLAT (human cell line activation test), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway (AOP) for the assessment of the skin sensitisation potential of chemicals.

LuSens-test

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).

Conclusion

The study results are used in a weight-of-evidence approach. As both results are negative, it is concluded that the test item is not sensitising to skin.

 

Justification for classification or non-classification

Based on the results obtained the test item was not classified for skin sensitisation according to Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/1182.