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Description of key information

In an integrated approach adressing two of three key events of the skin sensitisation AOP, results from an in chemico skin sensitisation study according to OECD guideline 442 C (DPRA, negative) and from an in vitro ARE-Nrf2 Luciferase Test according to OECD guideline 442 D (KeratinoSens, negative) were combined and predicted a non-skin sensitising potential of the test item.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2020-05-25 to 2020-07-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
adopted 2019-06-18
Deviations:
yes
Remarks:
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.
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 was conducted under these conditions.
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
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:
Positive controls: The positive control chemical (Cinnamaldehyde) was prepared at a concentration of 100 mM in acetonitrile.
Reference controls: The reference control A, B and C1 samples of both peptides were prepared at a concentration of 500 μM 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 controls: Sample prepared of the respective peptide buffer and the test item or the positive control without peptide. n=1, each

INCUBATION
- Incubation conditions: 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.
- Precipitation noted: No, the solubility of the test item in acetonitrile at a nominal concentration of 100 mM was achieved.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: Calibration standards of both peptides were prepared in a solution of 20% acetonitrile buffer using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide.The following calibration solutions were prepared from the peptide stock solution of each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A blank of the dilution buffer was also included in the standard calibration curve for both peptides. The blank is 25% acteonitrile:buffer solution with phosphate buffer pH 7.5 for the cysteine peptide and with ammonium acetate buffer pH 10.2 for the lysine peptide without peptide.
- Verification of the suitability of the HPLC for test chemical and control substances: Analysis was performed according to a proficiency study conducted at the testing facility.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm
Positive control results:
Cinnamic aldehyde showed 70.6 % ± 0.403 % mean cysteine peptide depletion and 43.3 % ± 2.92 % mean lysine peptide depletion.
Key result
Run / experiment:
mean
Parameter:
other: mean lysine depletion (migrated information)
Value:
0.743
Vehicle controls validity:
valid
Remarks:
Reference Control C1
Negative controls validity:
valid
Remarks:
Co-elution Control
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
mean
Parameter:
other: mean cystein depletion (migrated information)
Value:
2.74
Vehicle controls validity:
valid
Remarks:
Reference Control C1
Negative controls validity:
valid
Remarks:
Co-elution Control
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: Analysis was performed according to a proficiency study conducted at the testing facility.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- 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
Interpretation of results:
study cannot be used for classification
Remarks:
The 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 for the assessment of the skin sensitisation potential of chemicals.
Conclusions:
Under the conditions of this assay, the test item was not predicted to be a skin sensitizer by DPRA.
Executive summary:

The purpose of this GLP compliant study (based on the based on the OECD Guidelines for the Testing of Chemicals: Test Guideline No. 442C: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)) was to assess the reactivity and sensitizing potential of the test item. 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. Solutions of the test item were analyzed by the DPRA method in both the cysteine and lysine containing synthetic peptides. Based on this assay, with an overall depletion value of 1.74%, the test item was placed in the reactivity class of “no to minimal” and hence it is predicted by DPRA not to be a skin sensitizer.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2020-07-14 to 2020-10-28
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 Test Method)
Version / remarks:
2018-06
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
other: ARE-Nrf2 luciferase LuSens test method (migrated information)
Justification for non-LLNA method:
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.
Details on the study design:
PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The test item stock solution was prepared in DMSO with a concentration of 200 mM.
- Preparation of the test chemical serial dilutions: Dilutions were prepared by 1:2 serial dilutions from the highest soluble/dispersible concentration. The test item concentrations (three replicates for each concentration) were diluted 1:25 in treatment medium.
- Preparation of the positive controls: The positive control EGDMA was prepared in treatment medium including 1% (v/v) DMSO to reach a final concentration of 120 μM.
- Preparation of the solvent, vehicle and negative controls: The negative control lactic acid was prepared in treatment medium including 1% (v/v) DMSO to reach a final concentration of 5000 μM.
- Stable dispersion obtained: Yes

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 2000 µM
- Solubility in solvents: Precipitation not indicated
- Solubility in incubation medium: Precipitation not indicated
- Cytotoxicity assessment performed: Yes, the CV75 value of the cytotoxicity test was calculated as 1510.9 μM
- Final concentration range selected on basis of: Cytotoxicity (the highest concentration used was CV75 × 1.2)

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 24 (solvent control), 5 (positive control), 6 (negative control) and 3 (each test item concentration)
- Number of repetitions: 2
- Test chemical concentrations: 729, 874, 1049, 1259, 1511and 1813 µM
- Application procedure: 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.
- Exposure time: 48 hours ± 1 hour
- Study evaluation and decision criteria used: See "Any other information on materials and methods incl. tables"
- Description on study acceptance criteria: See "Any other information on materials and methods incl. tables"

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): The passage numbers of the used LuSens cells were 13 in the cytotoxicity test and 15 and 7 in the LuSens test for the main experiments 1 and 2, respectively. The seeding density was 9000-11000 LuSens cells per well.
- Incubation conditions: At 37 ± 1.5 °C and 5.0 ± 0.5 % CO2 (standard cell culture conditions)
- Washing conditions: After treatment and microscopic assessment of the cells, the cells were washed at least twice with 10 mL Ca2+/Mg2+ free DPBS including EDTA.
- Precipitation noted: Not indicated

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: Multimode Reader (TriStar2 LB 942) by Berthold Technologies GmbH Co KG, Germany. The technical proficiency of the LuSens with the OECD 442D guideline recommended proficiency substances was demonstrated.
- Plate used: 96 well microtiter plate
- Lysate preparation: After treatment and washing of the cells, 200 μL of the MTT working solution (0.5 mg/mL MTT in treatment medium) were added to each treatment well and the cells were incubated for 3 hours ± 30 min 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.

DATA EVALUATION
- Cytotoxicity assessment: A cell viability of ≥ 70% is considered non-cytotoxic.
- Prediction model used: See "Any other information on materials and methods incl. tables"
Positive control results:
The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (experiment 1: 8.53; experiment 2: 6.62).
Key result
Run / experiment:
mean
Parameter:
other: EC 1.5 [442D] (migrated information)
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
The EC1.5 was not determinable as 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.
Key result
Run / experiment:
mean
Parameter:
other: CV75 [442D and 442E] (migrated information)
Value:
1 510.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Cytotoxicity results
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Not indicated

DEMONSTRATION OF TECHNICAL PROFICIENCY: The technical proficiency of the LuSens with the OECD 442D guideline recommended proficiency substances was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: See "Attached background material"
Interpretation of results:
study cannot be used for classification
Remarks:
The ARE-Nrf2 Luciferase Test Method can be used as part of a testing battery (including e.g. h-CLAT (human cell line activation test) and DPRA) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.
Conclusions:
In conclusion, the test item did not activate the LuSens cells up to a concentration of 1813 μg/mL 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:

A GLP compliant 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 the test item according to OECD guideline 442D. In the cytotoxicity test, cytotoxic effects were observed following incubation with the test item concentration of 2000 μM (highest tested concentration, threshold of cytotoxicity: < 75%). The CV75 value of the cytotoxicity test was calculated as 1510.9 μM. The test item was tested in 2 independent main experiments. The following concentrations of the test item were tested in the main experiments: 729, 874, 1049, 1259, 1511, 1813 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:


The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (ME 1: 8.53; ME 2: 6.62), the positive control had a relative cell viability ≥ 70% as compared to the solvent control (ME 1: 92.46%; ME 2: 122.51%), the average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid, as well as the basal expression of untreated cells was < 1.5 fold as compared to the average solvent control (ME 1: 1.31; ME 2: 0.91), the average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) should be below 20% in each main experiment (ME 1: 7.9%; ME 2: 6.6%) and at least three test concentrations had a cell viability of at least 70% relative to the solvent controls. Moreover, since the result is considered negative, at least one concentration was cytotoxic, i.e. had a cell viability < 70%.


 


In conclusion, the test item did not activate the LuSens cells up to a concentration of 1813 μg/mL 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).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

WoE, Skin sensitisation in chemico (DRPA), RL1


The purpose of this GLP compliant study (based on the OECD Guidelines for the Testing of Chemicals: Test Guideline No. 442C: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)) was to assess the reactivity and sensitizing potential of the test item. 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. Solutions of the test item were analyzed by the DPRA method in both the cysteine and lysine containing synthetic peptides. Based on this assay, with an overall depletion value of 1.74%, the test item was placed in the reactivity class of “no to minimal” and hence it is predicted by DPRA not to be a skin sensitizer.


 


WoE, Skin sensitisation in vitro (ARE-Nrf2 Luciferase Test Method), RL1


A GLP compliant 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 the test item according to OECD guideline 442D. In the cytotoxicity test, cytotoxic effects were observed following incubation with the test item concentration of 2000 μM (highest tested concentration, threshold of cytotoxicity: < 75%). The CV75 value of the cytotoxicity test was calculated as 1510.9 μM. The test item was tested in 2 independent main experiments. The following concentrations of the test item were tested in the main experiments: 729, 874, 1049, 1259, 1511, 1813 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:


The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (ME 1: 8.53; ME 2: 6.62), the positive control had a relative cell viability ≥ 70% as compared to the solvent control (ME 1: 92.46%; ME 2: 122.51%), the average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid, as well as the basal expression of untreated cells was < 1.5 fold as compared to the average solvent control (ME 1: 1.31; ME 2: 0.91), the average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) should be below 20% in each main experiment (ME 1: 7.9%; ME 2: 6.6%) and at least three test concentrations had a cell viability of at least 70% relative to the solvent controls. Moreover, since the result is considered negative, at least one concentration was cytotoxic, i.e. had a cell viability < 70%.


 


In conclusion, the test item did not activate the LuSens cells up to a concentration of 1813 μg/mL 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 test item was not sensitising.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the available data, the test substance is not classified for skin sensitisation according to Regulation (EC) No 1272/2008 (CLP), as amended for sixteenth time in Regulation (EU) No 2021/743.