Registration Dossier
Registration Dossier
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EC number: 210-502-3 | CAS number: 617-04-9
Endpoint summary
Administrative data
Description of key information
The test item is predicted as negative and not to be a potential skin sensitizer in the DPRA (reference 7.4.1-1).
The test item is considered negative for the second key event of the skin sensitisation Adverse Outcome Pathway (reference 7.4.1-2).
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2021-01-26 to 2021-02-18
- 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: On the day of the experiment (immediately before treatment) the test item was dissolved in DMSO to prepare a stock solution with a concentration of 200 mM in accordance to the OECD Guideline 442D.
- Preparation of the test chemical serial dilutions: For the MTT test (dose finding assay) twelve concentrations of the test item were analysed. Therefore, dilutions were prepared by 1:2 serial dilutions from the highest soluble/dispersible concentration.
- Preparation of the positive controls: In Treatment Medium including 1 % (v/v) DMSO, final concentration 120 μM
- - Preparation of the solvent, vehicle and negative controls: Lactic acid with a final concentration of 5000 μM in treatment medium including 1 % (v/v) DMSO; DMSO with a final concentration of 1 % (v/v) in treatment medium
- Stable dispersion obtained: yes
DOSE RANGE FINDING ASSAY:
- Highest concentration used: The highest tested concentration for the dose finding assay was 2000 μM.
- Solubility in solvents: concentration of 200 mM in accordance to the OECD Guideline 442D was possible
- Solubility in incubation medium: not specified
- Cytotoxicity assessment performed: yes, to obtain a CV75
- Final concentration range selected on basis of: Since no CV75 could be determined, the maximum guideline recommended concentration of 2000 μM was used as highest test item concentration and 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
- Number of replicates: solvent: 24, positive control: 5, negative control: 6, medium control: 12, test item: 3 per concentration
- Number of repetitions: 3 independent main experiments. The second main experiment was not valid (the positive control and the average coefficient of variation did not fulfil the acceptance criteria) and therefore, is not be reported.
- Test chemical concentrations: 804, 965, 1157, 1389, 1667, 2000 μM
- 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.
- Exposure time: 48 ± 1 hours
- Study evaluation and decision criteria used:
MTT:
The quantity of formazan is presumably directly proportional to the number of viable cells, as monitored by the absorbance. The relative absorbance (= cell viability) as compared to the solvent control is calculated using this formula: relative absorbance [%]=100x ((absorbancesample – absorbanceblank)/(absorbancesolvent – absorbance blank))
absorbance sample is the MTT-absorbance reading in the sample well
absorbance blank is the MTT-absorbance reading in the blank well (no cells and treatment)
absorbance solvent control is the average MTT-absorbance reading in the wells with cells and solvent control
The arithmetic mean was calculated for each sample: test item concentrations, medium, solvent, negative and positive control.
The CV75 value, a concentration showing 75 % of LuSens cell survival (25 % cytotoxicity), is calculated by using the following equation: CV75=Conc. > 75-(((Conc. >75-Conc. <75)x(%>75-75))/(%>75-%<75))
Conc. >75 = max. measured concentration with the % of solvent control >75 % ≡ a)
Conc. <75 = min. measured concentration with the % of solvent control <75 % ≡ b)
% >75 = relative absorbance at a) in %
% <75 = relative absorbance at b) in %
Luciferase Fold Induction:
The fold induction is calculated using the following formula (OECD 442D): Fold induction= (Lsample-Lblank)/(Lsolvent-Lblank)
where
Lsample is the luminescence reading in the sample well (samples: test item concentrations, medium, negative and positive control)
Lblank is the luminescence reading in the blank well without cells and no treatment
Lsolvent is the average luminescence reading in the wells containing cells and solvent control
The arithmetic mean of the luciferase fold induction was calculated for each sample: test item concentrations, medium, negative and positive control.
- Description on study acceptance criteria:
The following acceptance criteria should be met in the LuSens test method (OECD 442D):
• 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, 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.
SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): 9000 to 11000 LuSens cells per well (96 well microtiter plate); The passage numbers of the used LuSens cells were 9 in the cytotoxicity test and 11 and 15 in the LuSens test for the main experiments 1 and 3, respectively.
- Incubation conditions: for 24 hours ± 30 minutes at 37 ± 1.5 °C and 5.0 ± 0.5 % CO2
- Washing conditions: 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+.
- Precipitation noted: No precipitation was detected.
LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: The absorption and luminescence measurement of the LuSens samples were conducted with a 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 plate
- Lysate preparation: After washing, 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.
DATA EVALUATION
- Cytotoxicity assessment: The CV75 value, a concentration showing 75 % of LuSens cell survival (25 % cytotoxicity), was calculated.
- Prediction model used:
If the luciferase induction is ≥ 1.5 fold and statistically significant compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations (cell viability ≥ 70 %) and at least 3 tested concentrations are non-cytotoxic, the main experiment of the LuSens prediction is considered positive.
If these conditions are met in 2 of 2 or in 2 of 3 main experiments, the LuSens prediction is considered positive, otherwise the LuSens prediction is considered negative.
A negative result obtained with a test item that does not form a stable dispersion and was not tested up to 2000 μM (or 2000 μg/mL for test items with no defined MW) and for which no cytotoxicity is observed in any of the tested concentration should be considered as inconclusive (OECD 442D).
If no clear dose-response curve or a biphasic dose-response curve is observed, the experiment should be repeated to verify whether this is specific to the test item or due to an experimental artefact. If the biphasic response (i.e. when the threshold of 1.5 is crossed twice) is reproducible in an independent verification experiment, the lower concentration of a ≥ 1.5 induction should be reported (i.e. when the threshold of 1.5 is crossed the first time).
Mono constituent test items with a Log Pow > 7 may be insoluble in the culture medium. However, if the test item is soluble or can be stably dispersed/suspended, the LuSens test may be performed. - Vehicle / solvent control:
- DMSO
- Negative control:
- DL-Lactic acid
- Positive control:
- EGDMA (120 M) [442D]
- Positive control results:
- The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (ME 1: 5.26; ME 3: 14.47).
The positive control had a relative cell viability ≥ 70 % as compared to the solvent control (ME 1: 115.86 %; ME 3: 116.99 %). - Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 3
- Parameter:
- other: Fold induction
- Value:
- 1.11
- Cell viability:
- 140.36 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 1
- Parameter:
- other: Fold Induction
- Value:
- 1.03
- Cell viability:
- 90.24 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Outcome of the prediction model:
- negative [in vitro/in chemico]
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
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. 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: 0.46; ME 3: 1.03).
- Acceptance criteria met for positive control: yes, see above
- Acceptance criteria met for variability between replicate measurements: yes. The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20 % in each main experiment (ME 1: 10.9 %; ME 3: 7.6 %). - Interpretation of results:
- GHS criteria not met
- Conclusions:
- The test item is considered negative for the second key event of the skin sensitisation Adverse Outcome Pathway.
- Executive summary:
An in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens) was performed according to OECD 442 D 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 the skin sensitization AOP) of the test item. In the cytotoxicity test, cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (2000 μM). Due to the lack of cytotoxicity, a CV75 value could not be calculated. In this case, the OECD 442D guideline recommends testing a test item concentration of 2000 μM. Five further test item dilutions were prepared by serial dilution with a dilution factor of 1.2. The test item was tested in 3 independent main experiments. The second main experiment was not valid (the positive control and the average coefficient of variation did not fulfil the acceptance criteria) and therefore, is not be reported. The concentrations of 804, 965, 1157, 1389, 1667, 2000 μM test item were tested in the main experiments. After treatment with the test item for 48 ± 1 hours the luciferase induction is < 1.5 fold compared to the solvent control in all tested concentrations. Since these conditions are met in 2 of 2 main experiments, the LuSens prediction is considered negative. All acceptance criteria were met. The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (ME 1: 5.26; ME 3: 14.47). The positive control had a relative cell viability ≥ 70 % as compared to the solvent control (ME 1: 115.86 %; ME 3: 116.99 %). 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: 0.46; ME 3: 1.03). The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20 % in each main experiment (ME 1: 10.9 %; ME 3: 7.6 %). A maximum concentration of 2000 μM has been tested. In conclusion, the test item did not activate the LuSens cells up to a concentration of 2000 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).
- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2021-01-25 to 2021-01-27
- 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:
- adopted 18 June 2019, corrected: 26 June 2020
- Deviations:
- yes
- Remarks:
- Please refer to "Principles of method".
- 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 of test system:
- other: Cysteine peptide: Ac-RFAACAA-OH Lysine peptide: Ac-RFAAKAA-OH
- Details on the study design:
- PREPARATION OF CONTROLS
- Positive control: 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 positive control (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=3
- Reference Control C2: Peptide stability control for the solvent used to dissolve the test item (samples containing only 0.5 mM peptide dissolved in the appropriate peptide buffer and water). 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 sample and the reference control B samples of both peptides were prepared at a concentration of 500 μM in acetonitrile. Reference control C samples were prepared at a concentration of 500 μM in acetonitrile (C1, the solvent used to dissolve the positive control) and in water (C2, the solvent used to dissolve the test item).
PREPARATION OF THE TEST ITEM
The test item was dissolved immediately before testing in deionised water to prepare a 100 mM stock solution.
PREPARATION OF THE PEPTIDE STOCK SOLUTIONS
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of the appropriate peptide in approximately 20 mL 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 PEPTIDE CALIBRATION STANDARDS
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 was 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.
PREPARATION OF POSITIVE CONTROL AND CYSTEINE PEPTIDE DEPLETION SAMPLES AND CO-ELUTION CONTROLS
Triplicate solutions each of the positive control and test item stock solutions were diluted with the cysteine peptide stock solution so as to prepare solutions containing 500 μM cysteine and 5 mM of Cinnamaldehyde or 5 mM of the test item. For the co-elution control, buffer solution was used in place of the cysteine stock solution.
PREPARATION OF POSITIVE CONTROL AND LYSINE PEPTIDE DEPLETION SAMPLES AND CO-ELUTION CONTROLS
Triplicate solutions each of the positive control and test item stock solutions were diluted with the lysine peptide stock solution to prepare solutions containing 500 μM lysine and 25 mM of Cinnamaldehyde or 25 mM of the test item. For the co-elution control, buffer solution was used in place of the lysine stock solution.
TREATMENT
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.
METHOD OF ANALYSIS
Pre-column: Security Guard C18; 4,0 x 2,0 mm ID
Column: Zorbax SB C18; 2,1 x 100 mm; 3,5 μm
Eluent A: 0.1 % trifluoroacetic acid (TFA) in water
Eluent B: 0.085 % trifluoroacetic acid (TFA) in acetonitrile (ACN)
Flow rate: 0.35 mL/min
Detector: DAD (diode-array detector)
Wave length: 220 nm und 258 nm
Oven temperature: 30 °C
Injection volume: 2 μL
Run time: 26 min
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.
- Calculation of peptide depletion: [1- (Peptide peak area in replicate injection/mean peptide peak area in reference control c)]x100
ACCEPTANCE CRITERIA
- Linearity of standard calibration curve: coefficient of determination (r2) > 0.99
- Mean peptide depletion value of positive control: 60.8% - 100% for the cysteine peptide, 40.2% - 69.0% for the lysine peptide
- Maximum standard deviation positive control: < 14.9 percent points for cysteine depletion, < 11.6 percent points for the lysine depletion
- Mean peptide concentration of the Reference Controls A: 0.45 - 0.55 mM
- CV of peptide peak areas of reference controls B and C1: < 15%
- Maximum SD for the test item: < 14.9 percent points for cysteine depletion, < 11.6 percent points for the lysine depletion
- Mean peptide concentration of Reference Controls C (C1 and C2): 0.45 to 0.55 mM - Vehicle / solvent:
- water
- Positive control:
- cinnamic aldehyde
- Positive control results:
- Positive control depletion (percent points)
Cysteine: 74.6 (SD, 0.966 %, n=3)
Lysine: 62.8 (SD, 0.868 %, n=3) - Key result
- Run / experiment:
- mean
- Parameter:
- other: Mean cysteine and lysine % depletion
- Value:
- 1.16 %
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- mean
- Parameter:
- lysine depletion
- Value:
- 0.783 %
- At concentration:
- 25 mM
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- mean
- Parameter:
- mean cystein depletion
- Value:
- 1.54 %
- At concentration:
- 5 mM
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Outcome of the prediction model:
- no or minimal reactivity [in chemico]
- Other effects / acceptance of results:
- OTHER EFFECTS: No other effects observed.
DEMONSTRATION OF TECHNICAL PROFICIENCY: A DPRA proficiency study was conducted at the laboratory under the same conditions as the study.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: not appicable
- 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
- Range of historical values: Please refer to "Any other information on results". - Interpretation of results:
- GHS criteria not met
- Conclusions:
- The results of this skin sensitization study according to OECD guideline 442C (DPRA) showed no to minimal mean depletion of both peptides (1.16 %) in the presence of the test item. It is therefore predicted as negative and not to be a potential skin sensitizer in the DPRA.
- Executive summary:
The purpose of this 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 water when incubated for 24 ± 2 hours in the range between 22.5 and 30 °C.
Solutions of the test item were analyzed by the DPRA method in both the cysteine and lysine containing synthetic peptides. There were no co-elution peaks in either the cysteine or lysine assays. With an overall depletion value of 1.16 %, based on this assay the test item is placed in the reactivity class of “no to minimal” and hence it is predicted by DPRA as negative and not to be a skin sensitizer.
Referenceopen allclose all
Table 1: Results of the main experiment 1 (Cell viability)
Treatment group | Concentration [µM] | Absorbance (OD570) | Mean OD570 | SD OD570 | Mean OD570 blank corr. | Cell viability [%] | |||||
Well 1 | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | ||||||
Blank | 0.013 | ||||||||||
Solvent control | 0.449 | 0.06 | 0.436 | 100.0 | |||||||
Medium control | 0.588 | 0.05 | 0.575 | 132.00 | |||||||
Positive Control | 120 | 0.493 | 0.489 | 0.587 | 0.505 | 0.516 | 0.518 | 0.04 | 0.505 | 115.86 | |
Negative Control | 5000 | 0.430 | 0.403 | 0.419 | 0.485 | 0.506 | 0.514 | 0.460 | 0.05 | 0.447 | 102.44 |
Test Item | 804 | 0.402 | 0.516 | 0.475 | 0.464 | 0.06 | 0.451 | 103.55 | |||
965 | 0.468 | 0.467 | 0.453 | 0.463 | 0.01 | 0.450 | 103.16 | ||||
1157 | 0.427 | 0.431 | 0.402 | 0.420 | 0.02 | 0.407 | 93.38 | ||||
1389 | 0.483 | 0.437 | 0.471 | 0.464 | 0.02 | 0.451 | 103.39 | ||||
1667 | 0.423 | 0.373 | 0.421 | 0.406 | 0.03 | 0.393 | 90.09 | ||||
2000 | 0.403 | 0.393 | 0.423 | 0.406 | 0.02 | 0.393 | 90.24 | ||||
Table 2: Results of the main experiment 1 (Fold induction)
Treatment group | Concentration [µM] | Luminescence | Mean Luminescence | SD Luminescence | Mean Luminescence blank corr. | Fold induction | |||||
Well 1 | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | ||||||
Blank | 214 | ||||||||||
Solvent control | 314.6 | 34.4 | 100.6 | 1.00 | |||||||
Medium control | 265.4 | 28.1 | 51.4 | 0.51 | |||||||
Positive Control | 120 | 857 | 746 | 776 | 695 | 643 | 743.4 | 81.2 | 529.4 | 5.26 | |
Negative Control | 5000 | 244 | 288 | 244 | 288 | 251 | 244 | 259.8 | 22.0 | 45.8 | 0.46 |
Test Item | 804 | 266 | 281 | 303 | 283.3 | 18.6 | 69.3 | 0.69 | |||
965 | 266 | 296 | 303 | 288.3 | 19.7 | 74.3 | 0.74 | ||||
1157 | 259 | 273 | 273 | 268.3 | 8.1 | 54.3 | 0.54 | ||||
1389 | 296 | 355 | 310 | 320.3 | 30.8 | 106.3 | 1.06 | ||||
1667 | 273 | 318 | 281 | 290.7 | 24.0 | 76.7 | 0.76 | ||||
2000 | 333 | 318 | 303 | 318.0 | 15.0 | 104.0 | 1.03 | ||||
Table 3: Results of the main experiment 3 (Cell viability)
Treatment group | Concentration [µM] | Absorbance (OD570) | Mean OD570 | SD OD570 | Mean OD570 blank corr. | Cell viability [%] | |||||
Well 1 | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | ||||||
Blank | 0.014 | ||||||||||
Solvent control | 0.804 | 0.10 | 0.790 | 100.0 | |||||||
Medium control | 1.148 | 0.14 | 1.134 | 143.65 | |||||||
Positive Control | 120 | 0.768 | 0.901 | 0.926 | 1.037 | 1.057 | 0.938 | 0.12 | 0.924 | 116.99 | |
Negative Control | 5000 | 0.874 | 0.910 | 0.931 | 0.903 | 0.905 | 0.930 | 0.909 | 0.02 | 0.895 | 113.32 |
Test Item | 804 | 1.100 | 0.977 | 1.023 | 1.033 | 0.06 | 1.019 | 129.09 | |||
965 | 1.185 | 0.948 | 0.932 | 1.022 | 0.14 | 1.008 | 127.61 | ||||
1157 | 1.243 | 1.100 | 1.162 | 1.168 | 0.07 | 1.154 | 146.19 | ||||
1389 | 1.238 | 1.345 | 1.160 | 1.248 | 0.09 | 1.234 | 156.23 | ||||
1667 | 1.288 | 0.968 | 1.213 | 1.156 | 0.17 | 1.142 | 144.67 | ||||
2000 | 1.175 | 1.150 | 1.042 | 1.122 | 0.07 | 1.108 | 140.36 | ||||
Table 4: Results of the main experiment 3 (Fold induction)
Treatment group | Concentration [µM] | Luminescence | Mean Luminescence | SD Luminescence | Mean Luminescence blank corr. | Fold induction | |||||
Well 1 | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | ||||||
Blank | 148 | ||||||||||
Solvent control | 174.3 | 13.2 | 26.3 | 1.00 | |||||||
Medium control | 179.3 | 16.2 | 31.3 | 1.19 | |||||||
Positive Control | 120 | 539 | 628 | 539 | 488 | 451 | 529.0 | 66.6 | 381.0 | 14.47 | |
Negative Control | 5000 | 200 | 155 | 170 | 163 | 170 | 192 | 175.0 | 17.4 | 27.0 | 1.03 |
Test Item | 804 | 170 | 163 | 192 | 175.0 | 15.1 | 27.0 | 1.03 | |||
965 | 170 | 170 | 170 | 170.0 | 0.0 | 22.0 | 0.84 | ||||
1157 | 163 | 170 | 185 | 172.7 | 11.2 | 24.7 | 0.94 | ||||
1389 | 163 | 170 | 155 | 162.7 | 7.5 | 14.7 | 0.56 | ||||
1667 | 170 | 185 | 185 | 180.0 | 8.7 | 32.0 | 1.22 | ||||
2000 | 185 | 177 | 170 | 177.3 | 7.5 | 29.3 | 1.11 | ||||
Table 5: Historical Control Data (2019)
| Solvent Control | Medium Control | Positive Control | Negative Control | ||||||||
| Luciferase induction | Relative viability [%] | Luciferase induction | Relative viability [%] | Luciferase induction | Relative viability [%] | Luciferase induction | Relative viability [%] | ||||
Min | 1 | 100 | 0.69 | 92.17 | 4.28 | 81.51 | 0.60 | 86.87 |
| |||
Max | 1 | 100 | 1.49 | 167.81 | 14.90 | 129.03 | 1.29 | 128.30 |
| |||
Mean | 1 | 100 | 0.99 | 130.62 | 7.29 | 100.55 | 0.95 | 104.10 |
| |||
SD | 0.0 | 0.0 | 0.25 | 17.65 | 3.09 | 15.81 | 0.15 | 9.61 |
| |||
n | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 |
| |||
Table 1: Analytical acceptance criteria for each peptide run were met:
| Peptide | Standard Linearity | Positive control depletion (percent points) | Reference controls (mean peptide concentration / coefficient of variation) | SD Test item depletion (percent points) |
Acceptance criteria | Cysteine | r2>0.99 | 60.8-100 (SD <14.9 %) | 450 - 550 μM (CV <15 %) | SD <14.9 % |
Lysine | r2>0.99 | 40.2-69.0 (SD <11.6 %) | 450 - 550 μM (CV <15 %) | SD <11.6 % | |
Achieved results | Cysteine | r2=0.9999 | 74.6 (SD, 0.966 %, n=3) | A: 502 μM (CV 0.0131 %, n=1) B: 500 μM (CV 0.654 %, n=6) C1: 497 μM (CV 0.754 %, n=3) C2: 494 μM (CV 1.06 %, n=3) | SD 1.16 % (n=3) |
Lysine | r2=0.9999 | 62.8 (SD, 0.868 %, n=3) | A: 497 μM (CV 0.629 %, n=1) B: 488 μM (CV 1.31 %, n=6) C1: 490 μM (CV 0.910 %, n=3) C2: 484 μM (CV 1.58 %, n=3) | SD 0.902 % (n=3) |
Table 2: Depletion of peptide in the presence test material
| Mean peak area of reference controls | Mean peak area of peptide with test item | Mean peptide depletion by test item (%) | Mean of cysteine and lysine% depletion by test item (%) |
Cysteine | Control B: 3292021 (n=6) Control C1: 3273801 (n=3) | 3204157 (n=3) | 1.54 | 1.16 |
Lysine | Control B: 2810468 (n=6) Control C1: 2826755 (n=3) | 2791274 (n=3) | 0.783 |
Historical control data (03/2018 – 10/2020)
Cysteine samples prepared at a concentration of 500 μM (376 μg/mL).
Lysine samples prepared at a concentration of 500 μM (388 μg/mL).
CV= Coefficient of Variation
Table 3: Historic Data for Reference Control A
| Cysteine peptide concentration (μM) | Lysine peptide concentration (μM) |
Maximum | 552 | 548 |
Minimum | 470 | 486 |
Mean | 507 | 507 |
Number | 54 | 54 |
CV (%) | 2.87 | 2.60 |
Table 4: Historic Data for Reference Control B
| Cysteine3peptide concentration (μM) | Lysine peptide concentration (μM) |
Maximum | 528 | 533 |
Minimum | 433 | 467 |
Mean | 493 | 497 |
Number | 108 | 108 |
CV (%) | 3.10 | 2.41 |
Table 5: Historic Data for Positive Controls
| Cysteine peptide concentration (μM) | Lysine peptide concentration (μM) |
Maximum | 163 | 310 |
Minimum | 120 | 178 |
Mean | 142 a | 272 b |
Number | 54 | 54 |
CV (%) | 7.27 | 11.8 |
a: Overall mean depletion 71.2 % (n=54)
b: Overall mean depletion 45.2 % (n=54)
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
For evaluation of the skin sensitising properties of the test item a weight of evidence approach using in vitro/ in chemico experimental studies was conducted.
DPRA (OECD guideline 442C; reference 7.4.1-1):
The purpose of this 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 (AOP) for the assessment of the skin sensitisation potential of chemicals. The test item was dissolved in water when incubated for 24 ± 2 hours in the range between 22.5 and 30 °C. Solutions of the test item were analyzed by the DPRA method in both the cysteine and lysine containing synthetic peptides. There were no co-elution peaks in either the cysteine or lysine assays. With an overall depletion value of 1.16 %, based on this assay the test item is placed in the reactivity class of “no to minimal” and hence it is predicted by DPRA as negative and not to be a skin sensitizer.
LuSens (OECD guideline 442D; reference 7.4.1-2):
An in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens) was performed according to OECD 442 D 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 the skin sensitization AOP) of the test item. In the cytotoxicity test, cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (2000 μM). Due to the lack of cytotoxicity, a CV75 value could not be calculated. In this case, the OECD 442D guideline recommends testing a test item concentration of 2000 μM. Five further test item dilutions were prepared by serial dilution with a dilution factor of 1.2. The test item was tested in 3 independent main experiments. The second main experiment was not valid (the positive control and the average coefficient of variation did not fulfil the acceptance criteria) and therefore, is not be reported. The concentrations of 804, 965, 1157, 1389, 1667, 2000 μM test item were tested in the main experiments. After treatment with the test item for 48 ± 1 hours the luciferase induction is < 1.5 fold compared to the solvent control in all tested concentrations. Since these conditions are met in 2 of 2 main experiments, the LuSens prediction is considered negative. All acceptance criteria were met. The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (ME 1: 5.26; ME 3: 14.47). The positive control had a relative cell viability ≥ 70 % as compared to the solvent control (ME 1: 115.86 %; ME 3: 116.99 %). 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: 0.46; ME 3: 1.03). The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20 % in each main experiment (ME 1: 10.9 %; ME 3: 7.6 %). A maximum concentration of 2000 μM has been tested. In conclusion, the test item did not activate the LuSens cells up to a concentration of 2000 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.
Conclusion
Based on the results of an in chemico/in vitro test strategy the test item is not peptide reactive (DPRA, OECD TG 442C) and does not activate keratinocytes (LuSens, OECD TG 422D). As two out of three key events of the Adverse Outcome Pathway showed no potential of the test item to cause effects related to skin sensitization and already allow classification and risk assessment, studies addressing the other key event need not be conducted. Therefore, the test item is predicted to be no skin sensitizer.
This is further supported with a (Q)SAR prediction.
Supporting, Derek Nexus v6.0.1 (reference 7.4.1-3):
Using Derek Nexus v6.0.1, the test item was predicted to be a non-sensitiser. The substance is not within the applicability domain of the model. Thus the estimation may be less accurate.
The adequacy of a prediction depends on the following conditions:
a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;
b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;
c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;
d) the (Q)SAR model is relevant for the regulatory purpose.For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.
Description of the prediction model
The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file.
Assessment of estimation domain
The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.
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 data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitisation, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.
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