Registration Dossier
Registration Dossier
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EC number: 214-333-6 | CAS number: 1121-60-4
Endpoint summary
Administrative data
Description of key information
As a weight of evidence approach, three in vitro assays were carried out, namely DPRA, KeratinoSens and h-CLAT assay.
The test item was postive regarding skin sensitisation in the DPRA assay, KeratinoSens assay and hCLAT assay.
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:
- 2018-01-08 to 2018-03-29
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guidelines for Testing of Chemicals, No. 442E: “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)”
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158
- Version / remarks:
- 2015
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of dendritic cells
- Justification for non-LLNA method:
- This test method is able to detect chemicals that have sensitisation potential by addressing the third molecular key event of the adverse outcome pathway, namely dendritic celll activation, and allows for hazard identification in accordance with UN GHS “Category 1”. Data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as Integrated Approach to Testing and Assessment (IATA), combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the adverse outcome pathway.
- Specific details on test material used for the study:
- Two batches of test item were used for this assay. The preliminary solubility test and dose range finding assays 1, 2 and 3 were performed using batch 2071700008 of the test item. Dose range finding assay 4 and the main experiments used batch 2901700033.
- Details on the study design:
- TEST SYSTEM:
Cell line: The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for dendritic cells (DC). Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 10^6 cells/mL.
Cells were cultured in 75 cm2 culture flasks (Greiner) RPMI-1640, supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin/ 100 µg/mL streptomycin at 37 ¿ 1°C and 5% CO2.
Stock: The test item was dissolved in 0.9% NaCl solution at a concentration of 100 mg/mL. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2. The working stock solutions were prepared by diluting each stock solution 50 times with cell culture medium. No precipitation, turbidity or phase separation was observed when diluted 1:50 in cell culture medium. The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent (0.9% NaCl solution) was present at a constant volume ratio of 1% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.
CD54 and CD86 Expression:
THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 10^6 cells/mL.
500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10^6 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at
37 °C ± 1 °C and 5% CO2.
Blocking solution: 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min.
Staining: 50 µL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement (final concentration of PI was 0.625 µg/mL).
Expression level and cell viability: The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of ¿ = 488 nm and an emission wavelength of ¿ = 530 nm ± 15 nm for FITC and ¿ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated
Concentrations:
Test item doses in experiments 1, 2 and 3: 237.58, 197.98, 164.98, 137.49, 114.57, 95.48, 79.56, 66.30 µg/mL.
Controls: A medium control, a solvent control, and a positive control were set up in parallel in order to confirm the validity of the test.
Medium Control: A medium control was included in the test. Since the test item was solubilized in either cell culture medium or 0.9% NaCl, the medium control served as a solvent control.
Positive Control: 2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 µg/mL and nickel sulphate at a final concentration of 100 µg/mL.
Negative control: Lactic acid at a final concentration of 1000 µg/mL. - Positive control results:
- Please refer to 'any other information on results incl. tables'.
- Key result
- Run / experiment:
- other: 1,2 and 3
- Parameter:
- other: RFI CD86 [%]
- Value:
- 150
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: The RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability = 50% in at least two independent runs
- Key result
- Run / experiment:
- other: 1,2 and 3
- Parameter:
- other: RFI CD54 [%]
- Value:
- 200
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: The RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability = 50% in at least two independent runs
- Interpretation of results:
- Category 1 (skin sensitising) based on GHS criteria
- Conclusions:
- In this study under the given conditions the test item did upregulate the cell surface markers in at least three independent experiment runs. Therefore, the test item is considered to be a skin sensitiser.
In the context of the IATA, combining the results of the three available in vitro tests (OECD 442 D, C and E) the test item is classified as a Category 1 skin sensitiser (H317) according to CLP criteria (Regulation EC No 1272/2008). - Executive summary:
The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.
Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.
In the present study Pyridine-2-Aldehyde was dissolved in 0.9% NaCl. For the dose finding assay stock solutions with concentrations ranging from 100 mg/mL to 0.78 mg/mL (experiment 1) and from 500 mg/mL to 3.91 mg/mL (experiment 2, 3 and 4) were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.
A CV75 of 197.98 ± 22.91 µg/mL was derived in the dose finding assay 1 -3 (batch 2071700008). Since the dose finding assay 1-3 were performed using an expired batch 2071700008 of the test item, a fourth dose finding was performed with the batch 2901700033, to verify the result. The CV75 derived from one independent run with batch 2901700033 was found to be 201.46 µg/mL which was in line with the CV75 derived from dose finding assays 1-3. Therefore, the concentration range for the main experiment that was calculated based on the CV75 of dose finding assay 2 and 3 were accepted for further testing.
Based on that CV75, the main experiment was performed covering the following concentration steps: 237.58, 197.98, 164.98, 137.49, 114.57, 95.48, 79.56, 66.30 µg/mL
In all experiments no precipitation or turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.
Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.
Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentrationwas reduced to 64.0%(CD86), 62.7% (CD54) and 62.0% (isotype IgG1 control) inthe first experiment, to 55.6% (CD86), 54.0% (CD54) and 54.1% (isotype IgG1 control) in thesecond experiment and to 51.0% (CD86), 52.4% (CD54) and 51.6% (isotype IgG1 control) in the third experiment.
In the first experiment, an increase in the expression of the cell surface marker CD86 was observed from 66.30 up to 114.57 µg/mL, with the highest increase of up to 238% observed at 95.48 µg/mL. In the second experiment, an increase in the expression of CD86 was observed from 66.30 up to 137.49 µg/mL, with the highest increase of up to 273% observed at 95.48 µg/mL. In the third experiment, an increase in the expression of CD86 was observed from 66.30 up to 137.49 µg/mL, with the highest increase of up to 260% observed at 79.57 µg/mL.
In the first experiment, an increase in the expression of CD54 was observed from 66.30 up to 164.99 µg/mL, with the highest increase of up to 689% observed at 95.48 µg/mL. In the second experiment, an increase in the expression of CD54 was observed from 66.30 up to 137.49 µg/mL, with the highest increase of up to 452% observed at 95.48 µg/mL. In the third experiment, an increase in the expression of CD54 was observed from 66.30 up to 137.49 µg/mL, with the highest increase of up to 500% observed at 95.48 µg/mL.
Since the expression of both cell surface markers clearly exceeded the threshold in three independent experiments the test item is considered to be a skin sensitiser.
The controls confirmed the validity of the study for all experiments.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.
- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2017-11-03 to 2018-03-30
- 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))
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154
- Version / remarks:
- 2013
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
- Justification for non-LLNA method:
- This test method is able to detect chemicals that cause skin sensitisation and allows for hazard identification in accordance with UN GHS “Category 1”. Data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the adverse outcome pathway.
- Details on the study design:
- Skin sensitisation (In chemico test system) - Details on study design:
Preparation of the test Item: The test item was freshly prepared immediately prior to use.
The test item was pre-weighed into a glass vial and was dissolved in acetonitrile by shortly inverting the tube. The test item was immediately soluble in acetonitrile without the need of vortex mixing or ultra-sonic treatment. Acetonitrile was confirmed to be an appropriate solvent in a preliminary experiment. A stock solution of a concentration of 100 mM was prepared
- Controls
Reference controls (RC), co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.
- Postive Control
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was dissolved in acetonitrile and was used as a positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
- Co-elution Control
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and were included in every assay run for both peptides.
- Reference Control
Reference controls were set up in parallel to sample preparation in order to verify the validity of the test run.
Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run.
Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and end of each HPLC run.
Reference control C was set up for the test item and the positive control and prepared using acetonitrile in order to verify the solvent does not impact the percent peptide depletion (PPD). In addition, reference control C was used to calculate PPD. Reference control C was included in every assay run for both peptides and was injected together with the samples.
Test System:
For HPLC system and HPLC mobile phase see table 1 and 2
Peptides:
19.85 mg cysteine peptide (>95% purity) with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (38.60 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
20.09 mg lysine peptide (>95% purity) with an amino acid sequence of Ac RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (38.16 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.
Dose Groups:
Reference Control C (solvent control): undiluted
Test Item: 100 mM stock solution
Positive Control: 100 mM stock solution
Preliminary Experiment:
Solubility of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100 mM). Solubility was investigated in the following solvent suitable for the test:
acetonitrile
Following a visual inspection the test item was confirmed to be completely soluble in acetonitrile. Therefore, acetonitrile was chosen as suitable solvent for the main experiments.
Experimental Procedure:
Incubation of the Test Item with the Cysteine and Lysine Peptide
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. Samples were prepared according to the scheme described in table 3.
Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 400x g) to force precipitates to the bottom of the vial.
After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using HPLC.
Data Analysis:
The concentration of the cysteine and lysine peptide was determined in each sample form absorbance at lambda = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions.
The percent peptide depletion (PPD) was calculated according to the following formula:
PPD=(1-((Peptide Peak Area in the Replicate Injection)/(Mean Peptide Peak Area in Reference Contr
ol C)))*100
Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive to be a skin sensitiser in accordance with UN GHS “Category 1”, if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as “0” when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration differently from 100 mM. By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model) shown in table 4 the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers. Application of the prediction model for assigning a test item to a
reactivity class (i.e. low, moderate or high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA. In the framework of an IATA the test substance may be considered as non-sensitiser to skin in accordance with UN GHS “No Category” if the mean depletion of both peptides is below 6.38% (see Table 4 and 5).
Acceptance Criteria:
The run meets the acceptance criteria if:
- the standard calibration curve has a r² >0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is <14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is <11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is <15.0%.
The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is <14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is <11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM. - Positive control results:
- Positive controls depleted cysteine and lysine peptides by 70.42 % (SD 0.1) and 63.12 % (SD 0.7) respectively, hence being positive according to evaluation criteria.
- Key result
- Run / experiment:
- other: Cysteine and Lysine peptide depletion
- Parameter:
- other: % mean peptide depletion
- Value:
- 19.27
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Other effects / acceptance of results:
- Pre-Experiments:
Since the test item gave a visibily acceptable solution in acetonitrile, acetonitrile was chosen as a suitable vehicle.
Precipitation and Phase Separation:
For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.
For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive control (including the co-elution control). No precipitation, turbidity or phase separation was observed for the samples of the test item.
Coe-elution with the peptide peaks:
Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.
No co-elution of test item with the peptide peaks was observed, therefore, data are interpreted using prediction model 1.
Results Calibration Curve:
Cysteine Peptide Calibration Curve: y = 9664.62x + 11.85 ; R² = 0.9997
Lysine Peptide Calibration Curve: y = 26.85x+0.01 ; R² = 1.0000
For detailed results see tables 6 to 10. - Interpretation of results:
- Category 1 (skin sensitising) based on GHS criteria
- Conclusions:
- In this study, under the given conditions, the test item showed 'low reactivity' towards both peptides. The test item is considered as a "sensitiser".
In the context of the IATA, combining the results of the three available in vitro tests (OECD 442 D, C and E) the test item is classified as a Category 1 skin sensitiser (H317) according to CLP criteria (Regulation EC No 1272/2008). - Executive summary:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.
The test item showed low reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was > 6.38% (19.27%). Based on Prediction Model 1, the test item can be considered as a "sensitiser".
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2018-01-08 to 2018-03-30
- 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)
- Qualifier:
- according to guideline
- Guideline:
- other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155
- Version / remarks:
- 2015
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of keratinocytes
- Justification for non-LLNA method:
- This test method is able to detect chemicals that have sensitisation potential by addressing the second molecular key event of the adverse outcome pathway, namely the activation of keratinocytes, and allows for hazard identification in accordance with UN GHS “Category 1”. Data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the adverse outcome pathway.
- Specific details on test material used for the study:
- Two batches of test item were used for this assay. The preliminary solubility test was performed using batch 2071700008 of the test item. For the main experiments batch 2901700033 of the test item was used. Since the test item was also well soluble in DMSO in the main experiment, the solubility test can be considered as acceptable for both batches.
- Details on the study design:
- TEST SYSTEM
Cell line: transgenic cell line KeratinoSens™ (Givaudan, Switzerland) derived from human keratinocytes (HaCaT)
TEST SUBSTANCE PREPARATION
Concentration:
- Positive control doses: 4, 8, 16, 32 or 64 µM.
- Test item doses: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 or 2000 µM.
- Negative control doses: Details not reported.
Stock: A stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent.
Vehicle: Dimethyl sulfoxide (DMSO)
CONTROLS:
Positive Control: Cinnamic aldehyde
Negative control : DMSO
Blank control: No cells and DMSO
MEDIUM
- Maintenance Medium:
Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and Na-PyruvateDulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and Na-Pyruvate.
The medium was supplemented with the following components:
-10% fetal bovine calf serum.
-1% geneticin (final concentration: 500 µg/mL).
- Assay Medium:
Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
-10% fetal bovine calf serum.
Luciferase activity:
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light. Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.
Cell viability:
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. The medium was then removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight. After the incubation period the plate was shaken for 10 min and the OD was measured at ¿ = 600 nm.
ANALYSIS
ACCEPTANCE CRITERIA
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC 1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition. - Positive control results:
- Please refer to 'any other information on results incl. tables'.
- Run / experiment:
- other: 1
- Parameter:
- other: Imax
- Value:
- 20.31
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 1
- Parameter:
- other: Cell viability
- Value:
- 107.3
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Run / experiment:
- other: 1
- Parameter:
- other: EC 1.5 (µM)
- Value:
- 53.83
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Run / experiment:
- other: 2
- Parameter:
- other: Imax
- Value:
- 29.94
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 2
- Parameter:
- other: Cell viability
- Value:
- 5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Run / experiment:
- other: 2
- Parameter:
- other: EC 1.5 (µM)
- Value:
- 34.61
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.20 (experiment 1); 4.97 (experiment 2)).
The calculated EC1.5 was between 7 and 34 µM (16.97 µM (experiment 1) and 15.91 µM (experiment 2)).
The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (10% (experiment 1); 13% (experiment 2)). - Interpretation of results:
- Category 1 (skin sensitising) based on GHS criteria
- Conclusions:
- In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered a sensitiser.
In the context of the IATA, combining the results of the three available in vitro tests (OECD 442 D, C and E) the test item is classified as a Category 1 skin sensitiser (H317) according to CLP criteria (Regulation EC No 1272/2008). - Executive summary:
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.
In the present study Pyridine-2-Aldehyde was dissolved in DMSO. Based on a molecular weight of 107.11 g/mol a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:
2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM
Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.
In the first experiment, a max luciferase activity (Imax) induction of 20.31 was determined at a test item concentration of 250.00 µM. The corresponding cell viability was 107.3%. The lowest tested concentration with a significant luciferase induction >1.5 (1.63) was found to be 62.50 µM. The corresponding cell viability was >70% (95.0%).The calculated EC1.5was<1000 µM (53.83 µM).
In the second experiment, a max luciferase activity (Imax) induction of 29.94 was determined at a test item concentration of 1000.00 µM. The corresponding cell viability was 5.0%. The lowest tested concentration with a significant luciferase induction >1.5 (2.39) was found to be 62.50 µM. The corresponding cell viability was >70% (111.7%).The calculated EC1.5was<1000 µM (34.61 µM).
A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.
Under the condition of this study the test item is therefore considered as a sensitiser.
Referenceopen allclose all
Results of the Cell Batch Activation Test
Sample |
Concentration |
CD86 |
CD54 |
Activated |
Pass /Fail |
||||
Cell Viability [%] |
RFI |
Threshold OECD TG 442E |
Cell Viability [%] |
RFI |
Threshold OECD TG 442E |
yes/no |
|||
DNCB |
4 µg/mL |
81.1 |
347 |
>150 |
79.7 |
269 |
>200 |
yes |
pass |
NiSO4 |
100 µg/mL |
82.1 |
347 |
>150 |
82.5 |
391 |
>200 |
yes |
pass |
LA |
1000 µg/mL |
96.7 |
89 |
</=150 |
96.4 |
109 |
</=200 |
no |
pass |
The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86.
CD54 and CD86 Expression Experiment 1
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Flourescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
CD86 |
CD54 |
||
Medium Control |
- |
96.5 |
96.2 |
95.2 |
3671 |
1717 |
662 |
3009 |
1055 |
100 |
100 |
555 |
259 |
Solvent Control |
0.20% |
95.5 |
95.7 |
95.5 |
3573 |
1670 |
639 |
2934 |
1031 |
98 |
98 |
559 |
261 |
DNCB |
4.00 |
78.8 |
77.5 |
78.5 |
11590 |
3661 |
639 |
10951 |
3022 |
373 |
293 |
1814 |
573 |
Pyridine-2-Aldehyde |
237.58 |
64.0 |
62.7 |
62.0 |
3560 |
1209 |
839 |
2721 |
370 |
90 |
35 |
424 |
144 |
197.98 |
71.5 |
71.3 |
70.7 |
3164 |
1471 |
731 |
2433 |
740 |
81 |
70 |
433 |
201 |
|
164.99 |
73.9 |
73.7 |
72.8 |
4367 |
2924 |
718 |
3649 |
2206 |
121 |
209 |
608 |
407 |
|
137.49 |
74.3 |
73.5 |
74.3 |
5228 |
4633 |
733 |
4495 |
3900 |
149 |
370 |
713 |
632 |
|
114.57 |
78.1 |
77.8 |
77.9 |
7672 |
6418 |
719 |
6953 |
5699 |
231 |
540 |
1067 |
893 |
|
95.48 |
79.9 |
79.7 |
79.0 |
7983 |
8078 |
811 |
7172 |
7267 |
238 |
689 |
984 |
996 |
|
79.57 |
82.1 |
82.9 |
81.5 |
7170 |
6649 |
731 |
6439 |
5918 |
214 |
561 |
981 |
910 |
|
66.30 |
83.4 |
83.0 |
83.4 |
6231 |
4588 |
729 |
5502 |
3859 |
183 |
366 |
855 |
629 |
|
CD54 and CD86 Expression Experiment 2
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Flourescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
IgG Isotype |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
C86 |
CD54 |
||
Medium Control |
- |
95.5 |
95.6 |
95.5 |
2692 |
1373 |
636 |
2056 |
737 |
100 |
100 |
423 |
216 |
Solvent Control |
0.20% |
95.9 |
95.6 |
95.4 |
2792 |
1412 |
610 |
2182 |
802 |
106 |
109 |
458 |
231 |
DNCB |
4.0 |
82.2 |
81.9 |
82.9 |
7243 |
2334 |
639 |
6604 |
1695 |
303 |
211 |
1133 |
365 |
Pyridine-2-Aldehyde |
237.58 |
55.6 |
54.0 |
54.1 |
2991 |
1140 |
784 |
2207 |
356 |
107 |
48 |
382 |
145 |
197.98 |
66.7 |
67.4 |
66.1 |
2620 |
1188 |
701 |
1919 |
487 |
93 |
66 |
374 |
169 |
|
164.99 |
71.2 |
72.4 |
71.4 |
3189 |
1857 |
666 |
2523 |
1191 |
123 |
162 |
479 |
279 |
|
137.49 |
70.9 |
72.9 |
73.2 |
4251 |
2620 |
685 |
3566 |
1935 |
173 |
263 |
621 |
382 |
|
114.57 |
76.3 |
76.4 |
75.9 |
5954 |
3605 |
840 |
5114 |
2765 |
249 |
375 |
709 |
429 |
|
95.48 |
79.5 |
78.8 |
79.4 |
6403 |
4120 |
792 |
5611 |
3328 |
273 |
452 |
808 |
520 |
|
79.57 |
80.2 |
80.8 |
80.1 |
6159 |
3630 |
829 |
5330 |
2801 |
259 |
380 |
743 |
438 |
|
66.30 |
82.5 |
82.9 |
83.3 |
5222 |
2634 |
1042 |
4180 |
1592 |
203 |
216 |
501 |
253 |
|
CD54 and CD86 Expression Experiment 3
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Flourescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
IgG Isotype |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
C86 |
CD54 |
||
Medium Control |
- |
96.2 |
96.2 |
95.5 |
2816 |
1265 |
613 |
2203 |
652 |
100 |
100 |
459 |
206 |
Solvent Control |
0.20% |
95.0 |
95.6 |
95.3 |
3107 |
1268 |
609 |
2498 |
659 |
113 |
101 |
510 |
208 |
DNCB |
4.0 |
81.3 |
81.7 |
81.8 |
6285 |
2166 |
677 |
5608 |
1489 |
225 |
226 |
928 |
320 |
Pyridine-2-Aldehyde |
237.58 |
51.0 |
52.4 |
51.6 |
3212 |
1259 |
923 |
2289 |
336 |
104 |
52 |
348 |
136 |
197.98 |
60.6 |
59.4 |
59.7 |
2840 |
1218 |
769 |
2071 |
449 |
94 |
69 |
369 |
158 |
|
164.99 |
65.5 |
66.2 |
65.7 |
2934 |
1784 |
838 |
2096 |
946 |
95 |
145 |
350 |
213 |
|
137.49 |
69.2 |
68.3 |
67.5 |
4302 |
3135 |
765 |
3537 |
2370 |
161 |
364 |
562 |
410 |
|
114.57 |
72.5 |
71.1 |
71.0 |
5636 |
3920 |
749 |
4887 |
3171 |
222 |
486 |
752 |
523 |
|
95.48 |
75.2 |
76.2 |
75.4 |
6429 |
4086 |
828 |
5601 |
3258 |
254 |
500 |
776 |
493 |
|
79.57 |
76.7 |
78.3 |
75.4 |
6553 |
3684 |
832 |
5721 |
2852 |
260 |
437 |
788 |
443 |
|
66.30 |
75.4 |
77.6 |
76.1 |
5978 |
2979 |
966 |
5012 |
2013 |
228 |
309 |
619 |
308 |
|
Acceptance Criteria
Acceptance Criterion |
range |
Experiment 1 |
pass/fail |
Experiment 2 |
pass/fail |
Experiment 3 |
pass/fail |
||||||
cell viability medium and solvent control [%] |
>90 |
95.2 |
- |
96.5 |
pass |
95.4 |
- |
95.9 |
pass |
95.0 |
- |
96.2 |
pass |
number of test dosed with viability >50% CD86 |
=4 |
8 |
pass |
8 |
pass |
7 |
pass |
||||||
number of test dosed with viability >50% CD54 |
=4 |
8 |
pass |
8 |
pass |
8 |
pass |
||||||
number of test dosed with viability >50% IgG1 |
=4 |
8 |
pass |
8 |
pass |
8 |
pass |
||||||
RFI of positive control of CD86 |
=150 |
373 |
pass |
303 |
pass |
225 |
pass |
||||||
RFI of positive control of CD54 |
=200 |
293 |
pass |
211 |
pass |
226 |
pass |
||||||
RFI of solvent control of CD86 |
<150 |
98 |
pass |
106 |
pass |
113 |
pass |
||||||
RFI of solvent control of CD54 |
<200 |
98 |
pass |
109 |
pass |
101 |
pass |
||||||
MFI ratio IgG1/CD86 for medium control [%] |
>105 |
555 |
pass |
423 |
pass |
459 |
pass |
||||||
MFI ratio IgG1/CD86 for solvent control [%] |
>105 |
559 |
pass |
458 |
pass |
510 |
pass |
||||||
MFI ratio IgG1/CD54 for medium control [%] |
>105 |
259 |
pass |
216 |
pass |
206 |
pass |
||||||
MFI ratio IgG1/CD54 for solvent control [%] |
>105 |
261 |
pass |
231 |
pass |
208 |
pass |
||||||
Table 6: Results of the Cysteine Peptide Depletion
Cysteine Peptide |
||||||
Sample |
Peak Area |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
1463.307 |
0.1502 |
70.30 |
70.42 |
0.10 |
0.15 |
1455.760 |
0.1494 |
70.46 |
||||
1453.604 |
0.1492 |
70.50 |
||||
Test Item |
3375.259 |
0.3480 |
31.50 |
35.18 |
3.39 |
9.63 |
3160.271 |
0.3258 |
35.87 |
||||
3046.368 |
0.3140 |
38.18 |
||||
Table 7: Results of the Lysine Peptide Depletion
Lysine Peptide |
||||||
Sample |
Peak Area |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
4.951 |
0.1842 |
63.28 |
63.12 |
0.70 |
1.11 |
4.891 |
0.1819 |
63.72 |
||||
5.077 |
0.1888 |
62.35 |
||||
Test Item |
13.028 |
0.4849 |
3.38 |
3.37 |
0.38 |
11.40 |
12.978 |
0.4831 |
3.74 |
||||
13.082 |
0.4870 |
2.98 |
||||
Table 8: Categorization of the Test Item
Prediction Model |
Prediction Model 1 |
Prediction Model 2 |
||||
Test Substance |
Mean Peptide Depletion [%] |
Reactivity Category |
Prediction |
Mean Peptide Depletion [%] |
Reactivity Category |
Prediction |
Test Item |
19.27 |
Low Reactivity |
Sensitiser |
35.18 |
Moderate Reactivity |
Sensitiser |
Positive Control |
66.77 |
High Reactivity |
Sensitiser |
70.42 |
Moderate Reactivity |
Sensitiser |
Table 9: Acceptance Criteria for Cysteine Peptide
Cysteine Peptide Run |
|||
Acceptance Criterion |
Range |
Value |
pass/fail |
Coefficient of determination |
R² > 0.99 |
0.9997 |
pass |
Mean peptide concentration of RC A |
0.45=x = 0.55 mM |
0.5072 |
pass |
Mean peptide concentration of RC C (PC) |
0.45=x = 0.55 mM |
0.5086 |
pass |
Mean peptide concentration of RC C (TI) |
0.45=x = 0.55 mM |
0.5086 |
pass |
CV of the peak area of RC B |
< 15% |
0.76 |
pass |
CV of the peak area of RC C (PC) |
< 15% |
0.27 |
pass |
CV of the peak area of RC C (TI) |
< 15% |
0.27 |
pass |
Mean peptide depletion of the PC |
60.8% < x < 100% |
70.42 |
pass |
SD of peptide depletion of the PC replicates |
< 14.9% |
0.10 |
pass |
SD of peptide depletion of the TI replicates |
< 14.9% |
3.39 |
pass |
Table 10: Acceptance Criteria for Lysine Peptide
Lysine Peptide Run |
|||
Acceptance Criterion |
Range |
Value |
pass/fail |
Coefficient of determination |
R² > 0.99 |
1.0000 |
pass |
Mean peptide concentration of RC A |
0.45=x = 0.55 mM |
0.5027 |
pass |
Mean peptide concentration of RC C (PC) |
0.45=x = 0.55 mM |
0.5019 |
pass |
Mean peptide concentration of RC C (TI) |
0.45=x = 0.55 mM |
0.5019 |
pass |
CV of the peak area of RC B |
< 15% |
0.45 |
pass |
CV of the peak area of RC C (PC) |
< 15% |
0.17 |
pass |
CV of the peak area of RC C (TI) |
< 15% |
0.17 |
pass |
Mean peptide depletion of the PC |
40.2% < x < 69.0% |
63.12 |
pass |
SD of peptide depletion of the PC replicates |
< 11.6% |
0.70 |
pass |
SD of peptide depletion of the TI replicates |
< 11.6% |
0.38 |
pass |
Induction of Luciferase Activity – Overall Induction
|
Concentration [µM] |
Fold Induction |
Significance |
|||
Experiment 1 |
Experiment 2 |
Mean |
SD |
|||
Solvent Control |
- |
1.00 |
1.00 |
1.00 |
0.00 |
|
Positive Control |
4.00 |
1.16 |
1.14 |
1.15 |
0.02 |
|
8.00 |
1.22 |
1.19 |
1.21 |
0.02 |
|
|
16.00 |
1.48 |
1.50 |
1.49 |
0.02 |
|
|
32.00 |
1.81 |
1.88 |
1.85 |
0.05 |
* |
|
64.00 |
4.20 |
4.97 |
4.58 |
0.55 |
* |
|
Test Item |
0.98 |
0.94 |
1.22 |
1.08 |
0.20 |
|
1.95 |
1.01 |
1.10 |
1.05 |
0.06 |
|
|
3.91 |
0.96 |
1.06 |
1.01 |
0.07 |
|
|
7.81 |
0.99 |
1.13 |
1.06 |
0.10 |
|
|
15.63 |
1.01 |
1.33 |
1.17 |
0.23 |
|
|
31.25 |
1.16 |
1.39 |
1.28 |
0.16 |
|
|
62.50 |
1.63 |
2.39 |
2.01 |
0.54 |
|
|
125.00 |
4.70 |
5.20 |
4.95 |
0.35 |
* |
|
250.00 |
20.31 |
22.75 |
21.53 |
1.73 |
* |
|
500.00 |
n.a. |
n.a. |
n.a. |
n.a. |
n.a. |
|
1000.00 |
14.18 |
29.94 |
22.06 |
11.15 |
|
|
2000.00 |
0.01 |
0.01 |
0.01 |
0.00 |
|
|
* = significant induction according to Student’s t-test, p<0.05
n.a.: not applicable. Values could not be calculated, due to an overflow during the luminescence measurement
Calculated Parameters
Parameter |
Experiment 1 |
Experiment 2 |
Mean |
SD |
EC1.5[µM] |
53.83 |
34.61 |
44.22 |
13.59 |
Imax |
20.31 |
29.94 |
25.13 |
6.81 |
IC30[µM] |
471.59 |
388.75 |
430.17 |
58.57 |
IC50[µM] |
622.43 |
474.67 |
548.55 |
104 .48 |
n.a.: not applicable
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (sensitising)
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
The available data include 3 in vitro assays as each considers different events of the adverse outcome pathway for skin sensitisation. It is considered that these tests can be used in combination to support the discrimination between skin sensitisers and non-sensitisers. However, it is noted that the tests do not allow for the classification of skin sensitisers into subcategories 1A and 1B as defined by UN GHS/ CLP criteria.
Therefore, considering all of the available data (postive results in DPRA assay, KeratinoSens assay and hCLAT assay), the test substance is classified as a Category 1 skin sensitiser (H317) according to CLP criteria (Regulation EC No 1272/2008).
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
