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Key value for chemical safety assessment

Skin sensitisation

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Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Specific details on test material used for the study:
Expiry Date: 09 November 2018
Storage Conditions: at room temperature, protection from light
Description: Off-white solid
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 an appropriate solvent previously determined in a pre-experiment. A stock solution of 100 mM was prepared.
CONTROLS:
- Reference controls, 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.
- Positive Control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as 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 the positive control and were included in every assay run for both peptides.
- Reference Control: Reference controls (RCs) 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 in the end of each HPLC run. Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was the same as for the positive control because no (further) solvent was used. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples
TEST SYSTEM
- HPLC System: HPLC/DAD: Agilent, 1200 Series with Chemstation, Rev. B.04.01; Detection: 220 nm signal for quantitation, 258 nm signal used as indicator for co-elution; Analytical Column: Zorbax SB-C18, 100 mm x 2.1 mm, 3.5 µm, Agilent Art. Nr. 861753-902; Pre-Column: Phenomenex, AJO-4286, 4.0 x 2.0 mm; Column Temperature: 30°C; Sample Temperature: 25°C; Run Time: 20 minutes; Gradient: Time (0 min, 10 min, 11 min, 13 min, 13.5 min), Flow: 0.35 mL/min (all time points), %A: 90, 75, 10, 10, 90, %B: 10, 25, 90, 90, 10, End run at 20 min. Injection Volume: 10µL
- HPLC Mobile Phase A: 0.1% v/v trifluoroacetic acid in water; HPLC Mobile Phase B: 0.085% v/v trifluoroacetic acid in acetonitrile
- Peptides: 20.02 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (38.93 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM. 19.82 mg lysine peptide 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 (37.63 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.
PRE-EXPERIMENTS:
- 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
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. 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 the following HPLC procedure.
- Preparation of the HPLC Standard Calibration Curve: A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile : 80% buffer (v/ v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions covering the range 0-0.534 mM.
- HPLC Preparation and Analysis: Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30°C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected. HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days all test chemical solutions were freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.
DATA ANALYSIS
The concentration of the cysteine and lysine peptide was determined in each sample from absorbance at λ = 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 Control 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) 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%. In case of co-elution of the test item with a peptide peak, the peak cannot be integrated correctly and the calculation of the PPD is not possible. If severe co-elution occurs with both peptides then the analysis was reported as "inconclusive". In cases where the co-elution occurs only with the lysine peptide prediction model 2 can be applied (cysteine 1:10 prediction model).
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 < 1 1.6%, 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 < 1 1.6% 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:
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.21%.
Key result
Parameter:
other: Mean peptide depletion of both peptides
Value:
0.68
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
- Pre-Experiments: Solubility of the test item was determined prior to the main experiment. The test item was soluble in acetonitrile. No turbidity, precipitation and phase separation was observed for the test item solutions. All test item preparations of the main experiment were prepared using acetonitrile. All test item solutions were freshly prepared immediately prior to use.
- Precipitation and Phase Separation: All test item solutions were freshly prepared immediately prior to use. 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. Phase separation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis. For the 100 mM stock 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. No precipitation, turbidity or phase separation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.
- Co-elution with the Peptide Peaks: No co-elution of the test item with any of the peptide peaks was observed.
- The controls confirmed the validity of the study for both, the cysteine and lysine run. For the cysteine run the coefficient of determination for the calibration curve was > 0.99 (0.9992). The mean peptide depletion of the positive control was between 60.8% and 100% (69.31%). The mean peptide concentration of reference controls A and reference controls C was between 0.45 and 0.55 mM (RC A: 0.5119 mM, RC C acetonitrile. 0.5079 mM). The coefficient of variation of the peak areas of reference controls B and reference controls C was < 15%. (RC B: 0.48%, RC C acetonitrile: 0.30%). The standard deviation of the peptide depletion for the replicates of the positive control as well as for the test item samples was < 14.9% (PC: 0.45%; test item: 0.18%). For the lysine run the coefficient of determination for the calibration curve was > 0.99 (09999). The mean peptide depletion of the positive control was between 40.2% and 69.0% (63.12%). The mean peptide concentration of reference controls A and reference controls C was between 0.45 and 0.55 mM (RC A: 0.5008 mM, RC C acetonitrile 0.4926 mM). The coefficient of variation of the peak areas of reference controls B and reference controls C was < 15%. (RC B: 1.57%, RC C acetonitrile. 1.19%). The standard deviation of the peptide depletion for the replicates of the positive control as well as for the test item samples was < 11.6% (PC: 1.08%; test item: 0.74%).
Interpretation of results:
GHS criteria not met
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
Expiry Date: 09 November 2018
Storage Conditions: at room temperature, protection from light
Description: Off-white solid
Details on study design:
PREPARATION OF THE TEST ITEM:
- The test item was freshly prepared immediately prior to use. The test item was dissolved in dimethyl sulfoxide (DMSO). A stock solution of 200 mM was prepared by preweighing the test material into a glass vial. A stable suspension was formed when diluted 1:100 in cell culture medium. Vortex mixing was used to aid solubilisation. 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. These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.
CONTROLS:
- A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test.
- Blank: A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.
- Negative Control: DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.
- Positive Control: Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2) was used as positive control. CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 μM – 64 μM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.
CELL LINE
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 8 in experiment 1; P 10 in experiment 2) were used. Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37±1°C and 5% CO2. For test item exposure, cells were cultured in medium for test item exposure.
COMPOSITION OF MEDIA
- Maintenance Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with 10% fetal bovine calf serum and 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 10% fetal bovine calf serum
- Test Item Exposure Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the 1% fetal bovine calf serum.
DOSE GROUPS
1. Negative Control: 1% (v/v) DMSO in test item exposure medium; 2. Positive Control: CA: 4 μM, 8 μM, 16 μM; 32 μM; 64 μM; 3. Test Item: 12 concentrations of the test item (2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 μM). Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per plate in every independent run.
EXPERIMENTAL PROCEDURE
- A cell suspension of 8E4 cells/mL in assay medium was prepared. 125 μL of the cell suspension corresponding to 1E4 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability. After seeding cells were grown for 24 h ± 1 h in assay medium at 37°C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 μL test item exposure medium. 50 μL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item. All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and crosscontamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37°C ± 1°C and 5% CO2.
- 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. Afterwards the medium was 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 over the weekend (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.
DATA ANALYSIS
For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consisted of three replicates for every concentration step of the test item and the positive control. In case of discordant results a third independent run is performed. The following parameters were calculated: Cell Viability, Maximal Induction of the Luciferase Activity (Imax), EC1.5, IC50 and IC30. For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p <0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells. The lowest concentration with >1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30% reduction on cellular viability at the EC1.5 determining concentration.
PREDICTION MODEL
The test item is considered positive in accordance with UN GHS “Category 1” if the following conditions were met in at least two independently prepared test repetitions: 1) Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control; 2) cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5; 3) EC1.5 value is <1000 μM; 4) an apparent overall dose-response for luciferase induction. If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 μM is considered as inconclusive. A negative result for test items with a log KOW > 7 has to be interpreted with care due to the applicability of the test method.
ACCEPTANCE CRITERIA
The test meets acceptance criteria if: 1) 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; 2) the average induction in the three technical replicates for the positive control at a concentration of 64 μM is between 2 and 8; 3) the EC1.5 value of the positive control is within two standard deviations of the historical mean 4) 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:
EC1.5 was determined to be 20.4
Key result
Parameter:
other: EC1.5
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
not determinable
Parameter:
other: Imax
Run / experiment:
1
Value:
1.26
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Parameter:
other: Imax
Run / experiment:
2
Value:
1.44
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
In the first experiment, a max luciferase activity (Imax) induction of 1.26 was determined at a test item concentration of 1.95 μM. The corresponding cell viability was 97.0%. No significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.
In the second experiment, a max luciferase activity (Imax) induction of 1.44 was determined at a test item concentration of 0.98 μM. The corresponding cell viability was 98.9%. No significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.
No 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 non sensitiser.
Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In vitro

In the present chemico direct peptide reactivity assay (DPRA) study, performed in accordance with OECD guideline 442C and following GLP, the test substance was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 213.43 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5°C. Subsequently samples were analysed by HPLC. 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 phase separation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis. For the 100 mM stock 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 phase separation was observed for the samples of the positive control. Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant. Phase separation in the cysteine experiment was observed. Since it cannot be determined if the phase separation resulted from the test item or the peptide, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion. The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was 0.68%. According to the evaluation criteria in the guideline, if a precipitation or phase seperation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Due to the observed phase separation in the cysteine experiment no prediction can be made.

In the present KeratinoSens assay performed in accordance with OECD guideline 442D and following GLP, the test substance was dissolved in DMSO. Based on a molecular weight of 244.37 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 and a stable suspension was formed. 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 1.26 was determined at a test item concentration of 1.95 μM. The corresponding cell viability was 97.0%. No significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. In the second experiment, a max luciferase activity (Imax) induction of 1.44 was determined at a test item concentration of 0.98 μM. The corresponding cell viability was 98.9%. No significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. No 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 non sensitiser. The controls confirmed the validity of the study.

Human

Twenty-five volunteers were exposed to 4% test substance in petrolatum. The test substance is applied under occlusive dressing to the same site on the volar forearms of all subjects for five alternate-day 48-hour periods. Prior to each exposure, the site was pre-treated with 5% aqueous sodium lauryl sulfate under occlusive dressings for a 24-hour period. The challenge sites were read at the patch removal and 24 hours after the patch removal. No effects were observed.

Conclusion

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by addressing the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the AOP, namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. In the DPRA, the mean depletion of both peptides was 0.68%. However, phase separation in the cysteine experiment was observed. As a result, it cannot be determined if the phase separation resulted from the test item or the peptide. Thus the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion. Nonetheless, the observed peptide depletion is well below the cut-off of 6.38%. Suggesting a lack of reactivity of the test item towards the peptide. From the KeratinoSens assay, the test item is considered to be non-sensitising. Although not completely conclusive, these in chemico/vitro tests strongly suggest that the test substance is a non-sensitiser. Furthermore, a 4% solution does not cause a sensitising effects in humans. Overall, it can be concluded that the test substance is not a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test substance does not have to be classified as skin sensitiser according to Regulation (EC) No 1272/2008.