Registration Dossier
Registration Dossier
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EC number: 253-379-1 | CAS number: 37172-53-5
Endpoint summary
Administrative data
Description of key information
The test substance was not considered to be sensitising to the skin in an OECD 442C study and an OECD 442D study. Based on the current data-set, there are no indications that the test substance has skin sensitising properties. Therefore it is concluded that it is scientifically not necessary to conduct further testing for skin sensitising properties and that the test substance is not classified for this endpoint.
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- Version / remarks:
- Adopted 4 February 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154
- Version / remarks:
- 12 January 2013
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
- Justification for non-LLNA method:
- The DPRA has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-lead validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and was considered scientifically valid to be used as part of an IATA to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
- Specific details on test material used for the study:
- Description: Colourless to pale yellow liquid
Storage conditions: Room temperature, protected from light
Expiry date: 18 January 2020 - Details on the study design:
- TEST SYSTEM
- Test system: Synthetic peptides containing cysteine (SPCC) (Ac RFAACAA) or synthetic peptides containing lysine (SPCL) (Ac RFAAKAA).
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Source: JPT Peptide Technologies GmbH
- Batch SPCC: > 95%; Lot. No.: 111016HS_MHeW1017
- Batch SPCL: > 95%; Lot. No.: 120514HSDW_W1117
- Storage: All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.
TEST ITEM PREPARATION
- The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage.
- The test item was pre-weighed into a glass vial and was dissolved in acetonitrile, an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.
- 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 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.
EXPERIMENTAL DESIGN
- 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 1 in ‘Any other information on materials and methods incl. tables’.
- 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 a HPLC procedure.
PREPARATION OF SOLUTIONS FOR CYSTEINE REACTIVITY ASSAY
- Synthetic Peptide Containing Cysteine (SPCC) Stock Solution: 20.07 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (38.92 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
- SPCC Reference Control Solutions: Three SPCC reference control (RC) solutions (RC A, RC B and RC C) were prepared according to Table 1 in ‘Any other information on materials and methods incl. tables’.
- SPCC Calibration Curve: A SPCC calibration curve was prepared as described in Table 2 under ‘Any other information on materials and methods incl. tables’.
- Co-elution Control, Test Item and Positive Control Samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in Table 1 under ‘Any other information on materials and methods incl. tables’.
PREPARATION OF SOLUTIONS FOR LYSINE REACTIVITY ASSAY
- 20.42 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 (38.78 mL) to reach a concentration of 0.667 mM.
- SPCL Reference Control Solutions: Three SPCL reference control (RC) solutions (RC A, RC B and RC C) were prepared according to Table 1 in ‘Any other information on materials and methods incl. tables’.
- SPCL Calibration Curve: A SPCL peptide calibration curve was prepared as described in Table 2 under ‘Any other information on materials and methods incl. tables’.
- Co-elution Control, Test Item and Positive Control Samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in Table 1 under ‘Any other information on materials and methods incl. tables’.
HPLC-PDA 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.
ACCEPTABILITY 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.
ANALYSIS
DATA EVALUATION
- 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 absorbance at λ = 258 nm was also monitored for the samples of the test item and the reference controls as a co-elution control. The ratio of the peak areas (220 nm / 258 nm) was checked for consistency between reference control and test item samples. If this ratio was not consistent a co-elution was assumed and the evaluation would be adjusted accordingly.
- 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, 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.
DATA INTERPRETATION
- By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model) shown in Table 3 in ‘Any other information materials and methods incl. tables’, 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, if the mean depletion of both peptides is below 6.38%. See Table 3 in ‘Any other information on materials and methods incl. tables’.
- 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) see Table 4 in ‘Any other information materials and methods incl. tables’. - Positive control results:
- CYSTEINE REACTIVITY ASSAY (Table 1 and 3 in 'Any other information on results incl. tables')
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 69.51% ± 0.61%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
LYSINE REACTIVITY ASSAY (Table 2 and 4 in 'Any other information on results incl. tables')
The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 61.12% ± 1.28%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%). - Key result
- Parameter:
- other: Mean of SPCC and SPCL depletion (%)
- Value:
- 0
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Remarks on result:
- no indication of skin sensitisation
- Parameter:
- other: Cysteine reactivity
- Value:
- 0
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Parameter:
- other: Lysine reactivity
- Value:
- 0
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Other effects / acceptance of results:
- PRE-EXPERIMENT
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 solution. All test item preparations of the main experiment were prepared using acetonitrile. All test item solutions were freshly prepared immediately prior to use.
MAIN EXPERIMENT
- Precipitation: For the 100 mM stock 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.
- Phase separation: For the 100 mM stock solution of the test item phase separation 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 test item and the positive control (including the co-elution control).
- Co-elution with peptide peaks: No co-elution of the test item with any of the peptide peaks was observed.
- Depletion of the Cysteine and Lysine Peptides: The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptide. See Table 1 and 2 in ‘Any other information on results incl. tables’. - Interpretation of results:
- GHS criteria not met
- Remarks:
- In combination with KeratinoSens assay (OECD 442D)
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
- Version / remarks:
- Adopted 4 February 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155
- Version / remarks:
- 1 July 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of keratinocytes
- Justification for non-LLNA method:
- The in vitro ARE-Nrf2 luciferase KeratinoSens™ test method (hereafter called the KeratinoSens™ test method) underwent validation studies followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSens™ test method was considered scientifically valid to be used as part of an IATA, to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard identification.
- Specific details on test material used for the study:
- Description: Colourless to pale yellow liquid
Storage conditions: Room temperature, protected from light
Expiry date: 18 January 2020 - Details on the study design:
- SOLVENT
Tetrahydrofuran (CAS 109-99-9)
CONTROLS
- negative control: DMSO (Final concentration 1%)
- positive control: Cinnamic aldehyde (CAS 104-55-2, Purity>98%)
PREPARATION OF TEST ITEM STOCK, SPIKING AND WORKING SOLUTIONS
- The test item was dissolved in Tetrahydrofuran. A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial.
- 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. Since the test item was dissolved in THF, DMSO was added at a final concentration of 4% (v/v).
- 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.
- In total 12 test concentrations were tested, ranging from 0.98 to 2000 µM.
- All test item solutions were freshly prepared immediately prior to use.
- Each concentration step of the test item was assessed in three replicates in every independent run.
PREPARATION OF THE POSITIVE CONTROL
- Cinnamic aldehyde 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.
- Each concentration step of the positive control was assessed in three replicates in every independent run.
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.
- The negative control was assessed using six replicates per 96-well plate in every independent run.
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.
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 11 in experiment 1; P 02 in experiment 2) were used.
CELL CULTURE
- Maintenance medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and 1 mM 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 1 mM Na-Pyruvate. The medium was supplemented with 10% fetal bovine calf serum.
- Exposure medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate. The medium was supplemented with 1% fetal bovine calf serum.
- Environmental conditions: Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 ± 1°C and 5% CO2 in a humidified incubator. For test item exposure, cells were cultured in medium for test item exposure.
EXPERIMENTAL DESIGN
- Two independent experiments were performed, consisting of three replicates for every concentration step of the test item and positive control.
- Plating of Cells: 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 (MTT assay).
- Treatment of Cells: 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 Measurement: 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 sec. before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.
- Cytotoxicity Assessment: 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 overnight (experiment 2) or over the weekend (experiment 1). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.
ACCEPTABILITY 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 EC1.5 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.
DATA ANALYSIS
The following parameters are calculated in the KeratinoSensTM test method:
- Cell viability in %
- The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the test item.
- The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
- The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
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.
DATA INTERPRETATION
A KeratinoSens™ prediction is considered positive if the following conditions will be met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is < 1000 μM
- 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. - Positive control results:
- EXPERIMENT 1
- Induction at 64 µM: 3.13
- EC1.5: 12.24 μM
EXPERIMENT 2
- Induction at 64 µM: 7.05
- EC1.5: 13.22 μM - Run / experiment:
- other: Experiment 1 and 2
- Parameter:
- other: Viability (%)
- Value:
- 70
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: Viability was 42.5% at 2000 µM
- Remarks:
- see Table 1 in 'Any other information on results incl. tables'
- Key result
- Run / experiment:
- other: Experiment 1 and 2
- Parameter:
- other: The maximal average fold induction of luciferase activity
- Value:
- 1.17
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Remarks:
- Imax value for 2000 µM does not fall within the predicition model criteria / No further luciferase induction > 1.5 was found for the other tested concentrations. The calculated EC1.5 was > 1000 μM (1688.91 μM) / see Table 2 in 'Any other information on results incl. tables'
- Other effects / acceptance of results:
- ACCEPTANCE OF RESULTS (See Table 3 in 'Any other information on results incl. tables')
- Acceptance criteria for negative control are met.
- Acceptance criteria for positive control are met.
- Acceptance criteria for variability between replicate measurements are met. - Interpretation of results:
- GHS criteria not met
- Remarks:
- In combination with DPRA assay (OECD 442C)
Referenceopen allclose all
Table 1. Depletion of the Cysteine Peptide
Cysteine Peptide |
||||||
Sample |
Peak Area at 220 nm |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
1471.0265 |
0.1596 |
69.33 |
|
|
|
1486.4678 |
0.1613 |
69.01 |
69.51 |
0.61 |
0.88 |
|
1429.7356 |
0.1551 |
70.19 |
|
|
|
|
Test Item |
4806.7178 |
0.5218 |
0.00 |
|
|
|
4824.4810 |
0.5238 |
0.00 |
0.00 |
0.00 |
- |
|
4837.3389 |
0.5252 |
0.00 |
|
|
|
|
Table 2. Depletion of the Lysine Peptide
Lysine Peptide |
||||||
Sample |
Peak Area at 220 nm |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
1548.8514 |
0.1877 |
62.22 |
|
|
|
1581.2108 |
0.1916 |
61.43 |
61.12 |
1.28 |
2.09 |
|
1651.4639 |
0.2002 |
59.72 |
|
|
|
|
Test Item |
4150.5811 |
0.5047 |
0.00 |
|
|
|
4159.9512 |
0.5058 |
0.00 |
0.00 |
0.00 |
- |
|
4154.1421 |
0.5051 |
0.00 |
|
|
|
|
Table 3. Results of the Reference Controls for the Cysteine Peptide
Cysteine Peptide Run |
||||||||
Sample |
Peptide Peak Area |
Peptide Concentration [mM] |
||||||
PA |
Mean |
SD |
CV [%] |
Peptide Concentration |
Mean |
SD |
CV [%] |
|
Reference A 1 |
4794.1187 |
4762.6523 |
74.2400 |
1.56 |
0.5205 |
0.5170 |
0.0081 |
1.56 |
Reference A 2 |
4815.9771 |
0.5228 |
||||||
Reference A 3 |
4677.8613 |
0.5078 |
||||||
Reference B 1 |
4733.2197 |
4788.7638 |
81.7780 |
1.71 |
0.5138 |
0.5199 |
0.0089 |
1.71 |
Reference B 2 |
4766.4439 |
0.5175 |
||||||
Reference B 3 |
4777.8418 |
0.5187 |
||||||
Reference B 4 |
4763.3130 |
0.5171 |
||||||
Reference B 5 |
4739.6685 |
0.5145 |
||||||
Reference B 6 |
4952.0962 |
0.5376 |
||||||
Reference C 1 (PC solvent) |
4813.2861 |
4796.4543 |
14.5915 |
0.30 |
0.5225 |
0.5207 |
0.0016 |
0.30 |
Reference C 2 (PC solvent) |
4788.6934 |
0.5199 |
||||||
Reference C 3 (PC solvent) |
4787.3833 |
0.5197 |
||||||
Reference C 1 (TI solvent) |
4813.2861 |
4796.4543 |
14.5915 |
0.30 |
0.5225 |
0.5207 |
0.0016 |
0.30 |
Reference C 2 (TI solvent) |
4788.6934 |
0.5199 |
||||||
Reference C 3 (TI solvent) |
4787.3833 |
0.5197 |
||||||
Table 4. Results of the Reference Controls for the Lysine Peptide
Lysine Peptide Run |
||||||||
Sample |
Peptide Peak Area |
Peptide Concentration [mM] |
||||||
PA |
Mean |
SD |
CV [%] |
Peptide Concentration |
Mean |
SD |
CV [%] |
|
Reference A 1 |
4151.8887 |
4142.8311 |
11.9519 |
0.29 |
0.5049 |
0.5038 |
0.0015 |
0.29 |
Reference A 2 |
4147.3198 |
0.5043 |
||||||
Reference A 3 |
4129.2847 |
0.5021 |
||||||
Reference B 1 |
4154.4273 |
4131.4318 |
14.6986 |
0.36 |
0.5052 |
0.5024 |
0.0018 |
0.36 |
Reference B 2 |
4127.5840 |
0.5019 |
||||||
Reference B 3 |
4110.1455 |
0.4998 |
||||||
Reference B 4 |
4139.1934 |
0.5033 |
||||||
Reference B 5 |
4130.5562 |
0.5023 |
||||||
Reference B 6 |
4126.6846 |
0.5018 |
||||||
Reference C 1 (PC solvent) |
4123.4346 |
4099.5856 |
34.5874 |
0.84 |
0.5014 |
0.4985 |
0.0042 |
0.85 |
Reference C 2 (PC solvent) |
4115.4048 |
0.5004 |
||||||
Reference C 3 (PC solvent) |
4059.9175 |
0.4937 |
||||||
Reference C 1 (TI solvent) |
4123.4346 |
4099.5856 |
34.5874 |
0.84 |
0.5014 |
0.4985 |
0.0042 |
0.85 |
Reference C 2 (TI solvent) |
4115.4048 |
0.5004 |
||||||
Reference C 3 (TI solvent) |
4059.9175 |
0.4937 |
||||||
Table1. Results of the Cytotoxicity Measurement
|
Concentration [µM] |
Cell Viability [%] |
|||
Experiment 1 |
Experiment 2 |
Mean |
SD |
||
Solvent Control |
- |
100 |
100 |
100 |
0.0 |
|
4.00 |
96.6 |
102.3 |
99.5 |
4.0 |
Positive Control |
8.00 16.00 |
99.4 100.2 |
108.3 114.1 |
103.9 107.2 |
6.3 9.8 |
|
32.00 |
101.4 |
116.8 |
109.1 |
10.9 |
|
64.00 |
97.8 |
112.1 |
105.0 |
10.2 |
|
0.98 |
101.2 |
114.2 |
107.7 |
9.1 |
|
1.95 |
107.6 |
101.8 |
104.7 |
4.1 |
|
3.91 |
107.9 |
99.2 |
103.5 |
6.1 |
|
7.81 |
105.4 |
98.5 |
102.0 |
4.9 |
|
15.63 |
105.8 |
92.4 |
99.1 |
9.5 |
Test Item |
31.25 62.50 |
99.3 102.7 |
98.9 94.6 |
99.1 98.7 |
0.3 5.7 |
|
125.00 |
102.5 |
90.1 |
96.3 |
8.7 |
|
250.00 |
95.4 |
96.0 |
95.7 |
0.4 |
|
500.00 |
89.6 |
109.1 |
99.4 |
13.8 |
|
1000.00 |
74.1 |
79.7 |
76.9 |
4.0 |
|
2000.00 |
61.4 |
23.6 |
42.5 |
26.7 |
Table2. 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 |
|
|
4.00 |
1.28 |
1.05 |
1.17 |
0.16 |
|
|
8.00 |
1.36 |
1.24 |
1.30 |
0.08 |
|
Positive Control |
16.00 |
1.62 |
1.64 |
1.63 |
0.01 |
* |
|
32.00 |
1.92 |
2.56 |
2.24 |
0.46 |
|
|
64.00 |
3.13 |
7.05 |
5.09 |
2.77 |
|
|
0.98 |
1.08 |
1.26 |
1.17 |
0.13 |
|
|
1.95 |
0.92 |
0.99 |
0.96 |
0.05 |
|
|
3.91 |
0.96 |
0.92 |
0.94 |
0.02 |
|
|
7.81 |
0.99 |
0.84 |
0.91 |
0.11 |
|
|
15.63 |
0.92 |
0.93 |
0.93 |
0.01 |
|
Test Item |
31.25 62.50 |
0.93 0.92 |
0.92 0.79 |
0.92 0.86 |
0.01 0.10 |
|
|
125.00 |
0.93 |
0.74 |
0.84 |
0.13 |
|
|
250.00 |
0.79 |
0.94 |
0.86 |
0.10 |
|
|
500.00 |
1.00 |
0.92 |
0.96 |
0.05 |
|
|
1000.00 |
0.83 |
1.08 |
0.95 |
0.18 |
|
|
2000.00 |
1.15 |
1.69 |
1.42 |
0.39 |
|
* = significant induction according to Student’s t-test, p<0.05
Table3. Acceptance Criteria for positive and negative controls
Criterion |
Range |
Experiment 1 |
pass/fail |
Experiment 2 |
pass/fail |
CV Solvent Control PC (1% DMSO) |
< 20% |
12.1 |
pass |
14.6 |
pass |
CV Solvent Control TI (1% THF) |
<20% |
12.4 |
pass |
20.0 |
pass |
No. of positive control concentration steps with significant luciferase activity induction >1.5
|
≥ 1 |
3.0 |
pass |
3.0 |
pass |
EC1.5 PC
|
7 < x < 34 µM |
12.24 |
pass |
13.22 |
pass |
Induction PC at 64 µM |
2.00 < x < 8.00 |
3.13 |
pass |
7.05 |
pass |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
Skin sensitisation in chemico direct peptide binding assay
In a GLP compliant study according to OECD 442 the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine was assessed in chemico as a quantification measure of the sensitising potential of the test item using the direct peptide reactivity assay (DPRA). In the present study the test substance was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 226.31 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. All test item solutions were freshly prepared immediately prior to use. For the 100 mM stock 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 stock solution of the test item phase separation 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 test item and the positive control (including the co-elution control). 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 lysine experiment was observed. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation. Sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C-acetonitrile). The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was ≤ 13.89% (0.00%). Based on the prediction model 2 the test item can be considered as non-sensitiser.
In conclusion, the test item showed minimal reactivity towards the cysteine peptide under the given conditions. The test item is considered as “non-sensitiser”. The 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 integrated approach such as IATA.
Skin sensitisation in vitro activation of keratinocytes
In a GLP compliant study according to OECD 442D the ability of the test substance to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay was evaluated. 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 the test substance was dissolved in tetrahydrofuran. Based on a molecular weight of 226.31 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, 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 1.69 was determined at a test item concentration of 2000 μM. The corresponding cell viability was 23.6%. No further luciferase induction > 1.5 was found for the other tested concentrations. The calculated EC1.5 was >1000 μM (1688.91 μM). 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.
In conclusion, the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs under the given conditions. Therefore, the test item can be considered as non-sensitiser. The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Human patch test
Twenty-five volunteers were exposed to 2% test substance in petrolatum. The test substance is applied under occlusive dressing to the same site on the volar forearm for five 48-hour periods. Prior to each exposure, the site was pre-treated with 5% aqueous SLS under occlusion for 24 hours. Following a 10-day rest period, a challenge patch of the test substance is applied to a different site for 48 hours under occlusion. The challenge site is pre-treated for 1 hour with 10% aqueous SLS under occlusion. Observations are made immediately after removal of the challenge patch and 24 hours thereafter. No effects were observed.
Overall conclusion
A Weight of Evidence evaluation was prepared to reach an overall conclusion on the endpoint skin sensitisation based on all available relevant information. A DPRA assay and KeratinoSensTM assay were performed in accordance with the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a. The test substance was negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. Furthermore, the test substance gave a negative result in the KeratinoSensTM assay (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).
The results of these two tests do not indicate that the test substance has skin sensitizing properties and therefore it is concluded that it is scientifically not necessary to conduct further testing and that the test substance is not classified for this endpoint.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Based on the available information classification for skin sensitisation is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation 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.
