Registration Dossier
Registration Dossier
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EC number: 200-796-1 | CAS number: 73-24-5
Endpoint summary
Administrative data
Description of key information
Skin:
In an in chemico direct peptide reactivity assay (DPRA) according to OECD Guideline 442 C (reference 7.4.1-1), the test item showed no reactivity towards cysteine or lysine, respectively under the given conditions. In an in vitro skin sensitisation assay (ARE-Nrf2 Luciferase Test Method, KeratinoSens) according to OECD Guideline TG 442 D (reference 7.4.1 -2), the test item did not induce luciferase activity in at least two independent experimental runs. Based on the results from the in chemico and in vitro studies and considering the AOP “2 out of 3” the test item in predicted as non-skin sensitizer.
An in silico assessment of the test item did not indicate a skin sensitisation potential (reference 7.4.1 -3).
.
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:
- 2018-07-23 to 2018-09-21
- 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 June 25, 2018
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155
- Version / remarks:
- 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
- Type of study:
- activation of keratinocytes
- Details on the study design:
- 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.
Cell line used: KeratinoSensTM (Givaudan, Switzerland)
Technical material and conditions:
- Maintenance Medium: D-MEM (GlutaMAXTM) with 1.0 g/L D-glucose and Na-Pyruvate + 10 % FBS + 1 % Geneticin (final concentration: 500 µg/mL)
- Assay Medium: D-MEM (GlutaMAXTM) with 1.0 g/L D-glucose and Na-Pyruvate + 10 % FBS
- Test Item Exposure Medium: D-MEM with 1.0 g/L D-glucose and Na-Pyruvate + 1 % FBS
- Luciferace reagent: Luciferase Assay Substrate (Promega, Cat. No.: E1501)
- Assay Buffer: Luciferase Assay Buffer (Promega, Cat. No.: E1501)
- Lysisbuffer: Luciferase Cell Culture Lysis (Promega Cat. No.: E1531)
- MTT Solution: MTT stock solution: 5 mg/mL in DPBS
- SDS solution: 10% (w/v) SDS in dist. water
- DPBS solution: DPBS solution (without Ca2+/Mg2+)
Controls used:
- Vehicle control: DMSO: 1% (v/v) in test item exposure medium
- Positive control: Cinnamic aldehyde in DMSO, 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
- Blank control: vehicle control without cells
Test item: 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49 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.
Test procedure:
Each concentration step of the test item (twelve concentrations, 0.49 - 1000 µM) and the positive control (5 concentrations, 4 - 64 µM) was assessed in three replicates in every independent run. The negative control was assessed using six replicates per plate in every independent run. The test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 h at 37 °C and antioxidant response element (ARE) dependent luciferase activity was measured in a plate reader. Cell viability was determined by a MTT assay. The test substance was incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 10% SDS solution the plate was incubated at 37 °C ± 1 °C and 5% CO2 overnight. The OD was measured at a wavelength of 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 consists of three replicates for every concentration step of the test item and the positive control. In case of discordant results a third independent run should be performed.
Acceptance criteria:
- 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.
Evaluation of results:
The test item is considered positive if the following conditions were 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. - Positive control results:
- The luciferase activity induced by the positive control at a concentration of 64 µM was between 5 and 7 (6.43 in experiment 1; 5.78 in experiment 2).
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: luciferase activity; Imax
- Value:
- 1.7
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: concentration: 1000 µM
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: cell viability [%]
- Value:
- 67.1
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: concentration: 1000 µM
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: EC1.5 [µM]
- Value:
- 761.3
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: luciferase activity, Imax
- Value:
- 1.82
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: concentration: 1000 µM
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: cell viability [%]
- Value:
- 69.4
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: concentration: 1000 µM
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: EC1.5 [µM]
- Value:
- 551.97
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes - Interpretation of results:
- other: no activation of keratinocytes
- Remarks:
- 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.
- Conclusions:
- In an in vitro skin sensitisation assay (ARE-Nrf2 Luciferase Test Method, KeratinoSens) according to OECD Guideline 442D, the test item did not induce luciferase activity.
- Executive summary:
In an in vitro skin sensitisation assay (ARE-Nrf2 Luciferase Test Method, KeratinoSens) according to OECD TG 442D, the skin sensitising potential of the test item was determined. The test item was dissolved in distilled water. Based on a molecular weight of 135.13 g/mol a stock solution of 100 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: 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49 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 1.70 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 67.1%. The lowest tested concentration with a significant luciferase induction >1.5 (1.70) was found to be 1000 µM. The corresponding cell viability was < 70% (67.1%). The calculated EC1.5 was <1000 µM (761.30). In the second experiment, a max luciferase activity (Imax) induction of 1.82 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 69.4%. The lowest tested concentration with a significant luciferase induction >1.5 (1.82) was found to be 1000 µM. The corresponding cell viability was < 70% (69.4%). The calculated EC1.5 was <1000 µM (551.97). Due to cell viability < 70% at the highest test item concentration and due to a missing dose response for luciferase activity induction in each individual run as well as for an overall luciferase activity induction the test item showed no sensitising potential. The controls confirmed the validity of the study.
- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2018-06-21 to 2018-07-12
- 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 February 04, 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:
- 2013
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
- Details on the study design:
- Skin sensitisation (In chemico test system) - Details on study design
Synthetic peptides used:
- cysteine peptide with an amino acid sequence of Ac-RFAACAA, JPT Peptide Technologies GmbH; > 95%; Lot. No.: 111016HS MHeW0318
- lysine peptide with an amino acid sequence of Ac-RFAAKAA, JPT Peptide Technologies GmbH; > 95%; Lot. No.: 120514HSDW W0318
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.
Controls used:
- Positive control: Cinnamic aldehyde 100 mM in acetonitrile
- Co-elution control: test item or positive control without cysteine or lysine peptide
- Reference controls: cysteine or lysine peptide in acetonitrile with and without test item
Test substance preparation:
- The test substance was prepared as a 100 mM preparation in acetonitrile.
Peptide stock solution preparation:
- 20.09 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (38.21 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
- 18.97 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 (35.11 mL) to reach a concentration of 0.667 mM.
Experimental procedure:
Three samples of the test substance in acetonitrile were incubated with each peptide for 24h at room temperature in the dark. The incubation tubes were sealed. Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.
Sample preparation:
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).
HPLC conditions:
Peptide depletion was monitored by HPLC coupled with an UV detector at A = 220 nm (Agilent, Infinity 1260 II with Chromeleon 7.2 SR5) 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 (0.1% ( v/v) trifluoroacetic acid in water) and 50% phase B (0.085% ( v/v) trifluoroacetic acid in acetonitrile) 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.
Calculation and data evaluation:
The concentration of the cysteine and lysine peptide was determined in each sample form absorbance at A = 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.
PPD = (1- (Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in Reference Control C)) * 100
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.
Evaluation of results:
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.
By using the prediction model 2 (cysteine 1:10 prediction model) the threshold of 13.89% peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers. - 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 65.70%.
- Key result
- Run / experiment:
- other: Cysteine
- Parameter:
- other: mean peptide depletion [%]
- Value:
- 0
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: Lysine
- Parameter:
- other: mean peptide depletion [%]
- Value:
- 0
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: Yes
- Acceptance criteria met for positive control: Yes
OTHER EFFECTS:
For the cysteine and lysine peptide experiment no precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). - Interpretation of results:
- other: peptide depletion
- Remarks:
- The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
- Conclusions:
- In an in chemico direct peptide reactivity assay (DPRA) according to OECD Guideline 442C, the test item showed low reactivity towards cysteine or lysine, respectively under the given conditions.
- Executive summary:
In an in chemico direct peptide reactivity assay (DPRA) according to OECD Guideline 442 C, the direct peptide binding potential of the test item was determined. The test substance was solved in water, based on the results of the pre-experiments. Based on a molecular weight of 135.13 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. After the 24 h ± 2 h incubation period but prior to the HPLC analysis the cysteine and lysine peptide samples were inspected for precipitation, turbidity or phase separation. For the cysteine and lysine peptide experiment no precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control. Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria of the depletion range of the positive control were fulfilled, the observed precipitations were regarded as insignificant. No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C). The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was < 6.38% (0.00%). Based on the prediction model 1 the test item can be considered as non-sensitiser.
- Endpoint:
- skin sensitisation, other
- Remarks:
- in silico
- Type of information:
- (Q)SAR
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification
- Justification for type of information:
- Please refer to the QMRF and QPRF files provided under the section attached justification.
- Qualifier:
- according to guideline
- Guideline:
- other: Guidance on information requirements and chemical safety assessment, Chapter R.6: QSARs and grouping of chemicals
- Principles of method if other than guideline:
- Estimates the skin sensitising properties of chemicals using structural alert relationships.
- GLP compliance:
- no
- Specific details on test material used for the study:
- SMILES: Nc1ncnc2[nH]cnc12
- Key result
- Parameter:
- other: alerts
- Value:
- 0
- Remarks on result:
- other: Skin sensitisation in mammal is equivocal. No alert matched for QSAR predicted value. The substance is only partly within the applicability domain of the model.
- Interpretation of results:
- other: Derek result: equivocal
- Conclusions:
- Using Derek Nexus v5.0 the skin sensitising potential of the test item was estimated to be equivocal. No alerts were matched. The substance is partly within the applicability domain of the model. Thus the estimation can be regarded as moderately accurate.
- Executive summary:
The skin sensitising properties were estimated using Derek Nexus v5.0. The skin sensitising potential of the test item was estimated to be equivocal based on the described QSAR method (Derek, 2017).
Further in vitro studies were initiated but were not completed by the CRO in time to complete the registration dossier. The study record will be updated as soon as the results will become available.
The adequacy of a prediction depends on the following conditions:
a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;
b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;
c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;
d) the (Q)SAR model is relevant for the regulatory purpose.
For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.
Description of the prediction Model
The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file.
Assessment of estimation domain
The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.
Referenceopen allclose all
Table: Additional parameters
| Parameter | Experiment 1 | Experiment 2 | Mean | SD |
| EC1.5[MM] | 761.3 | 551.97 | 656.63 | 148.02 |
| Imax | 1.7 | 1.82 | 1.76 | 0.09 |
| IC30[MM] | 898 | 947.11 | 922.55 | 34.72 |
| IC50[MM] | n/a | n/a | n/a | n/a |
n/a = not applicable
Table 2: Results of the Cytotoxicity Measurement
| Concentration [MM] |
Cell Viability [%] | ||||
| Experiment 1 | Experiment 2 | Mean | SD | ||
| Solvent Control | - | 100 | 100 | 100 | 0.0 |
| Positive Control | 4.00 | 86.4 | 103.6 | 95.0 | 12.2 |
| 8.00 | 92.4 | 105.9 | 99.2 | 9.5 | |
| 16.00 | 96 | 100 | 98.0 | 2.8 | |
| 32.00 | 86.6 | 101.3 | 93.9 | 10.4 | |
| 64.00 | 84.1 | 94.7 | 89.4 | 7.5 | |
| Test Item | 0.49 | 96.4 | 77.6 | 87.0 | 13.3 |
| 0.98 | 105.5 | 79.5 | 92.5 | 18.4 | |
| 1.95 | 99.7 | 68.4 | 84.0 | 22.2 | |
| 3.91 | 86.1 | 75.8 | 80.9 | 7.3 | |
| 7.81 | 85.1 | 72.8 | 79.0 | 8.7 | |
| 15.63 | 85.9 | 73.1 | 79.5 | 9.0 | |
| 31.25 | 90.9 | 72.8 | 81.8 | 12.8 | |
| 62.50 | 84.6 | 71.2 | 77.9 | 9.5 | |
| 125.00 | 87.2 | 69.7 | 78.5 | 12.3 | |
| 250.00 | 83.2 | 73.1 | 78.1 | 7.1 | |
| 500.00 | 81.2 | 74.8 | 78.0 | 4.5 | |
| 1000.00 | 67.1 | 69.4 | 68.3 | 1.6 | |
Table 3: Induction of Luciferase Activity Experiment 1
| Experiment 1 | Concentration [MM] | Fold Induction | Significance | ||||
| Rep. 1 | Rep. 2 | Rep. 3 | Mean | SD | |||
| Solvent Control | - | 1.00 | 1.00 | 1.00 | 1.00 | 0.00 | |
| Positive Control | 4.00 | 1.62 | 1.18 | 1.36 | 1.39 | 0.22 | |
| 8.00 | 1.27 | 1.20 | 1.14 | 1.20 | 0.07 | ||
| 16.00 | 1.49 | 1.63 | 1.53 | 1.55 | 0.08 | * | |
| 32.00 | 2.20 | 1.84 | 2.24 | 2.09 | 0.22 | * | |
| 64.00 | 7.00 | 6.59 | 5.69 | 6.43 | 0.67 | * | |
| Test Item | 0.49 | 1.15 | 1.11 | 1.16 | 1.14 | 0.03 | |
| 0.98 | 1.09 | 1.07 | 1.18 | 1.11 | 0.06 | ||
| 1.95 | 1.07 | 1.03 | 0.91 | 1.00 | 0.08 | ||
| 3.91 | 1.10 | 1.10 | 1.20 | 1.13 | 0.06 | ||
| 7.81 | 1.16 | 1.17 | 1.39 | 1.24 | 0.13 | ||
| 15.63 | 1.19 | 1.14 | 1.22 | 1.18 | 0.04 | ||
| 31.25 | 1.26 | 1.32 | 1.25 | 1.28 | 0.04 | ||
| 62.50 | 1.31 | 1.26 | 1.20 | 1.26 | 0.05 | ||
| 125.00 | 1.28 | 1.16 | 1.28 | 1.24 | 0.07 | ||
| 250.00 | 1.41 | 1.46 | 1.20 | 1.36 | 0.14 | ||
| 500.00 | 1.30 | 1.20 | 1.36 | 1.29 | 0.08 | ||
| 1000.00 | 1.86 | 1.60 | 1.63 | 1.70 | 0.14 | * | |
* = significant induction according to Student's t-test, p<0.05
Table 4: Induction of Luciferase Activity Experiment 2
| Experiment 2 | Concentration [MM] | Fold Induction | Significance | ||||
| Rep. 1 | Rep. 2 | Rep. 3 | Mean | SD | |||
| Solvent Control | - | 1.00 | 1.00 | 1.00 | 1.00 | 0.00 | |
| Positive Control | 4.00 | 1.34 | 1.28 | 1.24 | 1.29 | 0.05 | |
| 8.00 | 1.36 | 1.32 | 1.24 | 1.31 | 0.06 | ||
| 16.00 | 1.54 | 1.62 | 1.62 | 1.60 | 0.05 | * | |
| 32.00 | 2.02 | 2.16 | 2.21 | 2.13 | 0.10 | * | |
| 64.00 | 7.06 | 4.69 | 5.58 | 5.78 | 1.19 | * | |
| Test Item | 0.49 | 1.14 | 1.50 | 1.14 | 1.26 | 0.21 | |
| 0.98 | 0.86 | 1.01 | 1.01 | 0.96 | 0.09 | ||
| 1.95 | 0.97 | 1.19 | 1.13 | 1.10 | 0.12 | ||
| 3.91 | 1.01 | 1.24 | 1.10 | 1.11 | 0.11 | ||
| 7.81 | 1.19 | 1.54 | 1.41 | 1.38 | 0.18 | ||
| 15.63 | 1.17 | 1.33 | 1.54 | 1.35 | 0.19 | ||
| 31.25 | 1.13 | 1.21 | 1.36 | 1.24 | 0.12 | ||
| 62.50 | 1.29 | 1.32 | 1.46 | 1.36 | 0.09 | ||
| 125.00 | 1.18 | 1.44 | 1.30 | 1.31 | 0.13 | ||
| 250.00 | 1.19 | 1.49 | 1.38 | 1.35 | 0.16 | ||
| 500.00 | 1.35 | 1.63 | 1.41 | 1.46 | 0.15 | ||
| 1000.00 | 1.82 | 1.73 | 1.92 | 1.82 | 0.10 | * | |
* = significant induction according to Student's t-test, p<0.05
Table 5: Induction of Luciferase Activity - Overall Induction
| Concentration [MM] | Fold Induction | Significance | ||||
| Experiment 1 | Experiment 2 | Mean | SD | |||
| Solvent Control | - | 1.00 | 1.00 | 1.00 | 0.00 | - |
| Positive Control | 4.00 | 1.39 | 1.29 | 1.34 | 0.07 | |
| 8.00 | 1.20 | 1.31 | 1.26 | 0.07 | ||
| 16.00 | 1.55 | 1.60 | 1.57 | 0.03 | * | |
| 32.00 | 2.09 | 2.13 | 2.11 | 0.03 | * | |
| 64.00 | 6.43 | 5.78 | 6.10 | 0.46 | * | |
| Test Item | 0.49 | 1.14 | 1.26 | 1.20 | 0.09 | |
| 0.98 | 1.11 | 0.96 | 1.04 | 0.11 | ||
| 1.95 | 1.00 | 1.10 | 1.05 | 0.06 | ||
| 3.91 | 1.13 | 1.11 | 1.12 | 0.01 | ||
| 7.81 | 1.24 | 1.38 | 1.31 | 0.10 | ||
| 15.63 | 1.18 | 1.35 | 1.27 | 0.11 | ||
| 31.25 | 1.28 | 1.24 | 1.26 | 0.03 | ||
| 62.50 | 1.26 | 1.36 | 1.31 | 0.07 | ||
| 125.00 | 1.24 | 1.31 | 1.27 | 0.05 | ||
| 250.00 | 1.36 | 1.35 | 1.36 | 0.00 | ||
| 500.00 | 1.29 | 1.46 | 1.37 | 0.13 | ||
| 1000.00 | 1.70 | 1.82 | 1.76 | 0.09 | * | |
* = significant induction according to Student's t-test, p<0.05
Table 2: Depletion of the 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 | 4.1310 | 0.1469 | 70.73 | 71.32 | 0.53 | 0.74 |
| 4.0270 | 0.1433 | 71.47 | ||||
| 3.9870 | 0.1418 | 71.75 | ||||
| Test Item | 14.5500 | 0.5156 | 0.00 | 0.00 | 0.00 | n.a. |
| 14.0900 | 0.4994 | 0.00 | ||||
| 14.3050 | 0.5070 | 0.00 |
n.a.: not applicable
Table 3: Depletion of the 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 | 5.4360 | 0.2012 | 60.55 | 60.08 | 1.12 | 1.86 |
| 5.3900 | 0.1995 | 60.88 | ||||
| 5.6770 | 0.2101 | 58.80 | ||||
| Test Item | 13.5010 | 0.5007 | 0.00 | 0.00 | 0.00 | n.a. |
| 13.6360 | 0.5057 | 0.00 | ||||
| 13.6850 | 0.5075 | 0.00 |
n.a.: not applicable
Table 4: Categorization of the Test Item according to prediction model 1
| Prediction Model | Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50) | Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10) | ||||
| Test Substance | Mean Peptide Depletion [%] | Reactivity Category | Prediction | Mean Peptide Depletion [%] | Reactivity Category | Prediction |
| Test Item | 0 | Minimal Reactivity | no sensitiser | 0 | Minimal Reactivity | no sensitiser |
| Positive Control | 65.7 | High Reactivity | sensitiser | 71.32 | Moderate Reactivity | sensitiser |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
For the evaluation of the skin sensitisation potential of the test substance a Weight of Evidence approach was used. The protein reactivity of the test item was assessed in a Direct Peptide Reactivity Assay (DPRA). A Keratinocyte Activation Assay (KeratinoSens) was performed to evaluate the keratinocyte activation potential.
DPRA (reference 7.4.1 -1)
The direct peptide reactivity assay (DPRA) addresses the first key event in skin sensitisation, according to the defined adverse outcome pathway (AOP). Therefore, the direct peptide binding potential of the test item was determined in an in chemico DPRA according to OECD Guideline 442 C (reference 7.4.1-1). The test substance was solved in water, based on the results of the pre-experiments. Based on a molecular weight of 135.13 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. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C). The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was < 6.38% (0.00%). Based on the prediction model 1 the test item can be considered as non-sensitiser.
KeratinoSens (reference 7.4.1-2)
The KeratinoSens™ assay is supposed to address the second key event of the skin sensitisation process as defined by the AOP, the induction of cyto-protective signalling pathways in keratinocytes in response to electrophiles and oxidative stress. Accordingly, the skin sensitising potential of the test item was determined in an in vitro skin sensitisation assay (ARE-Nrf2 Luciferase Test Method, KeratinoSens) according to OECD Guideline 442 D. The test item was dissolved in distilled water. Based on a molecular weight of 135.13 g/mol a stock solution of 100 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: 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49 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 1.70 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 67.1%. The lowest tested concentration with a significant luciferase induction >1.5 (1.70) was found to be 1000 µM. The corresponding cell viability was < 70% (67.1%). The calculated EC1.5 was <1000 µM (761.30). In the second experiment, a max luciferase activity (Imax) induction of 1.82 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 69.4%. The lowest tested concentration with a significant luciferase induction >1.5 (1.82) was found to be 1000 µM. The corresponding cell viability was < 70% (69.4%). The calculated EC1.5 was <1000 µM (551.97). Due to cell viability < 70% at the highest test item concentration and due to a missing dose response for luciferase activity induction in each individual run as well as for an overall luciferase activity induction the test item showed no sensitising potential. The controls confirmed the validity of the study.
In silico assessment(reference 7.4.1 -3)
For in silico assessment, Derek Nexus v5.0 was used (reference 7.4.1 -3). No skin sensitising properties
of the test item were estimated. The substance is within the applicability domain of the model. Thus the
estimation can be regarded as accurate.
Conclusion:
The two assays (OECD TG 442 C and OECD TG 442 D) provided conclusive negative results. Considering the AOP “2 out of 3” the test item is predicted as non-skin sensitizer.
In addition, an in silico assessment (reference 7.4.1 -3) also showed no indication of skin sensitization.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Classification, Labeling, and Packaging Regulation (EC) No 1272/2008
The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this information, the substance is not considered to be classified under Regulation (EC) No 1272/2008, as amended for the eleventh time in Regulation (EU) No 2018/669.
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