Registration Dossier

Administrative data

Description of key information

Skin sensitisation:

A DEREK assessment, DPRA assay and a KeratinoSensTM assay were available.

A DEREK prediction on the skin sensitizing potential of the test item was negative.

In the DPRA the mean of cysteine or lysine depletion was 5.6% and therefore the DPRA was negative. Based on this outcome, the test item is concluded not to be reactive to proteins.

The KeratinoSensTM assay did not show a biologically relevant activation of keratinocytes when exposed to the test item in two out of three experiments and was therefore concluded to be negative.

The results obtained in the non-animal skin sensitization testing strategy represent sufficient evidence to conclude that the test item is not expected to lead to an allergic response following skin contact.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
2019-03-27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
See attached justification.
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Software tool(s) used including version: DEREK NEXUS version 6.0.1
- Model(s) used: DEREK NEXUS
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'
GLP compliance:
no
Key result
Remarks on result:
no indication of skin sensitisation

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization of the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. The test item is predicted to be not sensitizing to the skin.

Interpretation of results:
other: not sensitizing
Conclusions:
The test item is predicted to be not sensitizing to the skin.
Executive summary:

The objective of this study was to obtain a prediction on the potential for skin sensitization of the test item with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization of the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. The test item is predicted to be not sensitizing to the skin.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2019-04-23 to 2019-04-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
other: Direct Peptide Reactivity Assay (DPRA)
Specific details on test material used for the study:
Batch No.: A03-18-0081
Purity: ≥99.9%
Details on the study design:
Test System
- Test System: Synthetic peptides containing cysteine (SPCC) or synthetic peptides containing lysine (SPCL)
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Source: JPT Peptide Technologies GmbH

Experimental Design
- Preparation of Test Item: For both the cysteine and lysine reactivity assay 17.48 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1532 μL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
- Preparation of Solutions for Cysteine Reactivity Assay:
- Synthetic Peptide Containing Cysteine (SPCC) Stock Solution: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.5 mg of SPCC in 20.96 mL phosphate buffer pH7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
- SPCC Reference Control Solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL ACN.
- Preparation of Solutions for Lysine Reactivity Assay:
- Synthetic Peptide Containing Lysine (SPCL) Stock Solution: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.8 mg of SPCL in 20.85 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
- SPCL Reference Control Solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN.
- Sample Incubations: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25 ± 2.5 °C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.4 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence did not exceed 30 hours.
- HPLC Analysis: SPCC and SPCL peak areas in the samples were measured by HPLC.
Positive control results:
Cysteine Reactivity Assay
The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 100% ± 0.0%. 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
The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 55.1% ± 2.4%. 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
Run / experiment:
other: Cysteine Reactivity Assay
Parameter:
other: SPCC depletion%
Value:
0.6
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
The mean of the SPCC depletion
Key result
Run / experiment:
other: Lysine Reactivity Assay
Parameter:
other: SPCL depletion %
Value:
10.5
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
The mean of the SPCL depletion
Other effects / acceptance of results:
- Results Cysteine Reactivity Assay for the Test Item
Upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. No precipitate or phase separation was observed in any of the samples. For the 210160/A-cys samples, the mean SPCC A220/A258 area ratio was 37.87. Since this was within the 34.06 - 41.63 range, this indicated that there was no co-elution of the test item with SPCC. The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the test item was 0.6% ± 0.5%.
- Results Lysine Reactivity Assay for the Test Item
Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. No precipitate or phase separation was observed in any of the samples.
In the CC sample no peak was observed at the retention time of SPCL. This demonstrated that there was no co-elution of the test item with SPCL. For the 210160/A-lys samples, the mean SPCL A220/A258 area ratio was 31.70. Since this was within the 28.38 – 34.69 range, this again indicated that there was no co-elution of the test item with SPCL. The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the Test Item was 10.5% ± 0.4%.
DPRA Prediction and Reactivity Classification
Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.
In the cysteine reactivity assay the test item showed 0.6% SPCC depletion while in the lysine reactivity assay the test item showed 10.5% SPCL depletion. The mean of the SPCC and SPCL depletion was 5.6% and as a result the test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
- Test Acceptability: all acceptability criteria were met this DPRA is considered to be valid.
Interpretation of results:
other: negative
Conclusions:
The test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Executive summary:

The objective of this study was to determine the reactivity of the test item towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) according to OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)).

After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

 

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA.

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.

In the cysteine reactivity assay the test item showed 0.6% SPCC depletion while in the lysine reactivity assay the test item showed 10.5% SPCL depletion. The mean of the SPCC and SPCL depletion was 5.6%.

 

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. The test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2019-03-29 to 2019-04-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
Batch No.: A03-18-0081
Purity: ≥99.9%
Details on the study design:
VEHICLE
- Vehicle: dimethyl sulfoxide (DMSO)
- Justification for choice of vehicle: A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM (clear colorless solution).

Dose Formulation
- Preparation of Test Item Stock, Spiking and Working Solutions: The 100-fold dilution of the 200 mM DMSO stock in DMEM glutamax formed also a homogeneous solution (no precipitation). This concentration was selected as highest concentration for the main assay (highest dose required in the current guideline).
In the main experiments the test item was dissolved in DMSO at 200 mM (clear colorless solution). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution.
No precipitation was observed at the start and end of the incubation period in the 96-well plates.
Test item concentrations were used within 3 hours after preparation.
- Preparation of the Positive Control: The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol, for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described in paragraph 4.5.1, so that the final concentration of the positive control ranges from 7.8 to 250 μM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.
- Preparation of the Vehicle Control: The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.
- Blank: On each plate three blank wells were tested (no cells and no treatment).

Test System
- Test System: A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used.
- Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).
- Environmental conditions: All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 43 - 98%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.3 - 38.0 °C).
- Subculturing: Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).

Experimental Design
- Plating of Cells: For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+6 in experiment 1, P+8 in experiment 2 and P+10 in experiment 3.
- Treatment of Cells: The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37 ± 1.0 °C in the presence of 5% CO2. In total 3 valid experiments were performed.
- Luciferase Activity Measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 μL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 5 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).
- Cytotoxicity Assessment: For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 - 4 hours at 37 °C ± 1.0 °C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.
Positive control results:
Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.29 and the EC1.5 28 μM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.91 and the EC1.5 31 μM.
Experiment 3: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.15 and the EC1.5 40 μM.
Key result
Run / experiment:
other: Experiment 1
Parameter:
other: Imax
Remarks:
The maximum luciferase activity induction
Value:
1.28
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Experiment 2
Parameter:
other: Imax
Remarks:
The maximum luciferase activity induction
Value:
38.04
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: Experiment 3
Parameter:
other: Imax
Remarks:
The maximum luciferase activity induction
Value:
1.47
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
Experiment 1:
No precipitation was observed at the start and end of the incubation period in the 96-well plates. The test item showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated. No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.28 and therefore no EC1.5 could be calculated.
Experiment 2:
No precipitation was observed at the start and end of the incubation period in the 96-well plates. The test item showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated. A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 38.04 and the EC1.5 16 μM.
Experiment 3:
No precipitation was observed at the start and end of the incubation period in the 96-well plates. The test item showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated. No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with Vinyl ethylene carbonate. The Imax was 1.47 and therefore no EC1.5 could be calculated.

ACCEPTANCE OF RESULTS
All experiments passed the acceptance criteria:
The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
The EC1.5 of the positive control was within two standard deviations of the historical mean (28, 31 and 40 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.29-fold, 3.91-fold and 3.15-fold in experiment 1, 2 and 3, respectively).
Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (8.2%, 15% and 7.0% in experiment 1, 2 and 3, respectively).
Interpretation of results:
other: negative
Conclusions:
The test item is classified as negative (no biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the ability of the test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay according to OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method).

The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 μM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Three independent experiments were performed.

 

All experiments passed the acceptance criteria:

The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.

The EC1.5 of the positive control was within two standard deviations of the historical mean (28, 31 and 40 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.29-fold, 3.91-fold and 3.15-fold in experiment 1, 2 and 3, respectively).

Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (8.2%, 15% and 7.0% in experiment 1, 2 and 3, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

In the first experiment, the test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.28-fold leading to an individual run conclusion of negative.

In the second experiment, the test item showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 16 μM) was measured. The maximum luciferase activity induction (Imax) was 38.04-fold leading to an individual run conclusion of positive.

Since the first two experiments were not concordant a third experiment was performed to provide a final conclusion.

In the third experiment the test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.47-fold leading to an individual run conclusion of negative.

 

In conclusion, the test item is classified as negative (no biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

DEREK assessment:

The objective of this study was to obtain a prediction on the potential for skin sensitization of the test item with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization of the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. The test item is predicted to be not sensitizing to the skin.

 

DPRA assay:

The objective of this study was to determine the reactivity of the test item towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) according to OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)).

After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA.

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.

In the cysteine reactivity assay the test item showed 0.6% SPCC depletion while in the lysine reactivity assay the test item showed 10.5% SPCL depletion. The mean of the SPCC and SPCL depletion was 5.6%.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. The test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

KeratinoSensTM assay:

The objective of this study was to evaluate the ability of the test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay according to OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method).

The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 μM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Three independent experiments were performed.

All experiments passed the acceptance criteria:

The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.

The EC1.5 of the positive control was within two standard deviations of the historical mean (28, 31 and 40 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.29-fold, 3.91-fold and 3.15-fold in experiment 1, 2 and 3, respectively).

Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (8.2%, 15% and 7.0% in experiment 1, 2 and 3, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

In the first experiment, the test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.28-fold leading to an individual run conclusion of negative.

In the second experiment, the test item showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 16 μM) was measured. The maximum luciferase activity induction (Imax) was 38.04-fold leading to an individual run conclusion of positive.

Since the first two experiments were not concordant a third experiment was performed to provide a final conclusion.

In the third experiment the test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.47-fold leading to an individual run conclusion of negative.

In conclusion, the test item is classified as negative (no biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation:

Negative result in DEREK assessment, DPRA assay and KeratinoSensTM assay.

According to Regulation (EC) No 1272/2008, table 3.4.2, this substance is not classified for this endpoint.