Registration Dossier

Administrative data

Description of key information

- DPRA Assay

In the cysteine reactivity assay the test material showed 100 % SPCC depletion while in the lysine reactivity assay the test material showed 0.2 % SPCL depletion. The mean of the SPCC and SPCL depletion was 50.1 % and as a result the test material was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Therefore, the test material was considered to be positive in the DPRA.

- KeratinoSens Assay

Under the conditions of the study the test material was classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 February 2018 to 08 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide binding assay
Test material information:
Composition 1
Specific details on test material used for the study:
No correction for the purity/composition of the test material was performed.
Solubility of the test material in an appropriate solvent was assessed before performing the DPRA. The following solvents were evaluated: acetonitrile (ACN) and Milli-Q water (MQ).
Details on study design:
- Dose formulation
Test material stock solutions were prepared freshly for each reactivity assay. For both the cysteine and lysine reactivity assay 34.87 mg of test material was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1762 μL MQ 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 material was dissolved. The test material, 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.

- Test system
Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL (JPT Peptide Technologies GmbH, Berlin, Germany)

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 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
- 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 acetonitrile (ACN). In addition, a RCcysCMQ sample was included to evaluate the effect of the solvent that was used to dissolve the test material on the Percent Peptide Depletion. The RCcysCMQ sample was prepared by mixing 750 μL of the 0.667 mM SPCC stock solution with 200 μL ACN and 50 μL cosolvent.
- A SPCC calibration curve was prepared
- Co-elution Control, Test Material and Positive Control Samples
The co-elution control (CC) samples, test material samples and the cinnamic aldehyde positive control samples (PC) were prepared, as follows:
* Co-elution control (CC) - 1 replicate - (CCcys-209202/A): 750 μL Phosphate buffer pH 7.5, 200 μL ACN, 50 μL 209202/A test solution (100 mM)
* Cinnamic aldehyde (PC) - 3 replicates - (PCcys-1 to PCcys-3): 750 μL Stock solution of 0.667 mM SPCC, 200 μL ACN, 50 μL Cinnamic aldehyde solution (100 mM in ACN)
* Test mateiral 209202/A - 3 replicates - (209202/A-cys-1 to 209202/A-cys-3): 750 μL Stock solution of 0.667 mM SPCC, 200 μL ACN, 50 μL 209202/A test solution (100 mM)

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 mg of SPCL in 19.31 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. In addition, a RClysCMQ sample was included to evaluate the effect of the solvent that was used to dissolve the test material on the Percent Peptide Depletion. The RClysCMQ sample was prepared by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL
cosolvent.
- SPCL Calibration Curve
A SPCL peptide calibration curve was prepared
- Co-elution Control, Test Material and Positive Control Samples
The co-elution control (CC) samples, test material samples and the cinnamic aldehyde positive control samples (PC) were prepared, as follows:
* Co-elution control (CC) - 1 replicate - (CClys-209202/A): 750 μL Ammonium acetate buffer pH 10.2, 250 μL 209202/A test solution (100 mM)
* Cinnamic aldehyde (PC) - 3 replicates - (PClys-1 to PClys-3): 750 μL Stock solution of 0.667 mM SPCL, 250 μL Cinnamic aldehyde solution (100 mM in ACN)
* Test mateiral 209202/A - 3 replicates - (209202/A-lys-1 to 209202/A-lys-3): 750 μL Stock solution of 0.667 mM SPCL, 250 μL 209202/A test solution (100 mM)

> Sample Incubations
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test material 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 26 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.

> HPLC-PDA Analysis
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA:
Mobile phase A: 0.1 % (v/v) TFA in Milli-Q water B: 0.085 % (v/v) TFA in ACN
Gradient
Cysteine :
0 min 10 % B
10 min 25 % B
11 min 90 % B
13 min 90 % B
13.5 min 10 % B
20 min 10 % B

Lysine:
0 min 10% B
10 min 20% B
11 min 90% B
13 min 90% B
13.5 min 10% B
20 min 10% B

Flow: 0.35 mL/min
Injection volume: 3 μL
Sample tray temperature: Set at 25 °C
Column: Zorbax SB-C18, 100 mm x 2.1 mm, df = 3.5 μm (Agilent Technologies, Santa Clara, CA, USA)
Guard column: SecurityGuard™ cartridge for C18, 4 x 2.0 mm (Phenomenex, Torrance, CA, USA)
Column temperature: Set at 30 °C
Detection: Photodiode array detection, monitoring at 220 and 258 nm

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r² > 0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8 and 100 % for SPCC and between 40.2 and 69.0 % for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be < 14.9 % for the Percent Cysteine Peptide Depletion and < 11.6 % for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be < 15.0 %.
The following criteria had to be met for a test material’s results to be considered valid:
a) The maximum SD for the test material replicates had to be < 14.9 % for the Percent Cysteine Depletion and <11.6 % for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50 ± 0.05 mM.

ANALYSIS
- Data Evaluation
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion = [1 − (Peptide Peak Area in Replicate Injection (at 220 nm) / Mean Peptide Peak Area in Reference Controls (at 220 nm))] × 100
In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90 % < mean area ratio of control samples < 110 % gives a good indication that co-elution has not occurred. However, calculation of peak purity (area ratio 220 nm/258 nm) might not always be possible, particularly if the test material is highly reactive with the peptide.

- Data Interpretation
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test material. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38 % average peptide depletion was used to support the discrimination between a skin sensitiser and a non-sensitiser:
Negative: 0 % ≤ Mean % depletion ≤ 6.38 % (No or minimal reactivity)
Positive: 6.38 % < Mean % depletion ≤ 22.6 2% (Low reactivity), 22.62 % < Mean % depletion ≤ 42.47 % (Moderate reactivity) and 42.47 % < Mean % depletion ≤ 100 % (High reactivity)
Positive control results:
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 75.3 ± 2.1 %. This was within the acceptance range of 60.8 to 100 % with a SD that was below the maximum (SD < 14.9 %).
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 55.5 ± 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
Parameter:
other: Mean of SPCC and SPCL depletion
Run / experiment:
DPRA Prediction
Value:
50.1
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
- Solubility Assessment of the Test Material
At a concentration of 100 mM, the test material was not soluble in acetontrile (ACN), but was soluble in Milli-Q water (MQ). Therefore this solvent was used to dissolve the test material in this DPRA study.

- Cysteine Reactivity Assay
The reactivity of the test material towards SPCC was determined by quantification of the remaining concentration of SPCC using HPLC-PDA analysis, following 26 hours of incubation at 25 ± 2.5 °C.

The correlation coefficient (r^2) of the SPCC standard calibration curve was 0.997. Since the r^2 was >0.99, the SPCC standard calibration curve was accepted. The means of Reference Control samples A, C and CMQ were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve the test item did not impact the Percent SPCC Depletion. The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 5.8 %. This was within the acceptance criteria (CV <15.0 %) and confirms the stability of the HPLC run over time. The mean area ratio (A220/A258) of the Reference Control samples was 19.28. The mean A220/A258 ratio ± 10% range was 17.35-21.20. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

Preparation of a 100 mM test material stock solution in MQ showed that the test material was dissolved completely. Upon preparation and after incubation, both the co-elution control (CC) as well as the test material samples were visually inspected. No precipitate was observed in any of the samples after preparation, however, after incubation a precipitate (a gelatinous layer on the bottom of the vial) was observed in the co-elution control (CC) and test material samples. In the CC sample no peak was observed at the retention time of SPCC. This demonstrated that there was no co-elution of the test material with SPCC. For the 209202/A-cys samples, the mean SPCC A220/A258 area ratio could not be determined. Since the test material displayed high reactivity towards SPCC, accurate calculation of the peak purity was not possible due to the low SPCC signal at 258 nm.
Overall, it can be concluded that the test material did not co-elute with SPCC. The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls CMQ. The mean Percent SPCC Depletion for the test material was 100 ± 0.0 %.

- Lysine Reactivity Assay
The reactivity of the test material towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC-PDA analysis, following 26 hours of incubation at 25 ± 2.5 °C.

The correlation coefficient (r^2) of the SPCL standard calibration curve was 0.994. Since the r^2 was >0.99, the SPCL standard calibration curve was accepted. The means of Reference Control samples A, C and CMQ were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve the test material did not impact the Percent
SPCL Depletion. The CV of the peptide areas for the nine Reference Controls B and C was 2.5 %. This was within the acceptance criteria (CV <15.0 %) and confirms the stability of the HPLC run over time. The mean area ratio (A220/A258) of the Reference Control samples was 15.65. The mean A220/A258 ratio ± 10 % range was 14.08-17.21. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

Preparation of a 100 mM test material stock solution in MQ showed that the test material was dissolved completely. Upon preparation and after incubation, both the CC as well as the test material samples were visually inspected. No precipitate 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 material with SPCL. For the 209202/A-lys samples, the mean SPCL A220/A258 area ratio was 15.32. Since this was within the 14.08 - 17.21 range, this again indicated that there was no coelution of the test material with SPCL.
The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls CMQ. The mean Percent SPCL Depletion for the test material was 0.2 ± 0.3 %.

> DPRA Prediction and Reactivity Classification
Upon preparation of the SPCC and SPCL test material samples, no precipitate was observed in any of the samples, however, after incubation, a precipitate was observed for the SPCC test material samples.
In the cysteine reactivity assay the test material showed 100 % SPCC depletion while in the lysine reactivity assay the test material showed 0.2 % SPCL depletion. The mean of the SPCC and SPCL depletion was 50.1 % and as a result the test material was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Therefore, the test material was considered to be positive in the DPRA.
Interpretation of results:
other: Positive DPRA prediction
Conclusions:
In the cysteine reactivity assay the test material showed 100 % SPCC depletion while in the lysine reactivity assay the test material showed 0.2 % SPCL depletion. The mean of the SPCC and SPCL depletion was 50.1 % and as a result the test material was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Therefore, the test material was considered to be positive in the DPRA.
Executive summary:

The reactivity of the test material towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was investigated in accordance with the standardised guideline OECD 442C, under GLP conditions.

During the study, after incubation of the test materialwith either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) 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 material to one of four reactivity classes used to support the discrimination between sensitisers and nonsensitisers.

In the cysteine reactivity assay the test material showed 100 % SPCC depletion while in the lysine reactivity assay the test material showed 0.2 % SPCL depletion. The mean of the SPCC and SPCL depletion was 50.1 % and as a result the test material was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Therefore, the test material was considered to be positive in the DPRA.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 March 2018 to 18 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin sensitisation tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).
Test material information:
Composition 1
Details on study design:
DOSE FORMULATION
- Preparation of Test Material Stock, Spiking and Working Solutions
No correction was made for the composition/purity of the test material.
A solubility test was performed. The test material was not soluble in DMSO at a concentration of 200 mM and at 100 mM it formed a non-homogeneous suspension. Therefore, DMSO was considered not suitable as solvent for the test material. In Milli-Q water the test material was dissolved to a final concentration of 200 mM (colourless solution). The 100-fold dilution of the 200 mM Milli-Q water stock in DMEM glutamax formed also a clear solution (2000 µM). This concentration was selected as highest concentration for the main assay (highest dose required in the current guideline).
In the main experiments the test material was dissolved in Milli-Q water at 200 mM (colourless). From this stock 11 spike solutions in Milli-Q water were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold 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 Milli-Q water of 1 %). All concentrations of the test material 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 material concentrations were used within 3.5 hours after preparation.
Any residual volumes were discarded.

- Preparation of the Positive Control
The positive control used in the case of KeratinoSens™ is ethylene dimethacrylate glycol, for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted so that the final concentration of the positive control ranged 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 and 1 % Milli-Q water 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
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan. Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

CELL CULTURE
Basic medium
- Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1 % (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum (Life Technologies, Bleiswijk, The Netherlands).

Maintenance medium
- Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1 % (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum and geneticin (500 µg/mL).

Exposure medium
- Dulbecco’s minimal (DMEM glutamax) supplemented with 1 % (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum.

Environmental conditions
- All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 – 100 % (actual range 66 – 100 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.5 – 37.2 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

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+9 in experiment 1, P+12 in experiment 2 and P+17 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 materials 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 at 37 ± 1.0 °C in the presence of 5 % CO2. Initially, experiment 1 and 2 did not pass all the acceptability criteria and therefore these experiments were repeated. In total 3 valid experiments were performed.
Luciferase Activity Measurement
- The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilised) from Promega (Leiden, The Netherlands) 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 3 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 KeratinoSens™ 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 hours at 37 °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.

ACCEPTABILITY CRITERIA
The KeratinoSens™ test is considered acceptable if it meets the following criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
- The EC1.5 should be between 5 and 125 µM. Moreover, the induction for ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
- The average coefficient of variation of the luminescence reading for the solvent (negative) control Milli-Q water should be below 20 % in each repetition which consists of 18 wells. If the variability is higher, results should be discarded.
- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20 % in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.


INTERPRETATION

Data analysis
The following parameters are calculated in the KeratinoSensTM test method:
- The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
- 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.
Fold luciferase activity induction is calculated by Equation 1, and the overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.
Equation 1: Fold induction = (Lsample – Lblank) / (Lvehicle – Lblank)
Where:
Lsample is the luminescence reading in the test chemical well
Lblank is the luminescence reading in the blank well containing no cells and no treatment
Lvehicle is the average luminescence reading in the wells containing cells and vehicle (negative) control

The EC1.5 is calculated by linear interpolation according to Equation 2, and the overall EC1.5 is calculated as the mean of the individual repetitions.
Equation 2: EC1.5 = (Cb – Ca) x [(1.5 – Ia) / (Ib – Ia)] + Ca
Where:
Ca is the lowest concentration in μM with > 1.5 fold induction
Cb is the highest concentration in μM with < 1.5 fold induction
Ia is the fold induction measured at the lowest concentration with > 1.5 fold induction (mean of three replicate wells)
Ib is the fold induction at the highest concentration with < 1.5 fold induction (mean of three replicate wells)

Viability is calculated by Equation 3:
Equation 3: Viability = [(Vsample – Vblank) / (Vvehicle – Vblank)] x 100
Where:
Vsample is the MTT-absorbance reading in the test chemical well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vvehicle is the average MTT-absorbance reading in the wells containing cells and vehicle (negative) control

Control IC50 and IC30 are calculated by linear interpolation, and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.
Equation 4: ICx = (Cb – Ca) x [((100 – x) – Va) / (Vb – Va)] + Ca
x is the % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
Ca is the lowest concentration in μM (or µg/mL) with > x% reduction in viability
Cb is the highest concentration in μM (or µg/mL) with < x% reduction in viability
Va is the % viability at the lowest concentration with > x% reduction in viability
Vb is the % viability at the highest concentration with < x% reduction in viability

In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the vehicle (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30 % reduction in cellular viability at the EC1.5 determining concentration.

Data interpretation
A KeratinoSens prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the vehicle (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70 % at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction
Negative results obtained with concentrations <1000 µM or 200 µg/mL should be considered as inconclusive.
Positive control results:
Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.15 and the EC1.5 129 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.58 and the EC1.5 57 µM.
Experiment 3: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.78 and the EC1.5 75 µM.
Parameter:
other: EC1.5 (µM)
Run / experiment:
Experiment 1
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
not determinable
Parameter:
other: EC1.5 (µM)
Run / experiment:
Experiment 2
Value:
332
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Parameter:
other: EC1.5 (µM)
Run / experiment:
Experiment 3
Value:
67
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Parameter:
other: IMax
Run / experiment:
Experiment 1
Value:
1.46
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Parameter:
other: IMax
Run / experiment:
Experiment 2
Value:
1.66
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Parameter:
other: IMax
Run / experiment:
Experiment 3
Value:
3.23
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Other effects / acceptance of results:
RESULTS
- The test material was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway.
- Three independent experiments were performed. The cells were in these experiments incubated with test material in a concentration range of 0.98 – 2000 µM (2-fold dilution steps) for 48 hours. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

Experiment 1
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- The test material showed toxicity. The calculated IC30 was 548 µM and the calculated IC50 was 690 µM.
- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test material. The Imax was 1.46 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 material showed toxicity. The calculated IC30 was 870 µM and the calculated IC50 was 1230 µM.
- A dose related luminescence activity induction was observed after treatment with the test material. The Imax was 1.66 and the EC1.5 332 µM.

Experiment 3
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- The test material showed toxicity. The calculated IC30 was 1863 µM. Fifty percent toxicity was not reached and thus no IC50 could be calculated.
- A dose related luminescence activity induction was observed after treatment with the test material. The Imax was 3.23 and the EC1.5 67 µM.

All tests 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.
- A dose response was observed and the induction at 250 µM was higher than 2-fold in all experiments (2.15-fold, 2.58-fold and 2.78-fold in experiment 1, 2 and 3, respectively). The EC1.5 of the positive control was between 5 and 125 µM in experiment 2 and 3 (129 µM, 57 µM and 75 µM in experiment 1, 2 and 3, respectively).
- The average coefficient of variation of the luminescence reading for the vehicle (negative) control Milli-Q water was below 20 % (16.2, 10.3 and 11.1 % 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 % (19.9, 13.5 and 11.7 % in experiment 1, 2 and 3, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

DISCUSSION
The test material showed toxicity (IC30 values of 548 µM, 870 and 1863 µM in experiment 1, 2 and 3, respectively, and IC50 values of 690 µM and 1230 µM in experiment 1 and 2, respectively). In the first experiment 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 1.46-fold, leading to an individual run conclusion of negative. In the second experiment a biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 332 µM) was measured and a maximum luciferase activity induction (Imax) of 1.66-fold was measured. Since the EC1.5 was below IC30 this led to an individual run conclusion of positive. An additional third experiment was conducted to give a final conclusion. In the third experiment a biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 67 µM) was measured as well. The maximum luciferase activity induction (Imax) was 3.23-fold. Since EC1.5 was below IC30 this led to an individual run conclusion of positive.
The test material is therefore classified as positive in the KeratinoSens™ assay since positive results (>1.5-fold induction) were observed in 2 out of 3 experiments at test concentrations < 1000 µM with a cell viability of >70 % compared to the vehicle control.

Interpretation of results:
other: Positive in the Keratinosens assay
Conclusions:
Under the conditions of the study the test material was classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).
Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442D, under GLP conditions.

The objective of this study was to evaluate the ability of the test material to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

During the study the test material was dissolved in Milli-Q water at 200 mM. From this stock 11 spike solutions in Milli-Q water 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. A dose response was observed and the induction at 250 µM was higher than 2-fold in all experiments (2.15-fold, 2.58-fold and 2.78-fold in experiment 1, 2 and 3, respectively).  The EC1.5 of the positive control was between 5 and 125 µM in experiment 2 and 3 (129, 57 and 75 µM in experiment 1, 2 and 3, respectively). The average coefficient of variation of the luminescence reading for the vehicle (negative) control Milli-Q water was below 20 % (16.2, 10.3 and 11.1 % 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 % (19.9, 13.5 and 11.7 % in experiment 1, 2 and 3, respectively). Overall it is concluded that the test conditions were adequate and that the test system functioned properly.  

Under the conditions of the study the test material showed toxicity (IC30 values of 548, 870 and 1863 µM in experiment 1, 2 and 3, respectively, and IC50 values of 690 µM and 1230 µM in experiment 1 and 2, respectively). In the first experiment 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 1.46-fold, leading to an individual run conclusion of negative. In the second experiment a biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 332 µM) was measured and a maximum luciferase activity induction (Imax) of 1.66-fold was measured. Since the EC1.5 was below IC30 this led to an individual run conclusion of positive. An additional third experiment was conducted to give a final conclusion. In the third experiment a biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 67 µM) was measured as well. The maximum luciferase activity induction (Imax) was 3.23-fold. Since EC1.5 was below IC30 this led to an individual run conclusion of positive.

The test material is therefore classified as positive in the KeratinoSens™ assay since positive results (>1.5-fold induction) were observed in 2 out of 3 experiments at test concentrations < 1000 µM with a cell viability of >70 % compared to the vehicle control.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Skin Sensitisation

- DPRA Assay

The reactivity of the test material towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was investigated in accordance with the standardised guideline OECD 442C under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, after incubation of the test materialwith either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) 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 material to one of four reactivity classes used to support the discrimination between sensitisers and nonsensitisers.

In the cysteine reactivity assay the test material showed 100 % SPCC depletion while in the lysine reactivity assay the test material showed 0.2 % SPCL depletion. The mean of the SPCC and SPCL depletion was 50.1 % and as a result the test material was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Therefore, the test material was considered to be positive in the DPRA.

- KeratinoSens Assay

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442D, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to evaluate the ability of the test material to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

During the study the test material was dissolved in Milli-Q water at 200 mM. From this stock 11 spike solutions in Milli-Q water 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.  A dose response was observed and the induction at 250 µM was higher than 2-fold in all experiments (2.15-fold, 2.58-fold and 2.78-fold in experiment 1, 2 and 3, respectively).  The EC1.5 of the positive control was between 5 and 125 µM in experiment 2 and 3 (129, 57 and 75 µM in experiment 1, 2 and 3, respectively). The average coefficient of variation of the luminescence reading for the vehicle (negative) control Milli-Q water was below 20 % (16.2, 10.3 and 11.1 % 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 % (19.9, 13.5 and 11.7 % in experiment 1, 2 and 3, respectively). Overall it is concluded that the test conditions were adequate and that the test system functioned properly.  

Under the conditions of the study the test material showed toxicity (IC30 values of 548, 870 and 1863 µM in experiment 1, 2 and 3, respectively, and IC50 values of 690 µM and 1230 µM in experiment 1 and 2, respectively). In the first experiment 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 1.46-fold, leading to an individual run conclusion of negative. In the second experiment a biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 332 µM) was measured and a maximum luciferase activity induction (Imax) of 1.66-fold was measured. Since the EC1.5 was below IC30 this led to an individual run conclusion of positive. An additional third experiment was conducted to give a final conclusion. In the third experiment a biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 67 µM) was measured as well. The maximum luciferase activity induction (Imax) was 3.23-fold. Since EC1.5 was below IC30 this led to an individual run conclusion of positive.

The test material is therefore classified as positive in the KeratinoSens™ assay since positive results (>1.5-fold induction) were observed in 2 out of 3 experiments at test concentrations < 1000 µM with a cell viability of >70 % compared to the vehicle control.

Justification for classification or non-classification

In accordance with the Regulation (EC) No. 1272/2008, the substance requires classification as a skin sensitiser.