Registration Dossier

Administrative data

Description of key information

In silico, in chemico and in vitro data are used in a weight-of-evidence approach.

- In silico: A Derek Nexus assessment yielded an alert for skin sensitisation based on the presence of a epoxide (Alert 433) and predicted an EC3 of 1.8% (Van Gompel, 2017).

- In chemico (OECD 442C): The test item was considered to be positive in the DPRA and classified in the “high reactivity” when using the Cysteine 1:10 / Lysine 1:50 prediction model (Rijk, 2018).

- In vitro (OECD 442D): The KeratinoSensTM assay yielded a positive result (Westerink, 2018).

Based on the data available (a valid in chemico test, a valid in vitro test and an in silico assessment), a definite conclusion on classification cannot be made on the potency for skin sensitization. Although in vivo testing should be performed to determine the classification of the test item, further testing was considered but concluded to be not required.

The available data on the test substance T001597 itself, and data on a structurally similar substance T001598 and a mixture (T001596, mixture of T001598 and T001597) is deemed sufficient for endpoint coverage and conclusion on skin sensitization. T001597 is considered to be classified as skin sensitizer category 1B, as a worst case classification, based on available data and on the criteria in the CLP regulation 1272/2008. More details are included in the attached justification.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-05-24 to 2017-06-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16DB1993
- Expiration date of the lot/batch: 2017-10-03 (retest date)
- Purity (GC): 98.1%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvent was evaluated: acetonitrile (ACN). The test item should have been heated at 60°C for 3 days to liquefy before sampling. However, according to the laboratories internal procedures, general test item handling procedures were followed when preparing the test item and it was not heated but partly scraped off.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the cysteine and lysine reactivity assay 31.59 mg of the test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1627 µL ACN 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.

OTHER SPECIFICS:
- For preparation of the 100 mM test item stock solution a correction factor of 1.00 was used to correct for purity/composition of the test item. On request of the Sponsor this was corrected to 1.13 after the study was performed (study plan amendment). This change didn’t have an impact on the study results. In case more test item would be available to react with the peptides (by using a correction factor of 1.13) and/or by heating the test item before sampling and/or in absence of precipitation), only a difference in percentage peptide depletion would be observed, however the overall classification in the “high reactivity class” would be the same. Therefore, it was decided that this DPRA should not be repeated.
Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:

TEST SYSTEM
- 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.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Batch SPCC: 111016HS_MHeW0217
- Batch SPCL: 220114HSDW_W0217
- Storage: The peptides were stored in the freezer (≤ 15°C) for a maximum of 6 months.

EXPERIMENTAL DESIGN
TEST ITEM PREPARATION
see details under "Specific details on test material used for the study"

PREPARATION OF SOLUTIONS FOR CYSTEINE REACTIVITY ASSAY
- SPCC stock solution: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 20 mg of SPCC in 39.92 mL phosphate buffer pH 7.5. 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.
- SPCC calibration curve: A SPCC calibration curve was prepared as described under "Any other information on materials and methods incl. tables".
- Co-elution control, Test item and Positive control samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described under "Any other information on materials and methods incl. tables".

PREPARATION OF SOLUTIONS FOR LYSINE REACTIVITY ASSAY
- SPCL stock solution: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 20 mg of SPCL in 38.61 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.
- SPCL calibration curve: A SPCL peptide calibration curve was prepared as described under "Any other information on materials and methods incl. tables".
- Co-elution control, Test item and Positive control samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described under "Any other information on materials and methods incl. tables".

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 25 and 26 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 13 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. Sample analysis was performed using the following system:
System 1 (used for Cysteine Reactivity Assay):
- Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
- MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
- LC Column oven 300 (Thermo Scientific)
- Surveyor PDA detector (Thermo Scientific)
System 2 (used for Lysine Reactivity Assay):
- Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
- HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
- Column Oven #151006 (Grace, Worms, Germany)
- Surveyor PDA detector (Thermo Scientific)

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
- The standard calibration curve had to have an r²>0.99.
- 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.
- 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.
- The mean peptide concentration of Reference Controls A had to be 0.50±0.05 mM.
- 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 item’s results to be considered valid:
- The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
- The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50 ± 0.05 mM.
All results presented in the tables of the report were calculated using values as per the raw data rounding procedure and may not have been exactly reproduced from the individual data presented.

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:
% Peptide Depletion = [ 1 - (Peptide Peak Area in Replication injection at 220 nm / Mean Peptide Peak Area in Reference Controls at 220 nm) ] x 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%
DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. 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 sensitizer and a non-sensitizer.
There might be cases where the test item absorbed significantly at 220 nm and had the same retention time as the peptide (co-elution). If co-elution of a test item occurred with both the cysteine and the lysine peptide then the analysis was reported as “inconclusive”. In the case where co-elution occurred only with the lysine peptide, the Cysteine 1:10 prediction model was used.
Positive control results:
Cysteine reactivity assay:
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 73.1% ± 1.4%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%). This was also within the historical positive control data range.

Lysine reactivity assay:
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 58.7% ± 4.3%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%). This was also within the historical positive control data range.
Parameter:
other: % SPCC depletion
Run / experiment:
mean of 3 replicates
Value:
83.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: % SPCL depletion
Run / experiment:
mean of 3 replicates
Value:
12.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: % Mean of SPCC and SPCL depletion
Run / experiment:
mean of 3 replicates
Value:
47.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Positive: High reactivity
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
The DPRA assay was successfully validated at the laboratory and can be used to support the discrimination between sensitisers and non-sensitisers.

ACCEPTANCE OF RESULTS - Cysteine reactivity assay
- Correlation coefficient (r²) standard calibration curve: 0.994 was within the acceptance criteria (r²>0.99) (SPCC standard calibration curve accepted)
- Mean peptide concentration Reference Control A samples: 0.515 ± 0.012 mM was within the acceptance criteria of 0.50 ± 0.05 mM
- Mean peptide concentration Reference Control C samples: 0.474 mM ± 0.014 mM was within the acceptance criteria of 0.50 ± 0.05 mM
The mean of Reference Control Samples A and C were all within the acceptance criteria which confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
- Coefficient of Variation (CV) for Reference Control samples B and C: 3.9% was within the acceptance criteria (CV<15.0%) (confirms stability of the HPLC run over time)
- Mean peptide depletion cinnamic aldehyde: 73.1% was within the acceptance range of 60.8% to100%
- SD of peptide depletion cinnamic aldehyde: 1.4% was below the maximum (SD <14.9%)

ACCEPTANCE OF RESULTS - Lysine reactivity assay
- Correlation coefficient (r²) standard calibration curve: 0.991 was within the acceptance criteria (r²>0.99) (SPCL standard calibration curve accepted)
- Mean peptide concentration Reference Control A samples: 0.501 ± 0.006 mM was within the acceptance criteria of 0.50 ± 0.05 mM
- Mean peptide concentration Reference Control C samples: 0.484 ± 0.006 mM was within the acceptance criteria of 0.50 ± 0.05 mM
The mean of Reference Control Samples A and C were all within the acceptance criteria which confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.
- Coefficient of Variation (CV) for Reference Control samples B and C: 2.6% was within the acceptance criteria (CV<15.0%) (confirms stability of the HPLC run over time)
- Mean peptide depletion cinnamic aldehyde: 58.7% was within the acceptance range of 40.2% to 69.0%
- SD of peptide depletion cinnamic aldehyde: 4.3% was below the maximum (SD <11.6%)

Solubility assessment:

At a concentration of 100 mM, the test item was soluble in ACN. Therefore this solvent was used to dissolve the test item in this DPRA study.

Results cysteine reactivity assay for the test item:

Preparation of a 100 mM test item stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the co-elution control (CC) as well as the test item 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 SPCC. This demonstrated that there was no co-elution of the test item with SPCC. For the 207493/A-cys samples, the mean SPCC A220/A258 area ratio was 20.51. This was outside the 15.77-19.27 range. However, since the test item 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 item did not co-elute with SPCC.

Results lysine reactivity assay for the test item:

Preparation of a 100 mM test item stock solution in ACN showed that the test item dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. Upon preparation as well as after incubation a precipitate was observed in the CC and the test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide. Consequently, the peptide depletion might be underestimated.

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 207493/A-lys samples, the mean SPCL A220/A258area ratio was 13.42. Since this was within the 12.22-14.93 range, this again indicated that there was no co-elution of the test item with SPCL.

SPCC and SPCL depletion, DPRA prediction and reactivity classification of the test item:

SPCC depletion

SPCL depletion

Mean of SPCC and SPCL depletion

DPRA prediction and     reactivity classification

Mean

± SD

Mean

± SD

Cysteine 1:10 / Lysine 1:50 prediction model

83.5%

±3.2%

12.4%

±0.8%

47.9%

Positive:

High reactivity

SD = Standard Deviation; NA = not applicable              

Historical control data for DPRA studies

      Positive control - Cinnamic aldehyde
   SPCC depletion SPCL depletion 
 Range  71.8 - 77.9% 43.5 - 65.2% 
 Mean  74.4% 59.5% 
 SD  1.6% 5.5% 
 n 19 19

SD = Standard Deviation, n = Number of observations

The above mentioned historical control data were collected over the period of January 2017 to April 2017.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In conclusion, since all acceptability criteria were met the DPRA is considered to be valid. The test item was positive in the DPRA and was classified in the “high 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:
2017-08-09 to 2017-09-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I17EB1781
- Expiration date of the lot/batch: 2018-04-26 (retest date)
- Purity (GC): 98.8%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM (clear colorless solution). The 100-fold dilution of the 200 mM DMSO stock in DMEM 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).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: IIn the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM. From the stock 11 spike solutions in DMSO were prepared (2-fold dilution series).

OTHER SPECIFICS:
- For the preparation of the 200 mM test item stock no correction was made for the purity/composition of the test item. On request of the Sponsor this was corrected to 1.13 after the study was performed (study plan amendment). This change did not have an impact on the study results. In case more test item would be available to induce luciferase activity (by using a correction factor of 1.13), only a difference in EC1.5 and IC30 and IC50 would be observed, however the overall classification of “positive” would be the same. Therefore, it was decided that this Keratinosens should not be repeated.
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:

CONTROLS
- solvent control: 1% DMSO in exposure medium
- positive control: Ethylene dimethacrylate glycol

PREPARATION OF TEST ITEM STOCK, SPIKING AND WORKING SOLUTIONS
- A solubility test with dimethyl sulfoxide (DMSO) was performed as described in Specific details on test material used for this study
- In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM. From the 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 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 2.5 hours after preparation. Any residual volumes were discarded..

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, 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 SOLVENT CONTROL
The solvent 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
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 (Duebendorf, Switserland). 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 of 25 passages from the stock (p+25) and are employed for routine testing using the appropriate maintenance medium. Once a year the cell line is checked for infection with a mycoplasma detection test.

CELL CULTURE
- Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
- Maintenance medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL).
- Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal 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 81 – 98 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 – 37.1°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations, of less than an hour 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.

EXPERIMENTAL DESIGN
- Two experiments were conducted
- Subculturing: Cells were subcultured upon reaching 80-90% confluency in maintenance medium. 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).
- 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, one white 96-well plate (3 replicates per concentration) was used for the luciferase activity measurements, and one parallel 96-well transparent plate (3 replicates per concentration) was used for the MTT cell viability assay. The cells were incubated overnight in the incubator 24 ± 1 hours. The passage number used was p+13 in experiment 1 and p+15 in experiment 2.
- Treatment of cells: The medium was removed and replaced with fresh culture medium (150 μL exposure medium) 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 at 37±1.0oC in the presence of 5% CO2. Initially, experiment 1 did not pass all the acceptability criteria of the positive control (not reported) and therefore this part of the study (including the test item, positive and negative control) was repeated . In total 2 valid experiments were conducted.
- Luciferase activity measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together at room temperature. Prior to addition to the cells the Steady-Glo Luciferase substrate was mixed 1:1 with exposure medium. The assay plates were removed from the incubator and the medium was removed. Then 100 μL of PBS was added to rinse the cells. After removing the PBS, 200 µL of the Steady-Glo Luciferase substrate solution 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 DMEM-glutamax containing 5 mg/mL 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 KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM. Moreover, the average induction in the three replicates 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.
c) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
The coefficient of variation is calculated with the following formula:
Coefficient of Variation = [(Standard deviation / Mean luminescence reading) x 100%]

DATA EVALUATION AND STATISTICAL PROCEDURES
The following parameters were 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.
Imax, EC1.5, IC50 and IC30 were calculated based on the equations stated in the OECD Guideline 442D.
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 solvent (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. ToxRat Professional will be used for statistical analysis of the data.
Additional points for data analysis:
- A graph was produced to help visually check the data. If no clear dose response curve was observed, or if the dose-response curve obtained was biphasic (i.e. crossing the threshold of 1.5 twice), the experiment was repeated to verify whether this was specific to the test chemical or due to an experimental artefact. In case the biphasic response was reproducible in an independent experiment, the lower EC1.5 value (the concentration when the threshold of 1.5 is crossed the first time) should be reported.
- In the rare cases where a statistically non-significant induction above 1.5 fold was observed followed by a higher concentration with a statistically significant induction, results from this repetition were only considered as valid and positive if the statistically significant induction above the threshold of 1.5 was obtained for a non-cytotoxic concentration.
- Finally, for test chemicals generating a 1.5 fold or higher induction already at the lowest test concentration of for example 0.977 μM, the EC1.5 value of <0.977 was set based on visual inspection of the dose-response curve.

DATA INTERPRETATION
A minimum of two experiments was conducted, in case of two not concordant results, a third experiment was performed. A KeratinoSensTM 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 KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (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.
4. There is an apparent overall dose-response for luciferase induction
Negative results obtained with concentrations < 1000 µM should be considered as inconclusive.
Positive control results:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 μM (47 μM and 64 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.45-fold and 2.38-fold in experiment 1 and 2, respectively).
Parameter:
other: lmax
Run / experiment:
Experiment 1
Value:
12.13
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: lmax
Run / experiment:
Experiment 2
Value:
11.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC30 (in μM)
Run / experiment:
Experiment 1
Value:
181
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC30 (in μM)
Run / experiment:
Experiment 2
Value:
180
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: EC 1.5 (µM)
Run / experiment:
Experiment 1
Value:
20
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: EC 1.5 (µM)
Run / experiment:
Experiment 2
Value:
19
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC50 (µM)
Run / experiment:
Experiment 1
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC50 (µM)
Run / experiment:
Experiment 2
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
The KeratinoSensTM assay was successfully implemented and validated, and lab proficiency has been shown by obtaining the expected KeratinoSensTM prediction for the 10 proficiency chemicals that are described in the OECD 442D guideline.

ACCEPTANCE OF RESULTS:
Both tests passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 µM (47 µM and 64 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.45-fold and 2.38-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (5.4% and 5.5% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Test item 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 toxicity. The calculated IC30 was 181 μM and the calculated IC50 was 200 µM.

- A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 12.13 and the EC1.5 20 μM.

- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.45 and the EC1.5 47 µM

Experiment 2:

- No precipitation was observed at the start and end of the incubation period in the 96-well plates.

-The test item showed toxicity. The calculated IC30 was 180 μM and the calculated IC50 was 200 µM.

- A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 11.20 and the EC1.5 19 μM.

- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.38 and the EC1.5 64 µM.

Historical Control Data for the KeratinoSensTM Studies:

      Positive control
   EC1.5 (µM) Imax (µM) 
Range 5.3 - 123.0  1.6 - 52.7 
Mean  50.9  3.7 
 SD 29.2  4.8 
 n 193  189 

SD = Standard Deviation; n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of May 2016 to December 2017.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In conclusion, the test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in the report.
Endpoint:
skin sensitisation, other
Remarks:
in silico
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Justification for type of information:
1. SOFTWARE
Derek Nexus

2. MODEL (incl. version number)
Derek Nexus: 5.0.1, Nexus: 2.1.0

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
FC2=CC=C1O[C@@]([H])(CCC1=C2)[C@@]3([H])CO3

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
see attached QMRF
Q13-46-0045
The corresponding QMRF named “Derek for Windows - Skin sensitization” has been downloaded from the JRC QSAR model Database.

5. APPLICABILITY DOMAIN
(see Derek report in field 'Attached background material')
Alert matched: 433 Epoxide
Skin sensitisation in mammal is PLAUSIBLE

6. ADEQUACY OF THE RESULT (see Derek report in field 'Attached background material')
- Alert 433 Epoxide
Predicted value: The predicted EC3 value of 1.8% corresponds to “moderate sensitizer”
Following EU CLP criteria, if measured experimentally, the predicted value would correspond to “Category 1A” in the CLP classification system as the predicted EC3 value is below 2%.
Principles of method if other than guideline:
- Software tool(s) used including version:
Derek Nexus: 5.0.1; Nexus: 2.1.0
- Model(s) used:
Knowledge Base: Derek KB 2015 1.0
- Model description: see QMRF in field 'Attached justification'
- Justification of QSAR prediction: the QSAR prediction for skin sensitisation is used as part of the weight-of-evidence approach to cover the information requirements for this endpoint. The justification is further elaborated in the weight-of-evidence justification attached to the skin sensitisation endpoint summary in this dossier.
Specific details on test material used for the study:
SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: FC2=CC=C1O[C@@]([H])(CCC1=C2)[C@@]3([H])CO3
- Average Mol Mass: 194.2
- Exact Mol Mass: 194.0743
- Log Kp: -2.43
- Log P: 2.21
- Composition / purity: other information: not applicable for in silico study
Parameter:
EC3
Value:
1.8
Test group / Remarks:
Predicted EC3 value (Derek EC3 Model 1.0.5)
Remarks on result:
positive indication of skin sensitisation based on QSAR/QSPR prediction

Reasoning summary:

Skin sensitisation in mammal is PLAUSIBLE

Alert matched: 433 Epoxide

Comments:

Potential mechanism: Hapten acting as an alkylating agent [Payne and Walsh]

Compounds such as 3-methyl-3-phenyl-oxiranecarboxylic acid ethyl ester and 3-phenyl-oxirane-2-carboxylic acid ethyl ester have been reported to be strong sensitisers in the GPMT [Cronin and Basketter].

The activity of glycidyl ethers, amines, esters and amides is described in a separate alert

References:

- Cronin MTD and Basketter DA. (1994)

Multivariate QSAR analysis of a skin sensitization database., SAR and QSAR in Environmental Research, 2 , 159-179

DOI: 10.1080/10629369408029901

- Betso JE, Carreon RE and Miner VM. (1991)

The use of nuclear magnetic resonance spectrometry (1H NMR) for monitoring the reaction of epoxides with butylamine and predictive capabilities of the reactive alkylation index (RAI) for skin sensitization by epoxides., Toxicology and Applied Pharmacology, 108 , 483-488

- Payne MP and Walsh PT. (1994)

Structure-activity relationships for skin sensitization potential: development of structural alerts for use in knowledge-based toxicity prediction systems., Journal of Chemical Information and Computer Sciences, 34 , 154-161

DOI: 10.1021/ci00017a019

Validation comments:

1) Cronin and Basketter: 2 compounds activate this alert of which 2 are reported positive. (Positive predictivity: 100%.)

2) Gerberick: 1 compound activates this alert of which 0 are reported positive. (Positive predictivity: 0%.)

3) Contact Dermatitis: 1 compound activates this alert of which 0 are reported positive. (Positive predictivity: 0%.)

1) A collection of guinea pig maximisation test data for 216 compounds from the following reference: Cronin MTD and Basketter DA. Multivariate QSAR analysis of a skin sensitization database. SAR and QSAR in Environmental Research, 1994, 2, 159-179, available at "http://dx.doi.org/10.1080/10629369408029901".

2) A collection of local lymph node assay data for 318 compounds derived from the following references: (i) Gerberick GF, Ryan CA, Kern PS, Schlatter H, Dearman RJ, Kimber I, Patlewicz GY and Basketter DA. Compilation of historical local lymph node data for evaluation of skin sensitization alternative methods. Dermatitis, 2005, 16, 157-202. Downloaded from "http://www.inchemicotox.org/results/" (3 September 2010); (ii) Kern PS, Gerberick GF, Ryan CA, Kimber I, Aptula A and Basketter DA. Local lymph node data for the evaluation of skin sensitization alternatives: a second compilation. Dermatitis, 2010, 21, 8-32, available at &ldquo;http://dx.doi.org/10.2310/6620.2009.09038&rdquo;.

3) A collection of local lymph node assay data for 137 compounds published in Contact Dermatitis which have been extracted from Vitic Nexus (13 September 2012).

EC3 Result for Derek EC3 Model - 1.0.5:

- Alert matched: 433 Epoxide

Predicted LLNA EC3: 1.8% (moderate sensitiser)

Experimental Match: No exact match found

Compounds used in calculation: 10/16

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
T001597 is predicted as a sensitiser to the skin with a predicted LLNA EC3 value of 1.8 (epoxide) and a prediction strength as PLAUSIBLE using Derek Nexus v5.0.1. Based on the predicted EC3 value of 1.8%, T001597 is classified as skin sensitiser category 1A. This substance triggered a skin sensitisation alert for 433 Epoxide.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In silico, in chemico and in vitro data are used in a weight-of-evidence approach. The studies are discussed in detail in the Weight-of-Evidence justification attached to this endpoint summary.

The available data on the test substance T001597 itself, and data on a structurally similar substance T001598 and a mixture (T001596, mixture of T001598 and T001597) is deemed sufficient for endpoint coverage and conclusion on skin sensitization.

All available studies with T001597 resulted in positive results and pointed out that T001597 is a skin sensitizer. Both structurally related substances were classified as category 1B. Based on the above, a positive outcome of an LLNA study with T001597 is to be expected. 

No in vivo skin sensitization testing with T001597 has been performed (OECD Guideline 429), also taking into consideration animal welfare and the 3R principles. For T001597, additional testing is not considered required and an equal classification is proposed based on available data and on the criteria in the CLP regulation 1272/2008. More details are included in the justification attached in the waiver for in vivo skin sensitiezation testing.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on the test substance T001597 itself, and data on a structurally similar substance T001598 and a mixture (T001596, mixture of T001598 and T001597) is deemed sufficient for endpoint coverage and conclusion on skin sensitization.

For T001597, additional testing is not considered required and an equal classification is proposed based on available data and on the criteria in the CLP regulation 1272/2008: skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.