Registration Dossier

Diss Factsheets

Administrative data

Description of key information

OECD 442C: Direct Peptide Reactivity Assay

Under the conditions of this study, the test material was considered to be negative in the Direct Peptide Reactivity Assay.

OECD 442D: ARE-Nrf2 Luciferase Test Method

Under the conditions of this study the test material was considered to be negative in the ARE-Nrf2 Luciferase Test.

OECD 442E: human Cell Line Activation Test

Under the conditions of this study, the test material was considered to be positive in the human Cell Line Activation Test.

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:
weight of evidence
Study period:
24 July 2017 to 26 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
The study was conducted to quantify the reactivity of the test material towards model synthetic peptides containing either lysine or cysteine. The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
Specific details on test material used for the study:
- The test material was dissolved in isopropanol. This was the first of the listed vehicles that produced a suitable, slightly hazy, solution at a concentration of 100 mM.
- Formulations were prepared shortly before testing.
Details on the study design:
METHODS
Test Material Incubation
- Each test solution was prepared at ratios of 1:10 and 1:50 with the cysteine and lysine stock solutions, respectively. The preparations were placed in an incubator set at 25°C for 24 ± 2 hours. At the end of the incubation period the all samples were centrifuged at 400 g for 5 minutes, due to precipitate noted in the co-elution and test material samples.

Analytical Method
The following HPLC conditions were applied:
Column: Agilent Zorbax SB-C18 2.1 mm x 100 mm, 3.5 µm or equivalent
Wavelength: 220 nm
Guard column: Phenomenex Security Guard c18 4 mm x 2 mm
Flow rate: 0.35 mL/min
Oven temperature: 30 °C
Sample temperature: 25 °C
Injection volume: 7 µL
Mobile Phase: Phase A: 0.1 % (v/v) of trifluoroacetic acid in MilliQ water and Phase B: 0.085 % (v/v) of trifluoroacetic acid in acetonitrile.
Gradient:
0 minutes: 90 % A, 10 % B
10 minutes: 75 % A, 25 % B
11 minutes: 10 % A, 90 % B
13 minutes: 10 % A, 90 % B
13.5 minutes: 90 % A, 10 % B
20 minutes: 90 % A, 10 % B

Reference and Co-elution Controls
- Reference controls were prepared for each peptide.
- Reference control A and B for each peptide were prepared by adding 750 µL of peptide stock solution to 250 µL of acetonitrile.
- Reference control C for cysteine was prepared by adding 750 µL of peptide stock solution to 200 µL of acetonitrile and 50 µL vehicle.
- Reference control C for lysine was prepared by adding 750 µL of peptide stock solution to 250 µL vehicle.
- Reference control A (in triplicate) was used to verify the HPLC system suitability prior to the analysis. Reference control B (six replicates) was used to verify the stability of the reference controls over time and reference control C (in triplicate) was used to verify that acetonitrile did not impact the percent peptide depletion.
- Co-elution controls were prepared to detect possible co-elution of the test material with the peptides. A mixture of 750 µL of 100 mM Phosphate Buffer pH 7.5, 200 µL of acetonitrile and 50 µL of test material solution was used to detect possible co-elution of the test material with cysteine. A mixture of 750 µL of 100 mM ammonium acetate buffer pH 10.2 and 250 µL of test material solution was used to detect possible co elution of the test material with lysine.

Calibration Curves for Peptides
- Calibration curves were prepared for each peptide using a range of concentrations from approximately 0.534 mM to 0.0167 mM (Standards 1 to 6).
- Standard 1 was prepared at approximatively 0.534 mM by dilution of 1600 µL of the peptide stock solution (0.667 mM) with 400 µL of acetonitrile.
- Standards 2 to 6 for cysteine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM Phosphate Buffer pH 7.5).
- Standards 2 to 6 for lysine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM ammonium acetate buffer pH 10.2).
- Samples of dilution buffer alone were also prepared.

Sample Analysis Sequence
The analysis sequence for each peptide was as follows:
- System suitability: Standard 1, Dilution buffer.
- Calibration standards and reference controls: Standard 1, Standard 2, Standard 3, Standard 4, Standard 5, Standard 6, Dilution Buffer, Reference Control A, rep 1, Reference Control A, rep 2, Reference Control A, rep 3.
- Co-elution controls: Co-elution control for test material.
- Reference controls: Reference Control B, rep 1, Reference Control B, rep 2, Reference Control B, rep 3.
- First set of replicates: Reference Control C (positive control), rep 1, Reference Control C (test material), rep 1, Positive Control, rep 1, Test sample, rep 1.
- Second set of replicates: Reference Control C (positive control), rep 2, Reference Control C (test material), rep 2, Positive Control, rep 2, Test sample, rep 2.
- Third set of replicates: Reference Control C (positive control), rep 3, Reference Control C (test material), rep 3, Positive Control, rep 3, Test sample, rep 3.
- Reference controls: Reference Control B, rep 4, Reference Control B, rep 5, Reference Control B, rep 6.

DATA EVALUATION
Data Analysis and Calculations
- The chromatographic data were collected and processed using Chromeleon, a validated data capture system.
- A calibration curve was generated based on the concentration of standards and the peak area. All the peaks of the samples were integrated (standards, samples and controls) “valley to valley” (when possible) and Cysteine and Lysine peptides concentrations were determined from absorbance at 220 nm using the respective calibration curves. Areas were determined for all the samples.
- The PPD for each individual test material and positive control samples was calculated as follows:
PPD = 1 – [(Peptide peak area in replicate injection) / (Mean peptide peak area in reference controls C)] x 100
- The mean peptide peak areas for the nine reference controls B and C, Standard Deviation (SD) and Coefficient of Variation (CV) were calculated.
- The mean peptide peak areas and the mean peptide concentrations (mM) for the three reference controls C were calculated.
- The chromatograms of co-elution controls were compared to the chromatograms of reference controls C.
- For the positive control and for the test material, the PPD in each replicate was calculated from the peptide area of the replicate injection and the mean peptide peak for reference control C. The PPD of every injected positive control and test chemical replicate was calculated. The mean PPD of the three replicate determinations and SD were calculated.
- The mean percent cysteine and percent lysine depletion values were calculated for each test chemical (negative depletion is considered as “0” when calculating the mean).

Assay Acceptance Criteria
The following criteria should be met for a run to be considered valid:
- The standard calibration curve should have a r² >0.99.
- The mean peptide concentration for reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C should be < 15.0 %.
- The mean PPD value (%) of the three replicates for the positive control and maximum standard deviation (SD) must fall within the following ranges:
Cysteine: 60.8 to 100% (SD < 14.9)
Lysine: 40.2 to 69.0% (SD < 11.6)

The following criteria should be met for the test material’s result to be considered valid:
- The maximum standard deviation for the test material replicates should 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 should be 0.50 ± 0.05 mM.

Prediction Model
The mean percent cysteine and percent lysine depletion value was calculated. By using the cysteine 1:10/lysine 1:50 prediction model below, the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of an IATA:

Negative DPRA Prediction:
0 % ≤ mean % depletion ≤ 6.38% (No or minimal reactivity)

Negative DPRA Prediction:
6.38 % < mean % depletion ≤ 22.62% (Low reactivity)
22.62 % < mean % depletion ≤ 42.47% (Moderate reactivity)
42.47 % < mean % depletion ≤ 100% (High reactivity)
Key result
Run / experiment:
other: Lysine Depletion
Parameter:
other: Mean PPD
Value:
0.45
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Run / experiment:
other: Cysteine Depletion
Parameter:
other: mean PPD
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
Lysine Depletion
- The percentage peptide depletion values are shown in Table 1.
- The r² value for the standard calibration curve was 0.9999.
- The mean peptide concentrations for the reference controls A and C were: A: 0.48 mM, C (Positive control): 0.47 mM and C (Test material): 0.34 mM.
- The mean peak area result for reference controls B and C (Positive control) was 32.02 (SD 1.34, CV 4.19).
- The mean peak area result for reference controls B and C (Test material) was 29.27 (SD 5.02, CV 17.16).

Cysteine Depletion
- The percentage peptide depletion values are shown in Table 2.
- The r² value for the standard calibration curve was 0.9951.
- The mean peptide concentrations for the reference controls A and C were: A: 0.53 mM, C (Positive control): 0.51 mM and C (Test material): 0.50 mM.
- The mean peak area result for reference controls B and C (Positive control) was 30.59 (SD 0.96, CV 3.14).
- The mean peak area result for reference controls B and C (Test material) was 30.54 (SD 1.04, CV 3.39).

Table 1: Lysine Depletion Percentage Peptide Depletion Values

Substance

Replicate Peptide Peak Areas

Reference Control C
Mean Peptide Peak Area

PPD

Mean PPD

SD

Test Material

23.75

22.750

0.00

0.45

0.8

23.26

0.00

22.44

1.36

Positive Control

11.50

31.020

62.93

57.54

4.7

14.18

54.29

13.83

55.42

Table 2: Cysteine Depletion Percentage Peptide Depletion Values

Substance

Replicate Peptide Peak Areas

Reference Control C
Mean Peptide Peak Area

PPD

Mean PPD

SD

Test Material

32.820

30.200

0.00

0.0

-

31.570

0.00

31.340

0.00

Positive Control

8.780

30.370

71.09

71.53

0.4

8.600

71.68

8.560

71.81

Interpretation of results:
other: Negative in the Direct Peptide Reactivity Assay.
Conclusions:
Under the conditions of this study, the test material was considered to be negative in the Direct Peptide Reactivity Assay.
Executive summary:

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

The study was conducted to quantify the reactivity of the test material towards model synthetic peptides containing either lysine or cysteine. The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test material was dissolved in isopropanol at a concentration of 100 mM. The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24 ± 2 hours in glass autosampler vials, protected from light and set at 25°C.

The remaining concentration of cysteine- or lysine-containing peptides following the 24 hour incubation period was measured by high performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

The cysteine depletion value was 0.00 %, the lysine depletion value was 0.45 % and the mean of the cysteine and lysine depletion values was 0.23 %.

Under the conditions of this study, the test material was considered to be negative in the Direct Peptide Reactivity Assay.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
25 July 2017 to 18 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
2015
Deviations:
yes
Remarks:
EC1.5 values for the positive control in Experiments 1 and 2 and average induction at 64 μM in Experiment 2 were outside the range specified. These deviations did not affect the integrity/ outcome as the positive control gave a satisfactory dose response.
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The study was conducted to investigate the potential of the test material to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
Specific details on test material used for the study:
- Test formulations were prepared using isopropanol. Preliminary solubility trials showed that the test material was not soluble in dimethylsulphoxide, water or culture medium. The maximum attainable concentration in isopropanol was 100 mM.
- Serial dilutions were made (from the 1000 mM stock) using the vehicle to obtain 12 master concentrations (0.049, 0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50 and 100 mM). The master concentrations were then further diluted 25 fold into culture medium containing serum and finally used for treatment with a further 4 fold dilution factor so that the final concentrations range from 0.49 to 1000 μM.
- Formulations were prepared shortly before testing.
Details on the study design:
TEST SYSTEM
- The ARE-Nrf2 luciferase test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers.
- The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.
- KeratinoSens™ cell line supplied by Givaudan Schweiz, Zurich, Switzerland.
- Preparation of Cultures: A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing serum and Geneticin.

METHODS
Treatment Plate Preparation
- The cells were 80-90 % confluent. On the day prior to treatment, cells were harvested and distributed into 96-well plates (10000 cells/well) and incubated at 37 ± 1 °C, 5 % (v/v) CO2, for 24 ± 1 hours.
- For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.

Treatment
- At the end of the 24-hour incubation period, the medium was removed and replaced with fresh culture medium (containing serum but without Geneticin) to which test material and control formulations were added.
- One well per plate was left empty (no cells and no treatment) to assess background values.
- Each plate was sealed and incubated at 37 ± 1°C, 5 % (v/v) CO2 in air, in a humidified environment for 48 ± 1 hours.
- For each test material and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of at two independent repetitions each containing three replicates of each concentration. Discordant results were obtained between the two repetitions, therefore a third repetition containing three replicates was performed.
- Each independent repetition was performed on a different day with fresh stock solutions of chemicals and independently harvested cells. The cells came from different culture batches.
- Two further repetitions were conducted, however, in both cases the data did not meet the acceptance criteria. The data from these repetitions was invalidated and has not been reported.

Cytotoxicity Assessment
- After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The plate was sealed and incubated for 4 hours at 37 ± 1°C, 5 % (v/v) CO2. The MTT medium was removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37 ± 1°C, 5 % (v/v) CO2 in air and left overnight.
- After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

Luciferase Activity Measurements
- After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 19 to 31 minutes at 25 ± 2°C, loaded into the luminescence plate reader and read using the following parameters: 100 μL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time.


DATA EVALUATION
Analysis of Results
The following parameters were calculated:
- The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the test material 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; and
- The IC50 and IC30 concentration values for 50 and 30% reduction of cellular viability.

Imax
Fold luciferase activity induction at each concentration of test material and positive control was calculated as follows:
Fold induction = (Lsample – Lblank) / (Lsolvent – Lblank)
where
Lsample is the luminescence reading in the test material well,
Lblank is the luminescence reading in the blank well containing no cells and no treatment,
Lsolvent is the average luminescence reading in the wells containing cells and solvent (negative control).
The overall maximal average fold induction (Imax) was calculated as the average of the individual repetitions.

EC1.5
EC1.5 was calculated by linear interpolation as follows:
EC1.5 = (Cb – Ca) x [(1.5 – Ia) / (Ib – Ib)] + 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).
The overall EC1.5 was calculated as the geometric mean of the individual repetitions.

Viability
Viability was calculated as follows:
Viability = [ (Vsample – Vblank) / (Vsolvent – Vblank) ] x 100
where
Vsample is the MTT-absorbance reading in the test material well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vsolvent is the MTT-absorbance reading in the wells containing cells and solvent (negative control).

IC50 / IC30
IC50 and IC30 were calculated by linear interpolation as follows:
ICx = (Cb – Ca) x [((100 – x) –Va) / (Vb – Va) ] + Ca
where
x is the % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
Ca is the lowest concentration in μM with > x% reduction in viability
Cb is the highest concentration in μM 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
The overall IC50 and IC30 were calculated as the geometric mean of the individual repetitions.

Assay Acceptance Criteria
The following acceptance criteria should be met:
- The luciferase activity induction obtained with the positive control should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations (from 4 to 64 μM).
- The EC1.5 value for the positive control should be within two standard deviations of the historical mean (e.g. between 7 and 30 μM based on the validation dataset) and the average induction in the three replicates for the positive control at 64 μM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamic aldehyde will be evaluated to determine acceptability.
- The average coefficient of variation of the luminescence reading for the negative control (DMSO) should be below 20% in each repetition which consists of 6 wells in triplicate.

Prediction Model
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 > 1.5 fold and statistically significantly different when compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s T-test);
2. The cell viability is > 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 < 1000 μM (or < 200 μg/mL for test materials with no defined MW);
4. There is an apparent overall dose response for luciferase induction or if the dose response curve is biphasic.
Positive control results:
- Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at a concentration of 64 μM in Experiment 1, at concentrations of 8 to 64 μM in Experiment 2 and at concentrations of 32 to 64 μM in Experiment 3.
- The EC1.5 values for the positive control were 32.07, 5.97 and 16.27 μM in Experiments 1, 2 and 3, respectively. The average induction in the three replicates for the positive control at 64 μM was 2.63, 60.84 and 5.58 in Experiments 1, 2 and 3, respectively.
- The EC1.5 values for the positive control in Experiments 1 and 2 and the average induction at 64 μM in Experiment 2 were outside the range specified in the Protocol. These deviations from Protocol did not affect the integrity or outcome as the positive control gave a satisfactory dose response.
Key result
Run / experiment:
other: Experiment 1
Parameter:
other: Imax
Value:
0.44
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not valid
Key result
Run / experiment:
other: Experiment 2
Parameter:
other: Imax
Value:
1.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not valid
Key result
Run / experiment:
other: Experiment 3
Parameter:
other: Imax
Value:
0.24
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not valid
Key result
Run / experiment:
other: Experiment 2
Parameter:
other: EC1.5 (µg/mL)
Value:
76.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not valid
Key result
Run / experiment:
other: Experiments 1 and 3
Parameter:
other: EC1.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not valid
Remarks on result:
other: There were no EC1.5 values for Experiments 1 and 3 as there were no statistically significant increases in induction.
Other effects / acceptance of results:
CALCULATION OF IMAX AND EC1.5 VALUES
- The maximal fold increases (Imax) were 0.44, 1.90 and 0.24 for Experiments 1, 2 and 3, respectively.
- The EC1.5 value for Experiment 2 was 76.10 μg/mL. There were no EC1.5 values for Experiments 1 and 3 as there were no statistically significant increases in induction.

VIABILITY
- Cell viability was 99% at the EC1.5 determining concentration in Experiment 2. This measurement was not applicable for Experiments 1 and 3 as there were no EC1.5 determining concentrations in these experiments.
- In Experiment 2 there was an overall dose response for lucipherase.
- In Experiments 1 and 3, there was no apparent overall dose response for lucipherase and the dose response curves were not biphasic.

ASSAY ACCEPTANCE
- Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at a concentration of 64 μM in Experiment 1, at concentrations of 8 to 64 μM in Experiment 2 and at concentrations of 32 to 64 μM in Experiment 3.
- The EC1.5 values for the positive control were 32.07, 5.97 and 16.27 μM in Experiments 1, 2 and 3, respectively. The average induction in the three replicates for the positive control at 64 μM was 2.63, 60.84 and 5.58 in Experiments 1, 2 and 3, respectively. The EC1.5 values for the positive control in Experiments 1 and 2 and the average induction at 64 μM in Experiment 2 were outside the range specified in the Protocol. These deviations from Protocol did not affect the integrity or outcome as the positive control gave a satisfactory dose response.
- The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 13.49, 15.06 and 9.72 % in Experiments 1, 2 and 3, respectively.
Interpretation of results:
other: Negative in the ARE-Nrf2 Luciferase Test.
Conclusions:
Under the conditions of this study the test material was considered to be negative in the ARE-Nrf2 Luciferase Test.
Executive summary:

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

The study was conducted to investigate the potential of the test material to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test material was dissolved in isopropanol to the final concentration (100 mM). Serial dilutions were then made using isopropanol to obtain 12 master concentrations of the test material (0.049, 0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5 25, 50 and 100 mM). The master concentrations were then further diluted 25 fold into culture medium containing serum and finally used for treatment with a further 4 fold dilution factor so that the final concentrations ranged from 0.49 to 1000 μM.

Aliquots of 50 μL of each of the final concentrations were transferred to each of three luciferase plates and a single viability plate. Each plate was sealed using a plate sealer and then incubated at 37 ± 1°C, 5 % (v/v) CO2 in air, in a humidified environment for 48 ± 1 hours.

After the 48-hour exposure period, the medium in the luciferase plates was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).The plate was sealed and incubated for 4 hours at 37 ± 1°C, 5 % (v/v) CO2. The MTT medium was then removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37 ± 1°C, 5 % (v/v) CO2 in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

After the 48-hour exposure period, the cells in the viability plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25 ± 2°C, loaded into the luminescence plate reader and read.

The maximal fold increases (Imax) were 0.44, 1.90 and 0.24 for Experiments 1, 2 and 3, respectively. The EC1.5 value for Experiment 2 was 76.10 μg/mL. There were no EC1.5 values for Experiments 1 and 3 as there were no statistically significant increases in induction. Cell viability was 99% at the EC1.5 determining concentration in Experiment 2. This measurement was not applicable for Experiments 1 and 3 as there were no EC1.5 determining concentrations in these experiments. In Experiment 2 there was an overall dose response for lucipherase. In Experiments 1 and 3, there was no apparent overall dose response for lucipherase and the dose response curves were not biphasic.

Under the conditions of this study the test material was considered to be negative in the ARE-Nrf2 Luciferase Test.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
16 August 2017 to 01 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals Method 442E
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Type of study:
other: human Cell Line Activation Test
Justification for non-LLNA method:
The human Cell Line Activation Test (h-CLAT) has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-coordinated validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and has been recommended to be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
Specific details on test material used for the study:
Test Material Formulation
- Dose Finding Assay: The test material was dissolved at 20 mg/mL in isopropanol then eight stock solutions were prepared by 2-fold serial dilutions using the corresponding solvent. The stock solutions were then further diluted 250-fold in culture medium (working solutions).
- CD86/CD54 Expression Measurement: Eight stock solutions of each test material were prepared by 1.2-fold serial dilutions using the corresponding solvent to give eight doses ranging from 0.335 x CV75 to 1.2 x CV75. The stock solutions were then further diluted 250-fold into the culture medium (working solutions).
Details on the study design:
METHODS

Test System
- Specifications: Human monocytic leukaemia cell line, THP-1 (an immortalised human monocytic leukaemia cell line, used as a surrogate for dendritic cells), supplied by American Type Culture Collection (ATCC), Manassas, VA 20110 USA.
- Cell Culture Maintenance: THP-1 cells were cultured, at 37°C under 5 % CO2 and humidified atmosphere, in RPMI0-1640 medium supplemented with 10% heat inactivated (HI) foetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin and 100 µg/mL streptomycin. The cells were passaged every 2-3 days at a density of 0.1 to 0.2 x 10^6 cells/mL and maintained at a density from 0.1 x 10^6 to 0.8 x 10^6 cells/mL. Cell density did not exceed 1 x 10^6 cells/mL.
- Reactivity Check: This was performed using DNCB (CAS no. 97-00-7, ≥ 99% purity), nickel sulphate (CAS no. 10101-97-0, 99% purity) and lactic acid (CAS no. 50-21-5, 85% purity) two weeks after thawing. DNCB and nickel sulphate should produce a positive response of both CD86 and CD54 and lactic acid should produce a negative response of both CD86 and CD54. Only cells which passed the reactivity check were used for the assay.
- Plate Preparation: THP-1 cells were pre-cultured in culture flasks either at a density of 0.2 x 10^6 cells/mL for 48 hours or at a density of 0.1 x 10^6 cells/ml for 72 hours. On the days of testing, cells were harvested from the flasks and were resuspended with fresh culture medium at 2 x 10^6 cells/mL. The cells were then distributed into a 24 well flat-bottom plate (500 µL/well) or a 96-well flat-bottomed plate (80 µL/1.6 x 10^5 cells per well).

Study Design
- Dose Finding Assay: The test material working solutions or solvent controls were mixed 1:1 (v/v) with the cell suspensions in the 96-well plates. The plates were sealed and then incubated for 24 hours at 37°C, 5 % CO2. After the 24-hour incubation period, all cells from a 96-well flat-bottomed plate were transferred into a 96-well round-bottomed plate. The cells were washed at least twice in 200 µL of phosphate buffered saline containing 0.1% bovine serum albumin (FACS buffer) and re-suspended in 190 µL of FACS buffer. 10 µL of propidium iodide solution (PI) was added just before FACS analysis (final concentration of PI = 0.625 µg/mL). PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of 10,000 viable cells were acquired. Cell viability was calculated using the following equation:
Cell viability = (number of living cells / total number of acquired cells) x 100
The CV75 value, i.e. a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), was calculated by log-linear interpolation using the following equation:
Log CV75 = [(75 – c) x Log(b) - (75 – a) x Log (d)] / (a – c)
Where:
a was the minimum value of cell viability over 75% in testing groups
c was the maximum value of cell viability below 75% in testing groups
b and d were the concentrations showing the value of cell viability a and c respectively.

- CD86/CD54 Expression Measurement: One experiment (consisting of two independent runs) was needed to drive a prediction. Each independent run was performed on a different day. On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium at 2 x 10^6 cells/mL. The cells were then distributed into a 24-well plate (500 µL/1 x 10^6 cells per well). The test material working solution or solvent control was mixed 1:1 (v/v) with the cell suspensions in the 24-well plates. The plates were sealed and then incubated for 24 ± 0.5 hours at 37°C, 5 % CO2. After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation (approximately 250 g, 5 minutes) and washed twice with 1 mL of FACS buffer. After washing, the cells were blocked with 600 µL of blocking solution (FACS buffer containing 0.01 % (w/v) globulin) at 4°C for 15 minutes. After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate and centrifuged (approximately 250 g, 3 minutes). After centrifugation, the cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies, incubated at 4 °C for 30 minutes and then centrifuged with 150 µL of FACS buffer. The stained cells were washed three times with an excess of FACS buffer, re-suspended in FACS buffer and 12.5 µg/mL PI solution was added (to give a final PI concentration of 0.625 µg/mL). The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.


DATA EVALUATION

Analysis of Results
- Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 for the positive control cells and test material-treated cells were calculated according to the following equation:
RFI = [(MFI of test material-treated cells – MFI of test material-treated isotype control cells) / (MFI of solvent-treated cells – MFI of solvent-treated isotype control cells)] x 100
The cell viability from the isotype control cells (stained with mouse IgG1 antibodies) was calculated using the following equation:
Cell viability = (number of living cells / total number of acquired cells) x 100

Prediction Model
An h-CLAT prediction is considered positive if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the h-CLAT prediction is considered negative:
- The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%)
- The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%)

Calculation of Effective Concentration (EC) Values
For test materials predicted as positive, two EC values, the EC150 for CD86 and the EC200 for CD54, are calculated as follows:
EC150 (for CD86) = Bconcentration + [(150 – BRFI) / (ARFI – BRFI) x (Adose – Bdose)]
EC200 (for CD54) = Bconcentration + [(200 – BRFI) / (ARFI – BRFI) x (Adose – Bdose)]
Where:
A concentration is the lowest concentration in µg/mL with RFI > 150 (CD86) or 200 (CD54)
B concentration is the highest concentration in µg/mL with RFI < 150 (CD86) or 200 (CD54)
ARFI is the RFI at the lowest concentration with RFI > 150 (CD86) or 200 (CD54)
BRFI is the RFI at the highest concentration with RFI < 150 (CD86) or 200 (CD54).

Assay Acceptance Criteria
- The cell viabilities of medium and solvent control should be higher than 90%.
- In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%).
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) and cell viability should be more than 50%.
- For the test material, the cell viability should be more than 50% in at least four tested doses in each run.
Positive control results:
For the positive control, RFI values were ≥ 150% for CD86 and ≥ 200% for CD54 in all each independent runs. Cell viability was > 50% in each independent run.
Key result
Run / experiment:
other: Experiment 1, 33.3 µg/mL
Parameter:
other: RFI (CD86)
Value:
157
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Positive result
Key result
Run / experiment:
other: Experiment 1, 40 µg/mL
Parameter:
other: RFI (CD86)
Value:
159
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Positive result
Key result
Run / experiment:
other: Experiment 2, 33.3 µg/mL
Parameter:
other: RFI (CD86)
Value:
156
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Positive result
Other effects / acceptance of results:
DOSE FINDING ASSAY
- No CV75 value was calculated as there was no effect on viability following treatment with the test material at the concentrations used in this study.

CD86/CD54 EXPRESSION RESULTS
- Geometric mean fluorescence intensity (MFI) and viability results are given in Tables 1 and 2.
- The relative fluorescence intensity (RFI) values for the test material were calculated as shown in Table 3.

ASSAY ACCEPTANCE CRITERIA
- The cell viabilities of medium and solvent control were higher than 90% in each independent run.
- In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%). The assay was considered valid as this did not impact on the results obtained with the test material.
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control was > 105% on all occasions.
- For the positive control, RFI values were ≥ 150% for CD86 and ≥ 200% for CD54 in all each independent runs. Cell viability was > 50% in each independent run.
- For the test material, the cell viability was more than 50% in all tested concentrations in each independent run.

CONCLUSION
- The test material, was considered to be positive in the human Cell Line Activation Test.

Table 1: Expression Assay: MFI and Cell Viability Values in Experiment 1

Concentration (µg/mL)

MFI (Geo Mean)

Corrected MFI

Viability

CD86

CD54

Isotype

CD86

CD54

IgG

CD86

CD54

11.1

1236

787

721

515

66

98.8

98.5

98.2

13.3

1296

811

739

557

72

98.6

98.0

98.4

16.0

1280

826

758

522

68

98.2

98.2

98.1

19.2

1318

837

760

558

77

98.4

98.3

98.3

23.1

1336

893

804

532

89

98.4

98.2

98.2

27.7

1507

1003

918

589

85

98.6

98.1

98.0

33.3

1507

1046

926

581

120

98.3

97.9

98.5

40.0

1679

1243

1090

589

153

98.7

98.3

98.4

Solvent

951

676

580

371

96

99.1

98.5

98.3

Positive control

2845

2446

864

1981

1582

72.1

67.0

67.5

Table 2: Expression Assay: MFI and Cell Viability Values in Experiment 2

Concentration (µg/mL)

MFI (Geo Mean)

Corrected MFI

Viability

CD86

CD54

Isotype

CD86

CD54

IgG

CD86

CD54

11.1

1225

798

718

507

80

98.6

97.8

98.2

13.3

1202

819

731

471

88

98.7

98.0

98.4

16.0

1289

835

760

529

75

98.2

97.5

97.8

19.2

1225

867

797

428

70

98.7

97.9

98.2

23.1

1334

883

796

538

87

98.1

97.6

97.9

27.7

1467

970

899

568

71

98.0

97.7

97.5

33.3

1527

1026

935

592

91

98.3

97.6

97.4

40.0

1527

1084

984

543

100

98.4

98.2

97.9

Solvent

974

694

594

380

100

98.6

98.6

98.6

Positive control

2422

1793

751

1671

1042

79.6

78.3

77.9

Table 3: Calculation of the Relative Fluorescence Intensity (RFI) Values for the Test Material

Concentration (µg/mL)

RFI (CD86)

RFI (CD54)

Exp 1

Exp 2

Exp 1

Exp 2

11.1

139

133

69

80

13.3

150

124

75

88

16.0

141

139

71

75

19.2

150

113

80

70

23.1

143

142

93

87

27.7

159

149

89

71

33.3

157 #

156 #

125

91

40.0

159 #

143

159

100

# Positive result

Interpretation of results:
other: Positive in the human Cell Line Activation Test.
Conclusions:
Under the conditions of this study, the test material was considered to be positive in the human Cell Line Activation Test.
Executive summary:

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

The study was conducted to investigate the potential of the test material to activate monocytes and dendritic cells in the human monocytic leukaemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test material was dissolved in isopropanol and a dose finding assay was conducted to determine a concentration showing 75% THP-1 cell survival (CV75). No CV75 value was calculated as there was no effect on viability up to a concentration of 40 µg/mL.

Eight stock solutions were prepared by 1.2-fold serial dilutions using isopropanol to give eight doses ranging from 5.6 to 20 mg/mL. These stock solutions were then diluted 250-fold into the culture medium (working solutions). Aliquots of 500 µL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 10^6 cells per well. Each plate was sealed using a plate sealer and then incubated at 37 ± 1°C, 5 % (v/v) CO2 in air, in a humidified environment for 24 ± 0.5 hours.

After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation, washed with FACS buffer and then blocked with 600 µL of blocking solution (FACS buffer containing 0.01 % (w/v) globulin) at 4°C for 15 minutes.

After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate (or sample tubes), centrifuged and then stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4°C for 30 minutes.

The stained cells were washed with FACS buffer and re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

The relative fluorescence intensity (RFI) for CD54 was less than 200% at all tested concentrations. The RFI value for CD86 was ≥ 150% in both of the independent runs at one or more concentrations.

Under the conditions of this study, the test material was considered to be positive in the human Cell Line Activation Test.

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

OECD 442C: Direct Peptide Reactivity Assay

The skin sensitisation potential of the test material cause 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).

The study was conducted to quantify the reactivity of the test material towards model synthetic peptides containing either lysine or cysteine. The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test material was dissolved in isopropanol at a concentration of 100 mM. The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24 ± 2 hours in glass autosampler vials, protected from light and set at 25°C.

The remaining concentration of cysteine- or lysine-containing peptides following the 24 hour incubation period was measured by high performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

The cysteine depletion value was 0.00 %, the lysine depletion value was 0.45% and the mean of the cysteine and lysine depletion values was 0.23%.

Under the conditions of this study, the test material was considered to be negative in the Direct Peptide Reactivity Assay.

OECD 442D: ARE-Nrf2 Luciferase Test Method

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 study was conducted to investigate the potential of the test material to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test material was dissolved in isopropanol to the final concentration (100 mM). Serial dilutions were then made using isopropanol to obtain 12 master concentrations of the test material (0.049, 0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5 25, 50 and 100 mM). The master concentrations were then further diluted 25 fold into culture medium containing serum and finally used for treatment with a further 4 fold dilution factor so that the final concentrations ranged from 0.49 to 1000 μM.

Aliquots of 50 μL of each of the final concentrations were transferred to each of three luciferase plates and a single viability plate. Each plate was sealed using a plate sealer and then incubated at 37 ± 1°C, 5 % (v/v) CO2 in air, in a humidified environment for 48 ± 1 hours.

After the 48-hour exposure period, the medium in the luciferase plates was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).The plate was sealed and incubated for 4 hours at 37 ± 1°C, 5 % (v/v) CO2. The MTT medium was then removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37 ± 1°C, 5 % (v/v) CO2 in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

After the 48-hour exposure period, the cells in the viability plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25 ± 2°C, loaded into the luminescence plate reader and read.

The maximal fold increases (Imax) were 0.44, 1.90 and 0.24 for Experiments 1, 2 and 3, respectively. The EC1.5 value for Experiment 2 was 76.10 μg/mL. There were no EC1.5 values for Experiments 1 and 3 as there were no statistically significant increases in induction. Cell viability was 99% at the EC1.5 determining concentration in Experiment 2. This measurement was not applicable for Experiments 1 and 3 as there were no EC1.5 determining concentrations in these experiments. In Experiment 2 there was an overall dose response for lucipherase. In Experiments 1 and 3, there was no apparent overall dose response for lucipherase and the dose response curves were not biphasic.

Under the conditions of this study the test material was considered to be negative in the ARE-Nrf2 Luciferase Test.

OECD 442E: human Cell Line Activation Test

The skin sensitisation potential of the test material cause investigated in accordance with the standardised guideline OECD 442E, 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 study was conducted to investigate the potential of the test material to activate monocytes and dendritic cells in the human monocytic leukaemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test material was dissolved in isopropanol and a dose finding assay was conducted to determine a concentration showing 75% THP-1 cell survival (CV75). No CV75 value was calculated as there was no effect on viability up to a concentration of 40 µg/mL.

Eight stock solutions were prepared by 1.2-fold serial dilutions using isopropanol to give eight doses ranging from 5.6 to 20 mg/mL. These stock solutions were then diluted 250-fold into the culture medium (working solutions). Aliquots of 500 µL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 10^6 cells per well. Each plate was sealed using a plate sealer and then incubated at 37 ± 1°C, 5 % (v/v) CO2 in air, in a humidified environment for 24 ± 0.5 hours.

After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation, washed with FACS buffer and then blocked with 600 µL of blocking solution (FACS buffer containing 0.01 % (w/v) globulin) at 4°C for 15 minutes.

After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate (or sample tubes), centrifuged and then stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4°C for 30 minutes.

The stained cells were washed with FACS buffer and re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

The relative fluorescence intensity (RFI) for CD54 was less than 200% at all tested concentrations. The RFI value for CD86 was ≥ 150% in both of the independent runs at one or more concentrations.

Under the conditions of this study, the test material was considered to be positive in the human Cell Line Activation Test.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

From the three in vitro skin sensitisation studies that are available, two negative results were observed. Taking a weight of evidence approach for addressing this endpoint it is considered that there is not evidence of a clear positive response throughout the whole adverse outcome pathway and as such the substance is considered not to be a skin sensitiser.