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Administrative data

Description of key information

Skin Sensitisation: Keratinosens, Bailey (2019)

Under the conditions of this study, the test material is negative according to the KeratinoSens prediction model.

Skin Sensitisation: h-CLAT, Roth (2019)

Under the conditions of this study, the test material with a log Pow of 0.93 activated THP-1 cells and is therefore considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Skin Sensitisation: DPRA, Walker (2019)

Under the conditions of this study, the test material is predicted by DPRA as negative and to not be a potential skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 November 2018 to 16 November 2018
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The KeratinoSens™ test is a method for which validation studies have been completed followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSens™ test method was considered scientifically valid to be used as part of an IATA (Integrated Approach to Testing and Assessment), to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
Details on the study design:
CONTROLS
- Negative Control: DMSO
- Positive Control: Cinnamic Aldehyde

TEST SYSTEM
- The KeratinoSens™ cell line (test system) is an immortalised adherent cell line derived from HaCaT human keratinocytes, stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is 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 test substances.
- Method of administration of test material: Per plate, a single application of 12 concentrations of the test material was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%. The top concentration was previously determined by solubility testing.
- Method of administration of reference materials: Per plate, a single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1% and a single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate). One well per plate was left empty (no cells).
- Exposure times of test materials and reference materials: Cells were incubated with the test or reference material for 48 ± 2h prior to endpoint measurements.
- Number of repetitions: Three repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence (n=9 overall) and 2 x 96-well plates for MTT (n=6 overall). The validity of each repetition was assessed following acceptance criteria.

OVERVIEW
- Preliminary testing: Determination of the top concentration by solubility testing
- Day 1: Cell seeding (3 x 96-well plates for Luminescence; 2 x 96-well plate for MTT); 10,000 cells per well, passage number 17.
- Day 2: 24 hours after seeding, the test and control materials were applied and plates were incubated at 37 °C, 5 % CO2, ≥ 95 % relative humidity for 48 ± 2 hours.
- Day 4: Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MTT testing (2 plates)

DATA ANALYSIS
- XCellR8 Form F0056: “KeratinoSens data processing” v.2 was used to analyse data. This form is a Microsoft Excel workbook, validated in-house (24JUL18) containing formulae to process the raw data as described in SOP L0057.
- The following parameters were calculated using the KeratinoSens™ test method:
• 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 at which the induction of luciferase activity was above the 1.5-fold threshold (i.e. 50% enhanced luciferase activity).
• For each concentration showing > 1.5-fold luciferase activity induction, statistical significance is calculated (e.g. by 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). 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.
• The percentage of viability as compared to the Negative control.

ACCEPTANCE OF THE ASSAY
Test results are acceptable if:
• The positive control (cinnamic aldehyde) produces positive results, i.e. the luciferase gene induction produced by this control is above the threshold of 1.5 in at least one of the tested concentrations and this induction is statistically significant compared to the solvent (negative) control (p < 0.05).
• The lMAX and the EC1.5 for cinnamic aldehyde is calculated and meet either or both of the following targets: Average induction in the three replicates for cinnamic aldehyde at 32 µM is within the XCellR8 historical range (currently 1.6 and 3) or EC1.5 value for cinnamic aldehyde is within the XCellR8 historical range (currently 6 µM and 39 µM). At least one of these criteria must be met, otherwise the run is discarded unless there is sufficient reason not to do this as determined by the Study Director. If only one criterion is met, it is recommended to check the dose-response curve of cinnamic aldehyde in order to decide on acceptability.
• CV % of blank values < 20 %

INTERPRETATION OF RESULTS
A test material is considered positive using the KeratinoSens prediction model if the following conditions are met in 2 of 3 repetitions:
• 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).
• 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). Test materials that only induce the gene activity at cytotoxic levels are not rated positive, as in the case for some non-sensitising skin irritants.
• The EC1.5 value is < 1000 µM or < 200 µg/mL for test materials with no defined MW.
• There is an apparent overall dose-response for luciferase induction (or a biphasic response).
Positive control results:
The acceptance criteria stated that the Positive Control (PC) (Cinnamic aldehyde) must have induction >1.5-fold in at least one concentration, it showed this at 4/5 concentrations and therefore met the acceptance criteria.
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
Imax [442D]
Value:
1.226
At concentration:
500 mM
Cell viability:
103.327 %
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The maximal average fold induction of luciferase activity (IMAX) value observed at any concentration.
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
Imax [442D]
Value:
0.854
At concentration:
1 000 mM
Cell viability:
94.869 %
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The maximal average fold induction of luciferase activity (IMAX) value observed at any concentration.
Key result
Group:
test chemical
Run / experiment:
run/experiment 3
Parameter:
Imax [442D]
Value:
1.443
At concentration:
1 000 mM
Cell viability:
108.383 %
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The maximal average fold induction of luciferase activity (IMAX) value observed at any concentration.
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
EC 1.5 [442D]
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: N/A - threshold of induction not crossed at any test material concentration in any repetition.
Other effects / acceptance of results:
SOLUBILITY ASSESSMENT
- The test concentrations of the test material used in the KeratinoSens™ method were selected on the basis of a solubility test carried out during the study: Solubility of the test material was confirmed up to 200 mM in DMSO. Subsequent dilution in cell culture medium gave a top concentration of 2000 µM.

SKIN SENSITISATION
- In this study, the test material was classified as a Negative using the KeratinoSens prediction model.
- The sensitisation potential of the test material was quantified by calculating 2 parameters known as the EC1.5 and the IMAX value:
• The EC1.5 value is the Effective Concentration (EC) of test material that yielded an induction of luciferase activity greater than 1.5-fold over untreated controls. If at least one concentration induces statistically significant luciferase activity > 1.5, then the product is classified as positive provided the cell viability measured by MTT is greater than 70%. The test material did not induce statistically significant luciferase induction > 1.5 in any of the 3 repetitions. The statistical significance, viability, dose response and dose acceptance criteria were all met and therefore: The test material was classified as negative using the KeratinoSens prediction model.
• The IMAX value is the maximum induction observed within the concentration range tested. Although the KeratinoSens™ test is not validated to predict potency, the IMAX value can provide a useful tool for preliminary comparison of sensitisation potential between test materials. The maximum induction for rep 1 was 1.226 at 500.000 µM, 0.854 for rep 2 at 1000.000 µM and for rep 3 the maximum induction was 1.443 at 1000.000 µM. For reference, during test validation, sensitising proficiency chemicals produced IMAX values of up to 36-fold over untreated controls.
- All of the formal acceptance criteria of the tests were met.

Table 1: Results of Repetition 1

 

Test Material Concentration (µM)

0.977

1.953

3.906

7.813

15.625

31.250

62.500

125.000

250.000

500.000

1000.000

2000.000

Mean fold induction

0.716

1.043

1.043

1.094

1.128

1.061

1.187

1.165

1.158

1.226

1.059

0.818

Viability %

104.281

85.624

89.579

93.600

88.757

86.403

98.080

93.171

102.439

103.327

102.210

107.861

T-test

3.48E-03

6.54E-01

6.37E-01

3.24E-01

1.78E-01

5.18E-01

4.94E-02

7.79E-02

9.33E-02

1.67E-02

5.25E-01

5.80E-02

SD

0.122

0.209

0.054

0.166

0.155

0.145

0.124

0.073

0.111

0.043

0.024

0.160

IMAX

1.226 at 500.000 µM

EC1.5

N/A - threshold of induction not crossed at any test material concentration

IC30

N/A- Cell viability did not fall below 70%

IC50

N/A- Cell viability did not fall below 50%

 

Table 2: Results of Repetition 2

 

Test Material Concentration (µM)

0.977

1.953

3.906

7.813

15.625

31.250

62.500

125.000

250.000

500.000

1000.000

2000.000

Mean fold induction

0.519

0.689

0.667

0.699

0.673

0.731

0.804

0.752

0.770

0.831

0.854

0.526

Viability %

88.937

72.463

86.221

97.222

96.247

101.615

109.586

92.829

105.862

90.815

94.869

93.426

T-test

2.59E-06

1.73E-03

6.08E-04

2.09E-03

8.45E-04

5.86E-03

3.81E-02

9.41E-03

1.53E-02

7.48E-02

1.19E-01

3.51E-06

SD

0.060

0.170

0.046

0.128

0.107

0.148

0.101

0.076

0.062

0.118

0.089

0.065

IMAX

0.854 at 1000.000 µM

EC1.5

N/A - threshold of induction not crossed at any test material concentration

IC30

N/A- Cell viability did not fall below 70%

IC50

N/A- Cell viability did not fall below 50%

 

Table 3: Results of Repetition 3

 

Test Material Concentration (µM)

0.977

1.953

3.906

7.813

15.625

31.250

62.500

125.000

250.000

500.000

1000.000

2000.000

Mean fold induction

0.793

0.825

0.853

1.006

0.958

0.975

1.031

1.099

1.122

1.203

1.443

1.271

Viability %

141.968

97.441

105.926

96.755

102.259

102.534

98.897

94.869

107.917

109.373

108.383

136.691

T-test

2.87E-02

6.22E-02

1.16E-01

9.53E-01

6.52E-01

7.87E-01

7.39E-01

2.96E-01

1.93E-01

3.25E-02

1.38E-05

4.65E-03

SD

0.086

0.069

0.062

0.133

0.037

0.006

0.061

0.160

0.087

0.111

0.113

0.069

IMAX

1.443 at 1000.000 µM

EC1.5

N/A - threshold of induction not crossed at any test material concentration

IC30

N/A- Cell viability did not fall below 70%

IC50

N/A- Cell viability did not fall below 50%

 

Interpretation of results:
other: Negative according to the KeratinoSens prediction model.
Conclusions:
Under the conditions of this study, the test material is negative according to the KeratinoSens prediction model.
Executive summary:

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

The human skin sensitisation potential of the test material was assessed using the validated in vitro method, the KeratinoSens™ assay, adapted to animal product-free conditions by the Test Facility, and validated in-house to determine keratinocyte activation.

After 48h exposure of cells with 12 concentrations of the test material, Luciferase measurements and MTT viability testing were performed.

The EC1.5 value is the Effective Concentration (EC) of test material that yielded an induction of luciferase activity greater than 1.5-fold over untreated controls. If at least one concentration induces statistically significant luciferase activity >1.5, then the product is classified as positive provided the cell viability measured by MTT is greater than 70%. The test material did not induce statistically significant luciferase induction >1.5 in any of the 3 repetitions. The statistical significance, viability, dose response and dose acceptance criteria were all met and therefore: The test material was classified as negative using the KeratinoSens prediction model.

The IMAX value is the maximum induction observed within the concentration range tested. Although the KeratinoSens™ test is not validated to predict potency, the IMAX value can provide a useful tool for preliminary comparison of sensitisation potential between test materials. The maximum induction for rep 1 was 1.226 at 500.000 µM, 0.854 for rep 2 at 1000.000 µM and for rep 3 the maximum induction was 1.443 at 1000.000 µM. For reference, during test validation, sensitising proficiency chemicals produced IMAX values of up to 36-fold over untreated controls. All of the formal acceptance criteria of the tests were met.

Under the conditions of this study, the test material is negative according to the KeratinoSens prediction model.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 September 2018 to 25 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for the Testing of Chemicals: OECD 442E
Version / remarks:
In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation. Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), June 2018.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
other: human Cell Line Activation Test (h-CLAT)
Justification for non-LLNA method:
Recommended test system in the international OECD guidelines.
Details on the study design:
CONTROLS
- Medium Control and Solvent Control for the Test Material: culture medium
- Positive Control (h-CLAT): DNCB (2,4-dinitrochlorobenzene, CAS No.: 97-00-7) final concentration: 2 and 3 μg/mL, Purity ≥ 99 %)
- Solvent Control for the Positive Control (h-CLAT): DMSO (Dimethyl sulfoxide, CAS No. 67-68-5) in culture medium, final concentration 0.2 %, Purity ≥ 99 %

TEST MATERIAL PREPARATION
- On the day of the experiment (prior to start) the test material was prepared in culture medium which formed a stable suspension/dispersion at a concentration of 100 mg/mL and was dissolved at a concentration of 10 mg/mL in culture medium, as tested by a solubility test.
- For the cytotoxicity test (dose finding assay) eight concentrations of the test material were analysed. For this, dilutions were prepared by 1:2 serial dilutions from 5000 μg/mL in culture medium.

TEST SYSTEM
- Reasons for the Choice of THP-1 Cells: THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as a surrogate for human myeloid dendritic cells, because the THP-1 cells also show enhanced CD86 and/or CD54 expression when treated with sensitisers.
- THP-1 Cell Cultures: Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH (aliquots of cells in freezing medium at 1 × 10^6 to 2 × 10^6 cells/mL) allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice weekly. The cell density should not exceed 1 × 10^6 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to two months after thawing (passage number should not exceed 30).
- The passage numbers of the used THP-1 cells were 12 in both cytotoxicity assays and 17 and 19 in the h-CLAT runs 1 and 2, respectively.
- Culture Medium: RPMI 1640 Medium, GlutaMAXTM Supplement including 25 mM HEPES, supplemented with 10% FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1 % (v/v) sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) is used to culture the cells during the assay. Medium with supplements has to be stored at 2 - 8 °C and used within one month. The culture medium has to be warmed to room temperature just before use.
- Preparation and Seeding of THP-1 Cells: On the day of the cytotoxicity experiment directly before the application of the test material, solvent and medium control, a volume of 500 μL with a cell density of 1.8 - 2 × 10^6 THP-1 cells/mL was seeded in each well of a 24-well flat bottom plate. For the main experiment (h-CLAT) 0.9 - 1 × 10^6 cells/well in a volume of 500 μL were seeded in a 24-well plate before the treatment.

EXPERIMENTAL DESIGN AND PROCEDURES OF THE CYTOTOXICITY TEST
- Dose Finding Assay (Flow cytometer): The test material concentrations investigated in the main experiment (h-CLAT) were determined with two cytotoxicity tests. Both cytotoxicity tests were repeated once for technical reasons. The cytotoxicity repetitions are reported as first and second cytotoxicity test. The tests were performed with independent cell cultures (cells are collected from different culture flasks). The test material was prepared separately for each run.
- Treatment of the Cells: The test material dilutions were prepared freshly before each experiment. Each volume (500 μL) of the dilutions of the test material, culture medium and solvent control (e.g. 0.2 % (v/v) DMSO in culture medium) was added to the cells. Only culture medium was tested as solvent control. The treated THP-1 cells were incubated for 24 ± 0.5 hours. All dose groups were tested in one replicate for each cytotoxicity test. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations. Each concentration of the test material, culture medium and solvent control was prepared for the 7-AAD staining.
Staining of the Cells: Test material-treated and non-test-material treated cells were collected in sample tubes centrifuged (approx. 250 × g, 5 min), washed twice (2 - 8 °C) with approx. 600 μL FACS buffer (PBS with 0.1 % (w/v) BSA) and re-suspended in a final volume of 1 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added in each sample tube.
Flow Cytometry Acquisition (Cytotoxicity Test): Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions. The cytotoxicity was analysed by flow cytometry using the software Cellquest Pro 6.0. The 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.
- Preparation of the acquisition (Cytotoxicity Test): The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of the FL-3 channel
The voltage of FSC and SSC was set to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot should be checked to make sure that a single population appears without contamination or excessive debris. The FL-3 voltage was set and compensate to appropriate position (FACSCalibur, Becton Dickinson GmbH). The cell viability was measured by gating-out dead cells stained with 7-AAD. A total of 10,000 living cells were analysed. The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow in the flow line.
- Flow Cytometry Analysis (Cytotoxicity Test): The cell viability is shown by the cytometry analysis program (% total) or is calculated according to the following equation:
Cell Viability (%) = (Number of living cells / number of acquired cells) x 100
The CV75 value, a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), is 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 is the minimum value of cell viability over 75 %
c is the maximum value of cell viability below 75 %
b and d are the concentrations showing the value of cell viability a and c respectively

Acceptability of the Cytotoxicity Assay: The cytotoxicity test is considered to be acceptable if it meets the following criteria:
- The cell viability of the medium and solvent control (if the test material is dissolved in DMSO) should be more than 90 %.

Calculation of the Test Doses for the Main Experiment (h-CLAT): Since a mean CV75 value could not be calculated, the OECD 442E guideline recommended maximal to be tested test material concentration (5000 μg/mL) was used as highest concentration for the h-CLAT runs. Seven further concentrations of the test material were prepared by serial 1:1.2 dilution.

EXPERIMENTAL DESIGN AND PROCEDURES OF THE h-CLAT
- The test material was tested in two independent h-CLAT runs. The runs were performed on different days. The test material was prepared separately for each run.
- Treatment of the Cells: Each volume (500 μL) of the dilutions of the test material, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations. Each concentration of the test material, medium control, positive and DMSO control was prepared in triplicate for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).
- Staining of the Cells: The triplicates of each of the test material-treated and non-test-material-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1 % (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control). All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures. The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).
- Sample Preparation for Measurement: After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added.
- Flow Cytometry Acquisition: Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions. The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.
Preparation of the acquisition: The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson GmbH). The cell viability was detected by setting an R1-gate (dead cells are gated-out by staining with 7-AAD). Therefore, the R1 gate was set approximately at the middle position between the peak of the negative fraction and the positive fraction in the FL-3 histogram using DNCB-treated cells. The negative fraction corresponds to the living cells and was kept for the subsequent analyses. For each control and all test material concentrations, the cell viability was recorded from the isotype control cell tube (stained with FITC labelled-mouse IgG1), the CD54 and CD86 cell tube, where only the isotype control cells were used for the cell viability evaluation. The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.
- Acquisition: Dead cells were gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was calculated, but excluded from the evaluation, if the cell viability was less than 50 % (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).
- Data Analysis and Interpretation
Flow Cytometry Analysis: The RFI is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration of every chemical:
RFI (%) = [((MFI of test material treated cells) – (MFI of test material treated isotype control cells) / ((MFI of solvent control cells) – (MFI of solvent isotype control cells))] x 100
MFI = Geometric Mean Fluorescence Intensity (GeoMean)
The cell viability from the isotype control cells, CD54 and CD86 cells is calculated according to the following equation:
Cell Viability (%) = (Number of living cells/ Total number of acquired cells) x 100
Where only the isotype control cells (which are stained with mouse IgG1 (isotype) antibodies) are used for the cell viability evaluation.

- Acceptance Criteria:
The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control and DMSO control should be more than 90 %.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of 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 ≥ 150 % and CD54 ≥ 200 %) and the cell viability should be > 50 % in at least one concentration of the two tested positive control concentrations.
• For the test chemical, the cell viability should be more than 50 % in at least four tested concentrations in each run.
Negative results are acceptable only for test materials exhibiting a cell viability of < 90 % at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90 % the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90 % (OECD 442E guideline).

- Prediction model:
For CD86/CD54 expression measurement, each test material is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). 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 (OECD 442E guideline):
− 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 %).
Otherwise, the h-CLAT prediction is considered NEGATIVE.
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. An h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline).
Positive control results:
The RFI value of the positive control (DNCB) for CD86 exceeded the positive criterion (≥ 150 %) and the cell viability was > 50 %. The RFI value of the positive control (DNCB) for CD54 (2.0 μg/mL DNCB) in the second h-CLAT run did not exceed the positive criterion (≥ 200 %). This is considered to be acceptable since the CD54 RFI value of the positive control (3.0 μg/mL DNCB) in the second h-CLAT run exceeded the positive criterion.
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
CV75 [442D and 442E]
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
Due to the lack of cytotoxicity in the flow cytometric evaluation of the cytotoxicity test of both cytotoxicity tests, a mean CV75 value could not be calculated.
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
RFI CD86>200 [442E]
Value:
212.3 %
At concentration:
2 894 other: µg/mL
Cell viability:
91.07 %
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
RFI CD86>200 [442E]
Value:
169.3 %
At concentration:
2 009 other: µg/mL
Cell viability:
93.07 %
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
RESULTS OF THE DOSE FINDING ASSAY (CYTOTOXICITY TEST)
- First cytotoxicity test: Due to the lack of cytotoxicity in the flow cytometric evaluation of the cytotoxicity test, a CV75 value could not be calculated (threshold of cytotoxicity: < 75 %). However, cytotoxicity could be observed at the highest tested concentration by microscopic evaluation.
- Second cytotoxicity test: Due to the lack of cytotoxicity in the flow cytometric evaluation of the cytotoxicity test, a CV75 value could not be calculated (threshold of cytotoxicity: < 75 %). However, cytotoxicity could be observed at the highest tested concentration by microscopic evaluation.
- Due to the lack of cytotoxicity in the flow cytometric evaluation of the cytotoxicity test of both cytotoxicity tests, a mean CV75 value could not be calculated.

MAIN EXPERIMENT (h-CLAT)
- The following concentrations of the test material were tested in the main experiments (h-CLAT): 1395, 1674, 2009, 2411, 2894, 3472, 4167 and 5000 μg/mL
- The test material (log Pow of 0.93) was tested in 2 independent runs. The RFI of CD86 was greater than 150 % in at least one concentration of both independent runs and a dose response could be observed for the RFI values of CD86. The prediction is therefore considered positive for the test material in this h-CLAT.
- In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %). The RFI value of the positive control (DNCB) for CD86 exceeded the positive criterion (≥ 150 %) and the cell viability was > 50 %. The RFI value of the positive control (DNCB) for CD54 (2.0 μg/mL DNCB) in the second h-CLAT run did not exceed the positive criterion (≥ 200 %). This is considered to be acceptable since the CD54 RFI value of the positive control (3.0 μg/mL DNCB) in the second h-CLAT run exceeded the positive criterion.

Table 1: Results of the First Cytotoxicity Test for the Test Material

Test Group

Concentration [μg/mL]

Microscopic Evaluation / Cytotoxicity

Flow Cytometric Evaluation / Cell Viability [%]

Medium Control

-

no

96.49

Test Material

39.1

no

96.33

78.1

no

96.79

156

no

96.62

313

no

96.72

625

no

96.28

1250

no

95.96

2500

no

94.55

5000

yes

83.63

 

Table 2: Results of the Second Cytotoxicity Test for the Test Material

Test Group

Concentration [μg/mL]

Microscopic Evaluation / Cytotoxicity

Flow Cytometric Evaluation / Cell Viability [%]

Medium Control

-

no

96.22

Test Material

39.1

no

95.86

78.1

no

96.17

156

no

96.55

313

no

96.74

625

no

96.42

1250

no

96.38

2500

no

94.10

5000

yes

78.87

Table 3: Results of the First h-CLAT Run for the Test Material

 

Concentration (μg/mL)

RFI (%) CD54 Antibody

RFI (%) CD86 Antibody

Cell Viability ISO (%)

Medium Control

-

100.0

100.0

95.38

DMSO Control

-

100.0

100.0

94.99

Positive Control (DNCB)

2.0

292.2*

576.9*

81.82

3.0

517.2*

545.9*

79.80

Test Material

1395

117.2

139.1

96.84

1674

114.1

103.8

94.87

2009

142.2

112.8

94.06

2411

164.1

149.7

93.18

2894

185.9

212.3*

91.07

3472

178.1

252.3*

88.39

4167

181.3

212.5*

84.80

5000

160.9

293.1*

79.91

* RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %).

Table 4: Results of the Second h-CLAT Run for the Test Material

 

Concentration (μg/mL)

RFI (%) CD54 Antibody

RFI (%) CD86 Antibody

Cell Viability ISO (%)

Medium Control

-

100.0

100.0

95.73

DMSO Control

-

100.0

100.0

95.37

Positive Control (DNCB)

2.0

165.0#

240.0*

89.85

3.0

292.2*

530.2*

84.43

Test Material

1395

74.8

102.4

94.41

1674

99.0

115.5

94.10

2009

126.2

169.3*

93.07

2411

139.8

238.4*

92.59

2894

166.0

280.0*

91.06

3472

183.5

275.6*

87.61

4167

175.7

313.5*

83.27

5000

153.4

369.6*

78.12

* RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %).

# CD54 RFI value of the positive control (2.0 μg/mL DNCB) did not fulfil the positive criteria (CD54 ≥ 200 %).

Interpretation of results:
other: Positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).
Conclusions:
Under the conditions of this study, the test material with a log Pow of 0.93 activated THP-1 cells and is therefore considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).
Executive summary:

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

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitisation AOP) of the test material dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test material concentration for the main experiment (h-CLAT) of test material was previously determined by two cytotoxicity tests.

Cytotoxic effects were not observed following incubation with the test material up to the highest tested concentration (5000 μg/mL). Since a mean CV75 value could not be calculated, the OECD 442E guideline recommended maximal to be tested test material concentration (5000 μg/mL) was used as highest concentration for the h-CLAT runs.

The following concentrations of the test material were tested in the main experiments (h-CLAT): 1395, 1674, 2009, 2411, 2894, 3472, 4167 and 5000 μg/mL. The test material (log Pow of 0.93) was tested in 2 independent runs. The RFI of CD86 was greater than 150 % in at least one concentration of both independent runs and a dose response could be observed for the RFI values of CD86. The prediction is therefore considered positive for the test material in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %). The RFI value of the positive control (DNCB) for CD86 exceeded the positive criterion (≥ 150 %) and the cell viability was > 50 %. The RFI value of the positive control (DNCB) for CD54 (2.0 μg/mL DNCB) in the second h-CLAT run did not exceed the positive criterion (≥ 200 %). This is considered to be acceptable since the CD54 RFI value of the positive control (3.0 μg/mL DNCB) in the second h-CLAT run exceeded the positive criterion.

Under the conditions of this study, the test material with a log Pow of 0.93 activated THP-1 cells and is therefore considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 August 2018 to 24 August 2018
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))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
ANALYTICAL PROCEDURE
High performance liquid chromatograph (HPLC)

- Assessment of Test Material Solubility: The solubility of the test material was assessed in acetonitrile.
- Preparation of Peptide Stock Solutions: Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).
- Preparation of Peptide Calibration Standards: Calibration standards of each peptide were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile, and contained the respective peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.
- Preparation of Reference Controls and Precision Controls: Stability controls (Reference Control B) and precision controls (Reference control A) of both peptides were prepared at a concentration of 0.5 mM in acetonitrile/buffer.
- Preparation of Positive Control Solution and Test Material Stock Solution: The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of the test material was prepared in acetonitrile.
- Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls: Accurate volume aliquots of the test material and the positive control were diluted with the Cysteine peptide stock solution to prepare solutions containing 0.5 mM Cysteine and 5 mM of the test material and 5 mM of the positive control. For the co-elution control, acetonitrile was used in place of the Cysteine stock solution.
- Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls: Accurate volume aliquots of the test material and the positive control were diluted with the Lysine peptide stock solution to prepare solutions containing 0.5 mM Lysine and 25 mM of the test material and 25 mM of the positive control. For the co-elution control, acetonitrile was used in place of the Lysine stock solution.
- Incubation: The appearance of the test material and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25 °C for a minimum of 22 hours incubation prior to injection of the samples as part of analytical run. Before initiation of the run the appearance of the samples in the vials was assessed and documented again.
- Analysis: The concentration of both the Cysteine and Lysine peptides in the presence of the test material and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section.

- Instrumentation Parameters:
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30 °C
Sample temperature: 25 °C
Mobile Phase (MP) A: 0.1 % TFA in Water
Mobile Phase (MP) B: 0.085 % TFA in ACN
Gradient:
0 minutes: 90 % MP A, 10 % MP B
20 minutes: 75 % MP A, 25 % MP B
21 minutes: 10 % MP A, 90 % MP B
23 minutes: 10 % MP A, 90 % MP B
23.5 minutes: 90 % MP A, 10 % MP B
30 minutes: 90 % MP A, 10 % MP B
Flow rate: 0.35 mL/minute
Stroke volume: 25 µL
Detector wavelength: UV, 220 nm
Injection volume: 2 µL (slow draw rate)
Run time: 30 minutes
Approximate retention time (Cysteine): 11 minutes
Approximate retention time (Lysine): 7 minutes

CALCULATIONS
- The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:
% Peptide depletion = 100 - Peptide peak area in replicate depletion samples (x 100) / Mean Peptide peak area of reference control samples B
Positive control results:
Historical data for positive controls indicates that Cinnamic Aldehyde produced high reactivity with overall mean depletion values of 72.6% (n=18) for Cysteine and 58.8% (n=18) got Lysine, respectively, indicating a positive prediction of the substance as a potential skin sensitiser.
Key result
Run / experiment:
other: Cysteine
Parameter:
other: Mean peptide depletion by the test material (%)
Value:
0.315
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: SD: 0.12 %
Key result
Run / experiment:
other: Lysine
Parameter:
other: Mean peptide depletion by the test material (%)
Value:
0.418
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: SD: 0.35 %
Other effects / acceptance of results:
SOLUBILITY ASSESSMENT
- The solubility of the test material in acetonitrile at a nominal concentration of 100 mM was achieved.

REACTIVITY ASSESSMENT
- All analytical acceptance criteria for each peptide run were met.
- Reactivity of the test material is classed as “No or Minimal” hence the DPRA prediction is negative and the test material is therefore predicted to not be a sensitiser
- There were no co-elution peaks in the Cysteine and Lysine assays.

Table 2: Acceptability of the Assay 

 

Peptide

Standard Linearity

Positive Control Depletion (%)

Reference Controls

Test Material

Acceptance Criteria

Cysteine

r² > 0.99

60.8 – 100

(SD < 14.9 %)

0.45 – 0.55 mM (CV < 15 %)

SD < 14.9 %

Lysine

r² > 0.99

40.2 – 69.0

(SD < 11.6 %)

0.45 – 0.55 mM (CV < 15 %)

SD < 11.6 %

Achieved Results

Cysteine

r² > 0.999

72.5

(SD 0.07 %, CV 0.26 %, n=3)

A: 0.495 mM (CV 0.77 %, n=3)

B: 0.501 mM (CV 0.53 %, n=6)

SD 0.12 %

CV 0.12 % (n=3)

Lysine

r² > 0.999

56.9

(SD 0.28 %, CV 0.65 %, n=3)

A: 0.504 mM (CV 0.43 %, n=3)

B: 0.505 mM (CV 0.31 %, n=6)

SD 0.35 %

CV 0.35 % (n=3)

CV: Coefficient of Variation

SD: Standard deviation

Table 3: Depletion of Peptide in the Presence of the Test Material

 

Mean Peak Area of Reference Control (µV.sec)

Mean Peak Area of Peptide with Test Material (µV.sec)

Mean Peptide Depletion by the Test Material (%)

Cysteine

Control B: 752490 (n=6)

750120 (n=3)

0.315

Lysine

Control B: 804260 (n=6)

800900 (n=3)

0.418

Interpretation of results:
other: Negative in the DPRA assay
Conclusions:
Under the conditions of this study, the test material is predicted by DPRA as negative and to not be a potential skin sensitiser.
Executive summary:

The reactivity and skin sensitisation potential of the test material was investigated in chemico, in a study which was conducted accordance with the standardised guideline OECD 442C, under GLP conditions.

During the study, solutions of the test material were successfully analysed by the validated DPRA analytical method in both Cysteine and Lysine containing synthetic peptides. No or minimal mean depletion of both peptides (0.367 %) in the presence of the test material was observed.

Under the conditions of this study, the test material is predicted by DPRA as negative and to not be a potential skin sensitiser.

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

Skin Sensitisation: Keratinosens, Bailey (2019)

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 human skin sensitisation potential of the test material was assessed using the validated in vitro method, the KeratinoSens™ assay, adapted to animal product-free conditions by the Test Facility, and validated in-house to determine keratinocyte activation.

After 48h exposure of cells with 12 concentrations of the test material, Luciferase measurements and MTT viability testing were performed.

The EC1.5 value is the Effective Concentration (EC) of test material that yielded an induction of luciferase activity greater than 1.5-fold over untreated controls. If at least one concentration induces statistically significant luciferase activity >1.5, then the product is classified as positive provided the cell viability measured by MTT is greater than 70%. The test material did not induce statistically significant luciferase induction >1.5 in any of the 3 repetitions. The statistical significance, viability, dose response and dose acceptance criteria were all met and therefore: The test material was classified as negative using the KeratinoSens prediction model.

The IMAX value is the maximum induction observed within the concentration range tested. Although the KeratinoSens™ test is not validated to predict potency, the IMAX value can provide a useful tool for preliminary comparison of sensitisation potential between test materials. The maximum induction for rep 1 was 1.226 at 500.000 µM, 0.854 for rep 2 at 1000.000 µM and for rep 3 the maximum induction was 1.443 at 1000.000 µM. For reference, during test validation, sensitising proficiency chemicals produced IMAX values of up to 36-fold over untreated controls. All of the formal acceptance criteria of the tests were met.

Under the conditions of this study, the test material is negative according to the KeratinoSens prediction model.

Skin Sensitisation: h-CLAT, Roth (2019)

The skin sensitisation potential of the test material was 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).

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitisation AOP) of the test material dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test material concentration for the main experiment (h-CLAT) of test material was previously determined by two cytotoxicity tests.

Cytotoxic effects were not observed following incubation with the test material up to the highest tested concentration (5000 μg/mL). Since a mean CV75 value could not be calculated, the OECD 442E guideline recommended maximal to be tested test material concentration (5000 μg/mL) was used as highest concentration for the h-CLAT runs.

The following concentrations of the test material were tested in the main experiments (h-CLAT): 1395, 1674, 2009, 2411, 2894, 3472, 4167 and 5000 μg/mL. The test material (log Pow of 0.93) was tested in 2 independent runs. The RFI of CD86 was greater than 150% in at least one concentration of both independent runs and a dose response could be observed for the RFI values of CD86. The prediction is therefore considered positive for the test material in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI value of the positive control (DNCB) for CD86 exceeded the positive criterion (≥ 150%) and the cell viability was > 50%. The RFI value of the positive control (DNCB) for CD54 (2.0 μg/mL DNCB) in the second h-CLAT run did not exceed the positive criterion (≥ 200%). This is considered to be acceptable since the CD54 RFI value of the positive control (3.0 μg/mL DNCB) in the second h-CLAT run exceeded the positive criterion.

Under the conditions of this study, the test material with a log Pow of 0.93 activated THP-1 cells and is therefore considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Skin Sensitisation: DPRA, Walker (2019)

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

The DPRA is a chemistry-based assay exploiting the fact that most chemical allergens have electrophilic properties and are therefore able to react with the nucleophilic sidechains of amino acids to form covalent bonds. The underlying rationale of the assay is that if a chemical is capable of reacting with proteins then it has the potential to act as a sensitiser. The endpoint measured in the assay is the percentage depletion over time of two synthetic peptides (containing respectively a cysteine and a lysine amino acid) from the peptide mixtures following an approximate 24 hour (22-26 hours) incubation with the test material. The percentage of peptide depletion is calculated by High Performance Liquid Chromatography using ultra-violet detection. The DPRA test allows quantification of a chemical’s reactivity and is used to categorise a substance in one of four classes of reactivity to allow discrimination between skin sensitising and non-sensitising chemicals and thus assesses their sensitisation potential.

Solutions of the test material were successfully analysed by the validated DPRA analytical method in both Cysteine and Lysine containing synthetic peptides. No or minimal mean depletion of both peptides (0.367 %) in the presence of the test material was observed.

Under the conditions of this study, the test material is predicted by DPRA as negative and to not be a potential skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin sensitisation.