Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin sensitisation (in vitro):

1. Direct Peptide Reactivity Assay (DPRA): Positive (OECD 442C/GLP)

2. ARE-Nrf2 luciferase test assay ( KeratinoSens): Negative (OECD 442D/GLP)

3. U937 Cell Line Activation Test (U-SENS™) assay: Positive (OECD442E/GLP)

The results of the in chemico (DPRA) and in vitro test (U-SENS)were positive, however the results did not allow discrimination between Skin sensitiser category 1A and 1B. There were no potential source candidates with relaible data found for read-across so, as a last resort, in vivo testing (OECD 429) was performed.

 

Skin sensitisation (in vivo): Not sensitising (OECD 429/GLP)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Remarks:
Direct Peptide Reactivity Assay (DPRA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
27 May 2020 - 18 August 2020
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))
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: test material provided by sponsor: 91112Y
- Expiration date of the lot/batch: 12 March 2021
- Purity: 92.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25℃, ≤70% relative humidity)
Details on the study design:
Skin sensitisation (In chemico test system) - Details on study design:

This test is part of a tiered strategy for skin sensitization assessment. OECD 442D, OECD 442E and OECD 429 were also performed.

Background
The Direct Peptide Reactivity Assay (DPRA) is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25°C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. Cysteine and lysine peptide Percent Depletion Values are calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

Solvent Selection
Based on the proportions of the main components of the test item together with their individual molecular weights, an apparent molecular weight of 373 g/mol was provided by the Sponsor and was used for preparation of the test item stock solutions.

Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e., by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol (IPA), acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), methanol (MeOH) and ethanol (EtOH). The dissolution of the test item in the SPCC and SPCL assay buffers was also evaluated by diluting the test item stock solution in the buffer based incubation mixtures. For the SPCC assay, a 20-fold dilution was prepared by mixing one volume of the test item stock solution with fifteen volumes of phosphate buffer pH 7.5 and four volumes of ACN. For the SPCL assay, a 4-fold dilution was prepared by mixing one volume of the test item stock solution with three volumes of ammonium acetate buffer pH 10.2. The presence of cloudiness, precipitate and/or phase separation was evaluated by visual inspection to aid solvent selection for the main study.
Test item stock solutions were prepared freshly for each reactivity assay.

For both the cysteine and lysine reactivity assay 62.62 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1679 μL EtOH after vortex mixing and 10 minute of sonication to obtain a 100 mM solution. Visual inspection of the clear solution being formed was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively. Any residual volumes were discarded.

Controls
Reference controls, co-elution controls, positive control and negative controls were set up in parallel to the test item in order to confirm the validity of the test.

SPCC Reference Control Solutions
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL ACN. In addition, a RCcysCEtOH sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCEtOH sample was prepared by mixing 750 μL of the 0.667 mM SPCC stock solution with 200 μL ACN and 50 μL EtOH.

SPCL Reference Control Solutions
Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN. In addition, a RClysCEtOH sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCEtOH sample was prepared by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL EtOH.

Co-elution control
The co-elution control (CC) samples were:
Cysteine:750 μL Phosphate buffer pH 7.5,200 μL ACN, 50 μL test item/A test solution (100 mM)
Lysine:750 μL Ammonium acetate buffer pH 10.2, 250 μL test item/A test solution (100 mM)

Positive control
The positive controls were:
Cysteine:750 μL Stock solution of 0.667 mM SPCC, 200 μL ACN, 50 μL Cinnamic aldehyde solution, (100 mM in ACN)
Lysine:750 μL Stock solution of 0.667 mM SPCL, 250 μL Cinnamic aldehyde solution (100 mM in ACN)

Peptides
Synthetic Peptide Containing Cysteine (SPCC) Stock Solution
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.0 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
Synthetic Peptide Containing Lysine (SPCL) Stock Solution
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.0 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.

Dose Groups
1. Co-elution control (CC)
2. Cinnamic aldehyde (PC) (100mM)
3. Test item
Cysteine: 750 μL Stock solution of 0.667 mM SPCC, 200 μL ACN, 50 μL test item/A test solution (100 mM)
Lysine:750 μL Stock solution of 0.667 mM SPCL, 250 μL 211324/A test solution (100 mM)

Experimental Procedure

Preparation of the HPLC Standard Calibration Curve: SPCC and SPCL Calibration Curves were prepared with 7 solutions from 0-0.534mM.

After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.3 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence did not exceed 30 hours. Prior to HPLC analysis the samples were visually inspected for precipitation. The test item samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature and supernatant was transferred to a new vial.

HPLC Preparation and Analysis

SPCC and SPCL peak areas in the samples were measured by HPLC. Sample analysis was performed using the following systems:
System 1 (used for Cysteine Reactivity Assay):
• Alliance separations module 2695 (Waters, Milford, MA, USA)
• Dual λ absorbance detector 2487 (Waters)
System 2 (used for Lysine Reactivity Assay):
• Alliance separations module 2695 (Waters, Milford, MA, USA)
• Dual λ absorbance detector 2487 (Waters)
All samples were analyzed according to the HPLC method presented in Table 1 (Appendix 1).

Data evaluation
The concentration of SPCC or SPCL was spectrophotometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards. The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:

Percent Peptide Depletion = [1 − (Peptide Peak Area in Replicate Injection (at 220 nm)/Mean Peptide Peak Area in Reference Controls (at 220 nm))] × 100

In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90%
Positive control results:
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 72.3% ± 1.0%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 63.0% ± 0.8%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).
Run / experiment:
other: Test item
Parameter:
other: mean % cysteine depletion
Value:
20.07 %
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
72.3%
Run / experiment:
other: Test item
Parameter:
other: mean % lysine depletion
Value:
15.6 %
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
63%
Run / experiment:
other: Test item
Parameter:
other: Mean of % cysteine and lysine depletion
Value:
18.2 %
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not applicable
Remarks on result:
positive indication of skin sensitisation
Remarks:
low reactivity class
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate was observed. The test item samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature. Supernatants were transferred to new vials and analyzed.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS: Refer to above

The SPCC standard calibration curve is presented in Figure 1 (Appendix 2). The correlation coefficient (r2) of the SPCC standard calibration curve was 0.998. Since the r2 was >0.99, the SPCC standard calibration curve was accepted. The results of the Reference Control samples A, C and CEtOH are presented in Table 4 (Appendix 3). The means of Reference Control samples A, C and CEtOH were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (EtOH) used to dissolve the test item did not impact the Percent SPCC Depletion. The results of the cysteine reactivity assay for the test item are presented in Table 8 (Appendix 3). In the CC sample no peak was observed at the retention time of SPCC (see chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test item with SPCC. For the 211324/A-cys samples, the mean SPCC A220/A258 area ratio was 37.02. Since this was within the 33.67-41.15 range, this again indicated that there was no co-elution of the test item with SPCC. The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls CEtOH. The mean Percent SPCC Depletion for the test item was 20.7% ± 0.9%.

The SPCL standard calibration curve is presented in Figure 2 (Appendix 2). The correlation coefficient (r2) of the SPCL standard calibration curve was 1.000. Since the r2 was >0.99, the SPCL standard calibration curve was accepted. The results of the Reference Control samples A , C and CEtOH are presented in Table 10 (Appendix 3). The means of Reference Control samples A, C and CEtOH were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (EtOH) used to dissolve the test item did not impact the Percent SPCL Depletion.   The results of the lysine reactivity assay for the test item are presented in Table 14 (Appendix 3). In the CC sample no peak was observed at 220 nm at the retention time of SPCL (see chromatogram in Appendix 4). A small response was observed at 258 nm at the retention time of SPCL but this response was evaluated as having no impact on the study outcome. For the 211324/A-lys samples, the mean SPCL A220/A258 area ratio was 28.87. Since this was within the 27.45-33.55 range, this indicated that there was no co-elution of the test item with SPCL. The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls CEtOH. The mean Percent SPCL Depletion for the test item was 15.6% ± 0.8%.

An overview of the obtained assay validation parameters is presented in the table below.

Acceptability of the Direct Peptide Reactivity Assay (DPRA)

  Cysteine reactivity assay  Lysine reactivity assay
Acceptability criteria Results for SPCC Acceptability criteria Results for SPCL
Correlation coefficient (r2) standard calibration curve  >0.99 0.998 >0.99 1
Mean peptide concentration RC-A samples (mM) 0.50 ± 0.05 0.506 ± 0.004 0.50 ± 0.05 0.498 ± 0.006
Mean peptide concentration RC-C samples (mM) 0.50 ± 0.05 0.490 ± 0.004 0.50 ± 0.05 0.478 ± 0.004
Mean peptide concentration RC-CEtOHsamples (mM) 0.50 ± 0.05 0.482 ± 0.007 0.50 ± 0.05 0.479 ± 0.006
CV (%) for RC  B and C samples <15.0 1.3 <15.0 2.9
Mean peptide depletion cinnamic aldehyde (%) 60.8-100 72.3 40.2-69.0 63
SD of peptide depletion cinnamic aldehyde (%) <14.9 1 <11.6 0.8
SD of peptide depletion for the test item (%) <14.9 0.9 <11.6 0.8

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table belo.w

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item 

SPCC depletion  SPCL depletion Mean of SPCC and SPCL depletion DPRA prediction and reactivity classification
Mean ± SD Mean ± SD Cysteine 1:10 / Lysine 1:50 prediction model
Reaction mass of 2-(palmitoylamino)ethyl acrylate and 2-(stearoylamino) ethyl acrylate 20.70% ±0.9% 15.60% ±0.8% 18.20% Positive: Low reactivity
Interpretation of results:
other: WoE classification with OECD 442D, OECD 442E and OECD 429 studies.
Conclusions:
This test is part of a tiered strategy for skin sensitization assessment. OECD 442D, OECD 442E and OECD 429 were also performed. Under the experimental conditions of this study, the DPRA prediction is considered as positive and the test item was considered to have low reactivity for both peptides.
Executive summary:

In an in chemico skin sensitization: direct peptide reactivity assay (DPRA; 20243911), the test item (92.4%) in ethanol was evaluated by monitoring peptide depletion between the test item and synthetic cysteine and lysine peptides (24 ± 2 h at 25 ± 2.5 °C). Subsequently samples were analysed by HPLC. Reference controls (A, B, C (solvent control)), co-elution controls and a positive control (Cinnamic aldehyde in acetonitrile) were set up in parallel to the test item in order to confirm the validity of the test.

The validation parameters, i.e., calibration curve, mean concentration of Reference Control (RC) samples A, C and CEtOH, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA as stated in the OECD 442C guideline.

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate was observed. The test item samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature. Supernatants were transferred to new vials and analyzed. In the cysteine reactivity assay the test item showed 20.7% SPCC depletion while in the lysine reactivity assay the test item showed 15.6% SPCL depletion. The mean of the SPCC and SPCL depletion was 18.2% and as a result the test item was considered to be positive in the DPRA and classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. The test item was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, the percentages of SPCC and SPCL depletion might be underestimated.

This test is part of a tiered strategy for skin sensitization assessment. OECD 442D, OECD 442E and OECD 429 were also performed. The data generated with this test will be considered in the context of an integrated approache such as IATA, combining the result with other complementary information from the other 3 tests.

Endpoint:
skin sensitisation: in vitro
Remarks:
KeratinoSens Assay
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
27 May 2020 - 18 August 2020
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)
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: test material provided by sponsor: 91112Y
- Expiration date of the lot/batch: 12 March 2021
- Purity: 92.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25℃, ≤70% relative humidity)
Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design:
This test is part of a tiered strategy for skin sensitization assessment. OECD 442C, OECD 442E and OECD 429 were also performed.

Test System
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The cell line is routinely checked for infection with a mycoplasma detection test. The KeratinoSens™ cell line was generated by and obtained from Givaudan. Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium. Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).

Test item preparation:
No correction was made for the purity of the test item. A solubility test was performed. The test item could not be dissolved or homogenously suspended in Milli-W water at 200 mM and Ethanol at 800 mM. The test item was suspended in DMSO to a final concentration of 200 mM (non-homogenous suspension). The test item was homogenously suspended in DMSO at a final concentration of 100 mM. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. The 100-fold dilutions in DMEM glutamax of the 25 to 100 mM DMSO stock formed moderate precipitate and were therefore not suitable to test. The 100-fold dilution in DMEM glutamax of 12.5 mM showed slight precipitation. This concentration was selected as highest concentration for the main assay (limit of solubility). In the first main experiment, the test item was suspended in DMSO at 12.5 mM (slightly glassy suspension). The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. In the second main experiment, the test item was dissolved in DMSO at 12.5 mM (clear colourless solution). From each stock, 11 spike solutions in DMSO were prepared (2- fold dilution series). The stock and spike solutions were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49, 0.24, 0.12 and 0.06 μM (final concentration DMSO of 1%). 125 μM was selected as top dose level due to the limit of solubility. All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. No precipitation was observed at the start and end of the incubation period in the 96-well plates. Test item concentrations were used within 2.5 hours after preparation. Any residual volumes were discarded.

Preparation of the Positive Control
The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol (EDMG), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described in paragraph 4.4.1, so that the final concentration of the positive control ranges from 7.8 to 250 μM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate. The formulation of the positive control was used in studies performed concurrently.

Preparation of the Vehicle Control
The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.

Blank
On each plate three blank wells were tested (no cells and no treatment).

Experimental Design
Plating of Cells
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. One plate was used for the luciferase activity measurements, and one parallel replicate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+6 in experiment 1 and P+8 in experiment 2.

Treatment of Cells
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0C in the presence of 5% CO2.

Luciferase Activity Measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 μL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 5 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity Assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, and cells were incubated for 3 - 4 hours at 37°C ± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.
Positive control results:
The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration (Table 2).

The EC1.5 of the positive control was within two standard deviations of the historical mean (57 μM and 77 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (3.46-fold and 2.49-fold in experiment 1 and 2, respectively; Table 2).

Run / experiment:
other: Experiment 1
Parameter:
other: Imax
Value:
1.9
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: viability of cells at 63 µM was 10.3 %; EC1.5 = 8.4µM; IC50=1.6 µM
Run / experiment:
other: Experiment 2
Parameter:
other: Imax
Value:
1.32
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: viability of cells at 31 µM was 19.5 %; EC1.5 = N/A; IC50= 0.8µM
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (4.3% and 7.2% in experiment 1 (Table 4) and 2 (Table 10), respectively).

- Acceptance criteria met for positive control:
Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.46 and the EC1.5 57 μM.

Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.49 and the EC1.5 77 μM.

The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration (Table 2). The EC1.5 of the positive control was within two standard deviations of the historical mean (57 μM and 77 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (3.46-fold and 2.49-fold in experiment 1 and 2, respectively; Table 2).
Interpretation of results:
other: WoE classification with OECD 442D, OECD 442E and OECD 429 studies.
Conclusions:
This test is part of a tiered strategy for skin sensitization assessment. OECD 442C, OECD 442E and OECD 429 were also performed. Under the experimental conditions of this study, the test item is negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.
Executive summary:

In an in vitro skin sensitisation: ARE-Nrf2 luciferase test method assay (KeratinoSens; 20243913), the test item (92.4%) was evaluated for its potential to activate the Nrf2 transcription factor in KeratinoSens cells. The test item (in 1% DMSO in DMEM culture medium) was applied to cells at concentrations from 0.06 to 125 µM (first and second experiments; top dose was the limit of solubility) for 48 h at 37°C. Luciferase production was measured by flash luminescence. Cytotoxicity was measured using an MTT assay. Two independent experiments were performed and cells were assayed for viability and luciferase activity in each experiment. The positive control was ethylene dimethacrylate glycol and the negative control was untreated cells and 1% DMSO.

The controls confirmed the validity of the study. The test item showed toxicity (IC30 values of 0.9 μM and 0.6 μM and IC50 values of 1.6 μM and 0.8 μM in experiment 1 and 2, respectively). The maximum luciferase activity induction (Imax) was 1.90-fold and 1.32-fold in experiment 1 and 2 respectively. However, since positive results (>1.5-fold induction) were only observed in the first experiment at toxic test concentrations with a cell viability of <70% compared to the vehicle control, the results are considered not biologically relevant in accordance with the OECD test guideline 442D for interpretation of results. The test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5- fold induction) were observed.

This test is part of a tiered strategy for skin sensitization assessment. OECD 442C, OECD 442E and OECD 429 were also performed. The data generated with this test will be considered in the context of integrated approached such as IATA, combining the result with other complementary information from the other 3 tests.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 September 2020-15 December 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: test material provided by sponsor: 91112Y
- Expiration date of the lot/batch: 12 March 2021
- Purity: 92.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25℃, ≤70% relative humidity)
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The 10% (w/v) in DMF after overnight mixing resulted a yellow, opalescent solution thus the quality of this formulation was considered to be acceptable as the highest achievable concentration in a vehicle for the LLNA test.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was weighed, and formulations prepared the day before the treatment on a weight: volume basis (as % (w/v)) in the Pharmacy of Charles River Laboratories Hungary Kft. The formulations were stirred with magnetic stirrer overnight and they were considered to be stable for this period.
Species:
mouse
Strain:
CBA
Remarks:
CBA/CaOlaHsd mice
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo, RMS B.V. Postbus 553, 5800 AN Venray, Netherlands.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 12 weeks old
- Weight at study initiation: 17.8 – 21.8 grams (the weight variation in animals in the study did not exceed ± 20 % of the mean weight)
- Housing: Group caging (Type II. polypropylene/ polycarbonate) during the observation period and individual caging in the radioactive proliferation test phase in the main experiment.
- Diet: Animals received ssniff® SM Rat/Mouse – "Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice" produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany, batch numbers: 56065984, expiry date: 31 October 2020, respectively), and Gel diet Transport (Scientific Animal Food & Engineering, Route de Saint Bris, 89290 Augy, France, batch number: 60200300010101, expiry dates: 29 January 2021, respectively),) ad libitum.
- Water: Animals received tap water, ad libitum.
- Acclimation period: 35 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9 – 23.8°C
- Humidity (%): 30 – 70%
- Air changes (per hr): 15-20 air exchange/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Vehicle:
dimethylformamide
Concentration:
10%, 5% and 2% (w/v)
No. of animals per dose:
Preliminary Irritation/Toxicity Test: 2 animals/dose
Main experiment: 4 animals/treatment group
Details on study design:
PRE-SCREEN TESTS:
1. Vehicle Selection (Preliminary Compatibility Test)
The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD No. 429 vehicles were assessed: AOO (acetone:olive oil 4:1 (v:v) mixture), N,N-dimethylformamide (DMF), Methyl ethyl ketone (MEK), Propylene glycol (PG), Dimethyl sulfoxide (DMSO) and 1% aqueous Pluronic® PE9200 (1% Pluronic). In addition, DOO (dioxane:olive oil mixture (4:1 (v/v)) and cyclohexane were also assessed.

The initial Preliminary Compatibility Test was performed with AOO (acetone:olive oil 4:1 (v:v) mixture), N,N-dimethylformamide (DMF), Methyl ethyl ketone (MEK), Propylene glycol (PG) and 1% aqueous Pluronic® PE9200 (1% Pluronic). The 100% (w/v) concentration technically was not feasible. The 50% and 25% (w/v) concentrations were not suitable for the test using the standard formulation method.

Then further Preliminary Compatibility Tests were performed with AOO (acetone:olive oil 4:1 (v:v) mixture), N,N-dimethylformamide (DMF), Methyl ethyl ketone (MEK), Propylene glycol (PG), Dimethyl sulfoxide (DMSO) and 1% aqueous Pluronic® PE9200 (1% Pluronic) using overnight mixing with magnetic stirrer or for PG and 1% Pluronic shorter mixing time was also checked . Furthermore, DOO (dioxane:olive oil 4:1 (v:v) mixture) and cyclohexane vehicles were also assessed. The 50% and 25% w/v concentrations were not suitable for the test in any of these solvents. The 10% (w/v) in DMF after overnight mixing resulted a yellow, opalescent solution thus the quality of this formulation was considered to be acceptable as the highest achievable concentration in a vehicle for the LLNA test.

Note: At 50% (w/v) using the standard formulation method, the test item was adhered in the containers in the tested vehicles, with the exception of 1% Pluronic where partial dissolution was observed using the standard formulation method then using overnight stirring.
At 25% (w/v) after overnight stirring partial dissolution and viscous formulation was observed and pieces of the undissolved test item were observed in the formulations using AOO, DMF, MEK or PG. Formulation in 1% Pluronic was also viscous and undissolved test item was observed.

2. Dose Selection (Preliminary Irritation/Toxicity Test)
The Preliminary Irritation/Toxicity Test was on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 10% and 5% (w/v) in DMF. The preliminary experiment was conducted in a similar experimental manner to the main test with the exception that it was terminated on Day 6 and the radioactive proliferation assay was not performed. In the Preliminary Irritation/Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test (Day 1) and prior to termination (Day 6). Both ears of each mouse were observed for erythema and scored using Table 1 [1,2,3]. Ear thickness measurements was taken using a thickness gauge on Day 1 (pre-dose), Day 3 (before treatment, approximately 48 hours after the first dose) and Day 6.

During the Preliminary Irritation / Toxicity Test no mortality or clinical signs were observed. Minimal amount of test item residue was observed on the ears of all animals of the 10% (w/v) and 5% (w/v) dose groups from Day 1 to Day 4. Clinical observations are summarized in Table 9 of Appendix 3. No marked body weight loss (>5% decrease in body weight) was observed in average body weight in any dose group in the preliminary test (Table 7 of Appendix 3).

Ear thickness of the animals was measured using a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after euthanasia of the experimental animals on Day 6. The ear thickness values and ear punch weights (2 per animal) are summarized in Table 8 of Appendix 3. No increased ear thickness value (≥25%) was detected. Ear punch weights of the animals were within the historical control range.
The draining auricular lymph nodes of the animals were visually examined: they were normal in both dose groups (subjective judgement by analogy with observations of former experiments).

The 10% (w/v) dose group was considered acceptable and was therefore selected as highest dose for the main test. No ear thickness measurements or ear punch weight determination was performed in the main test.


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index. The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.


TREATMENT PREPARATION AND ADMINISTRATION:
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR using a gauge 25Gx1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (±30 minutes) and then euthanized by asphyxiation. Once removed, the draining auricular lymph nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing. Determination of incorporated 3HTdR was via β-scintillation counting with results expressed as the number of radioactive disintegrations per minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No statistical evaluation was performed in this study.
Positive control results:
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 4.2)* was noted for HCA in the main experiment.

*The SI value of the positive control data was slightly below the minimal value of the HC data for DMF, however the DPN value was within the range and the SI value was well above the threshold limit of 3. Therefore, the positive control substance produced a significant lymphoproliferative response and confirmed the appropriate performance of the assay. Furthermore, the DPN values observed for the positive control substance in this experiment were in line with the historical control range.
Parameter:
SI
Value:
1.3
Test group / Remarks:
2%
Parameter:
SI
Value:
1.3
Test group / Remarks:
5%
Parameter:
SI
Value:
2
Test group / Remarks:
10%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION :Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. The test item was solid, which was formulated in DMF. The resulting stimulation indices observed under these test conditions were considered to be good evidence that the test item is a non-sensitizer. The size of lymph nodes was in good correlation with this conclusion.

EC3 CALCULATION : not applicable

CLINICAL OBSERVATIONS: There was no mortality or clinical signs observed during the main assay. Test item residue or a minimal amount of test item residue was observed on the ears of all animals in the 10% (w/v) group from Day 1 to Day 5 and minimal amount of test item residue of the was observed in the ears of all animals in the 5% (w/v) group from Day 1 to Day 3.


BODY WEIGHTS:Marked body weight loss (>5% decrease in the body weight) was observed in only one animal in the 10% (w/v) group. However, the mean body weight was within the acceptable range. Therefore, this value was considered as individual variability


SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness).:there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item not to cause lymphoproliferation in the Local Lymph Node Assay.
Interpretation of results:
GHS criteria not met
Remarks:
Not classified as sensitizer
Conclusions:
In an in vivo skin sensitisation LLNA assay, the test item was shown to have no sensitisation potential (non-sensitizer).
Executive summary:

In a dermal sensitization study (20/071-037E) with the test item (92.4%), young adult CBA/Ca/Ola/Hsd mice (4 females/group) were tested in the Local Lymph Node Assay. The doses tested were 0, 2, 5 and 10% (w/v) in DMF, based on the results of the vehicle compatibility and preliminary irritation/toxicity tests. The reliability of the test system was confirmed by a positive control test with 25% (w/v) α-Hexylcinnamaldehyde (HCA), formulated in DMF that was performed concurrently, using 4 animals.

No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response (stimulation index value of 4.2) was noted for HCA in the main experiment.

There was no mortality or clinical signs observed during the main assay. Test item residue or a minimal amount of test item residue was observed on the ears of all animals in the 10% (w/v) group from Day 1 to Day 5 and minimal amount of test item residue of the was observed in the ears of all animals in the 5% (w/v) group from Day 1 to Day 3. Marked body weight loss (>5% decrease in the body weight) was observed in only one animal in the 10% (w/v) group. However, the mean body weight was within the acceptable range. Therefore, this value was considered as individual variability.

The SI values were 2.0, 1.3, 1.3 at concentrations of 10%, 5% and 2% (w/v), respectively. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item not to cause lymphoproliferation in the Local Lymph Node Assay. The resulting stimulation indices observed under these test conditions were considered to be good evidence that the test item is a  non-sensitizer. The size of lymph nodes was in good correlation with this conclusion.

Endpoint:
skin sensitisation: in vitro
Remarks:
U937 Cell Line Activation Test (U-SENS) Assay
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
18 Aug 2020 - 08 Dec 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation) (U937 Cell Line Activation Test (U-SENS) Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: test material provided by sponsor: 91112Y
- Expiration date of the lot/batch: 12 March 2021
- Purity.:92.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25℃, ≤70% relative humidity)
Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design:

This test is part of a tiered strategy for skin sensitization assessment. OECD 442C, OECD 442D and OECD 429 were also performed.

Vehicle
The vehicle of the test item was 0.4 % dimethyl sulfoxide (DMSO) in complete medium (RPMI-1640).

Negative Control
Lactic Acid (CAS No. 50-21-5, ≥85% purity, LA; Sigma) is used as negative control. On the treatment day, a solution at 10 mg/mL was prepared in RPMI medium. This solution was diluted 1:25 in order to obtain a 0.4 mg/mL stock solution (final dose level 200 µg/mL).

Positive Control
2,4,6-Trinitrobenzenesulfonic acid (CAS No. 2508-19-2, ≥99%, TNBS; Sigma,) was provided as 1 M solution. On the treatment day a 10 mg/mL solution was prepared in RPMI. This solution was diluted 1:100 in order to obtain a 0.1 mg/mL stock solution (final dose level 50 µg/mL).

Preparation of Test Item Stock, Spiking and Working Solutions
No correction was made for the composition/purity of the test item.

A solubility test was performed. The test item was either dissolved or suspended in complete medium to a final concentration of 0.4 and 50 mg/mL and in DMSO to a final concentration of 50 mg/mL. The test item formed a non-homogenous suspension in complete medium at 0.2, 0.4, 25 and 50 mg/mL. In DMSO the test item formed a inhomogeneous suspension at 50 mg/mL and formed a homogenous suspension at 25 mg/mL. DMSO was selected as solvent for the main assay.
In the main experiments the test item was suspended in DMSO) at 25 mg/mL. The stock was diluted to a final test concentrations of 200, 100, 50, 20, 10 and 1 µg/mL in the 96-well plate (final concentration DMSO of 0.4%) in the first experiment and to a final test concentrations of 200, 100, 80, 50, 40, 20 and 10 µg/mL in the 96-well plate (final concentration DMSO of 0.4%) in the second experiment.

The test item precipitated at dose levels of 50 µg/mL and upwards.
Test item concentrations were used within 2 hours after preparation.
Any residual volumes were discarded.

Test System
U937 human monocytes (Inducible CD86-expressing cells; ATCC no.: CRL-1593.2TM).
Stock cultures of these cells are stored in the freezer. Cells were used after an acclimatisation period of approximately 8 days after thawing and were not sub-cultured more than 21 times. At least once a year the cell line is checked for infection with a mycoplasma detection test. Each batch of cells received from a supplier are submitted to a qualification process to guarantee their suitability (spontaneous CD86 level) for the test by comparison with the historical data or data from the literature. Stock and treatment cultures were performed in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (FCS), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively). All incubations were carried out in a humid atmosphere of 80 - 100% (actual range 62 – 94%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.8 – 36.8°C).

Experimental Design
Plating of Cells
Cultures were initiated in 96-well plates using 100 µL/well of a cell suspension adjusted at 5.0 x 105 viable cells/mL. Cell viability was > 90%. All assays were performed using two replicate culture-wells for the test item. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding. Three replicates of untreated control (RPMI), vehicle control (in case of DMSO as vehicle), negative (LA) and positive (TNBS) controls were tested.

Number of experiments
Two valid experiments were conducted per test item to demonstrate reproducibility of the results and conclusion. Initially, experiment 1 did not pass all the acceptability criteria and therefore this part of the study was not reported and repeated.

Treatment of Cells
Cells are treated for 45 ± 3 hours with the selected doses or controls (100 µL). The test item was in the first experiment evaluated up to 200 µg/mL using six doses: 1.0, 10, 20, 50, 100 and 200 µg/mL.
In the second experiment cells were treated with seven selected doses of test item. At least 2 concentrations were common with the previous experiment. The concentrations selected in the second experiment were 10, 20, 40, 50, 80, 100 and 200 µg/mL.

In all experiments, an untreated control (RPMI), vehicle control (in case of DMSO as vehicle) and the positive (TNBS) and negative control (LA) items were included. The final volume in the wells was 200 µL.
Precipitate evaluation

After 45 ± 3 hours of exposure, wells were checked for precipitate.

Cell antibodies staining for IgG1 and CD86
Cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200 g). The supernatant was discarded and cells were rinsed once with 100 µL/well Phosphate Buffered Saline (PBS) containing 5% FCS. After a second centrifugation step (5 min, 200 g) 100 µL/well of staining buffer (PBS containing 5% FCS) was applied to the cells.
FITC-conjugated antibodies were used for both IgG1 and CD86 staining:
-Mouse IgG1 of unknown specificity, for isotypic control (#555748; BD)
-Human CD86 specific mouse IgG1 (#555657; BD)
The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 µL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated (4-8℃) in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and re-suspended in 90 µL of PBS.

Flow cytometry method
Acquisition
Just before acquisition, 5 µL of a 0.5 µg/mL propidium iodide (PI) solution was added to each well. The size (FSC) was set linear and the granularity (SSC) parameter was set to logarithmic scale and a R1 region was defined in which approximately 10,000 events were acquired for each culture. The acquisition parameters remained unchanged for the acquisition of all the wells. For the acquisition the BD FACSCanto™ flow cytometer was used and for further analysis BD FACSDiva™ software was used.

Analysis
All analysis parameters were set on the RPMI wells for IgG1 and remained unchanged, for the analysis of all the other wells. The P1 region was adjusted if necessary in a SSC (X-axis) and FSC (Y-axis) plot. The P2 region was defined for the PI negative cells among P1 in a histogram with counts (Y-axis) and PI fluorescence (X-axis). The amount of cytotoxicity was analyzed as percentage of cells in P2. The P2 region was then plotted in a Dot-plot as fluorescence (X-axis) and SSC (Y-axis) and a quadrant was placed according acceptability criterion b. The percentage of cells in the UR quadrant was used to calculate the stimulation index.

Color Interferences
On IgG1 analysis
There is colour interference in the IgG1 evaluation when the X Median of the FITC-fluorescence in the UL Quad is 50% higher than the X Median fluorescence of the vehicle control IgG1 well (IgG1 X Median S.I. ≥ 150%).

Analysis
CV70 and stimulation index (S.I.) calculations according to the guideline

Acceptability criteria and data interpretation
According to the guideline






Positive control results:
Experiment 1: The positive control (TNBS) showed a S.I. ≥ 838% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Experiment 2: The positive control (TNBS) showed a S.I. ≥ 720% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Run / experiment:
other: Experiment 1
Parameter:
other: CV70
Remarks:
µg/mL
Value:
98 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Experiment 1
Parameter:
other: EC150
Remarks:
µg/mL
Value:
37 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Run / experiment:
other: Experiment 2
Parameter:
other: CV70
Remarks:
µg/mL
Value:
44 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Experiment 2
Parameter:
other: EC150
Remarks:
µg/mL
Value:
13 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The test item precipitated at dose levels of 50 µg/mL and upwards in both experiments.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control (LA) showed a S.I. ≤ 84% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). (experiment 1) The negative control (LA) showed a S.I. ≤ 60% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). (experiment 2).

The mean viability of the triplicate DMSO vehicle control cells was above the threshold of 90% (100% in experiment 1 and 2). The DMSO vehicle control mean value of its triplicate CD86-IgG1 S.I. was smaller than 250% of the mean of the triplicate CD86-IgG1 S.I. of untreated U937 cells in both experiments.

- Acceptance criteria met for positive control: The positive control (TNBS) showed a S.I. ≥ 838% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%) (experiment 1). The positive control (TNBS) showed a S.I. ≥ 720% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%) (experiment 2).

In both experiments the positive and negative control were considered valid and the positive control fell within the historical control data (Appendix 3).

The test item showed colour interference at 100 µg/mL and upwards (experiment 1) and 20 and 80 µg/mL (experiment 2). However the EC150 in both experiments (37 and 13 µg/mL in experiment 1 and 2, respectively) was lower than the interference level, so this had no impact on the study.

Table 1          
Overview Stimulation index of CD86 and Cell Viability in Experiment 1 and 2 of Test Item

Test items

Dose
(µg/mL)

% Viability (Mean)*

CD86-IgG1 S.I.*(%)

Colour Interference S.I.* (%)

Experiment

Experiment

Experiment

1

2

1

2

1

2

Test item

 

 

 

 

 

 

 

1

99

-

92

-

96

-

 

10

99

100

88

85

100

117

 

20

99

99

81

300

115

150

 

40

-

77

-

945

-

99

 

50

98

59

201

2330

135

103

 

80

-

32

-

2645

-

167

 

100

69

37

600

2690

150

137

 

200

60

35

1062

2300

157

114

*Red values are either below 70% viability or above 150 S.I.

Table 2          
Overview Stimulation index of CD86 and Cell Viability in Experiment 1 and 2 of the Positive, Negative and Vehicle Control

Controls

% Viability (Mean)*

CD86-IgG1 S.I.*

 

 

Experiment

Experiment

 

1

2

1

2

 

LA1

100

100

59

60

 

LA2

99

100

84

60

 

LA3

99

100

72

50

 

TNBS1

99

100

934

720

 

TNBS2

99

100

1094

535

 

TNBS3

99

100

838

500

 

DMSO (mean)

100

100

135

100

 

 

IgG1 value (%)

CD86 basal expression (%)

CD86-IgG1 expression (%)

Experiment

Experiment

Experiment

1

2

1

2

1

2

RPMI1

1.2

 0.71

4.8

 1.51

3.6

 0.81

RPMI2

0.7

1.0

3.4

2.7

2.7

1.7

RPMI3

0.9

0.7

4.2

3.0

3.3

2.3

DMSO1

 

 

 

 

4.6

1.8

DMSO2

 

 

 

 

4.7

2.2

DMSO3

 

 

 

 

3.7

2.0

RPMI Mean Viability

 

99

100

 

RPMI Drift

 

5%

35%

 

LA Drift

 

0%

-17%

 

 

  *Red values are either below 70% viability or above 150 S.I.

Table 3          
Overview EC150 and CV70 Values

 

EC150(µg/mL)

CV70(µg/mL)

Test item Experiment 1

37

98

Test item Experiment 2

13

44

       

Table 6          
Historical Control Data for the USENS Assay

Positive control

Negative control

Viability (%)

S.I. (%)

Viability (%)

S.I. (%)

Mean

98

627

99

92

SD

2.6

349

1.2

26

Lower control limit (95% control limits)

93

-58

97

40

Higher control limit (95% control limits)

103

1311

101

143

n

732

732

733

731

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of Nov 2017 to Jun 2020.

                     




Interpretation of results:
other: WoE classification with OECD 442C, OECD 442D and OECD 429 studies.
Conclusions:
This test is part of a tiered strategy for skin sensitization assessment. OECD 442C, OECD 442D and OECD 429 were also performed. In a GLP-compliant guideline study, the test substance was found to be positive in the in vitro U-SENS test, indicating a possible skin sensitizing potential of the substance.
Executive summary:

In an in vitro skin sensitisation: U937 Cell Line Activation Test (U-SENS; 20243914), the test item (92.4%) was evaluated by the ability to induce an increase in cell surface marker expression (CD86) in U937 cells. The test item (in 0.4% DMSO in RPMI culture medium) was applied to U937 cells at concentrations of 1 -200 μg/mL (first experiment) and 10 -200 µg/mL (second experiment) for 45 ± 3 hrs. Flow cytometry analysis was carried out on IgG1-FITC and CD86-FITC antibody labelled cells. The stimulation index (S.I.) for CD86 expression for each tested concentration was then calculated. CV70 was used for viability discrimination. The positive control was 2,4,6-Trinitrobenzenesulfonic acid (TNBS) and the negative control was lactic acid.  

All acceptance criteria for controls and the test item were reached in each run; therefore, the study was considered to be valid. The test item precipitated at dose levels of 50 µg/mL and upwards in both experiments. The test item showed toxicity (CV70 of 98 µg/mL and 44 µg/mL in experiments 1 and 2, respectively). A biologically relevant induction of CD86 activity (EC150 of 37 µg/mL and 13 µg/mL in experiments 1 and 2, respectively) was measured. The test item showed colour interference at 100 µg/mL and upwards (experiment 1) and 20 and 80 µg/mL (experiment 2). However, the EC150 in both experiments was lower than the interference level, so this had no impact on the study. The test item is classified as Positive in the U-Sens assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.

This test is part of a tiered strategy for skin sensitization assessment. OECD 442C, OECD 442D and OECD 429 were also performed. The data generated with this test will be considered in the context of integrated approached such as IATA, combining the result with other complementary information from the other 3 tests.

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

A tiered strategy for skin sensitization assessment was used. OECD 442C, OECD 442D, OECD 442E and OECD 429 were performed. The data generated with these tests was considered in the context of an integrated approach, and the final result was decided by combining the results from all tests.  

Skin sensitisation (in vitro):

One in chemico and two in vitro skin sensitisation assays are available: Direct Peptide Reactivity Assay (DPRA), ARE-Nrf2 luciferase test assay (KeratinoSens) and U937 Cell Line Activation Test (U-SENS) Assay.

Direct Peptide Reactivity Assay (DPRA)

In an in chemico skin sensitization: direct peptide reactivity assay (DPRA; OECD 442C/GLP), the test item (92.4%) in ethanol was evaluated by monitoring peptide depletion between the test item and synthetic cysteine and lysine peptides (24 ± 2 h at 25 ± 2.5 °C). Subsequently samples were analysed by HPLC. Reference controls (A, B, C (solvent control)), co-elution controls and a positive control (Cinnamic aldehyde in acetonitrile) were set up in parallel to the test item in order to confirm the validity of the test. The validation parameters, i.e., calibration curve, mean concentration of Reference Control (RC) samples A, C and CEtOH, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA as stated in the OECD 442C guideline. Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate was observed. The test item samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature. Supernatants were transferred to new vials and analyzed. In the cysteine reactivity assay the test item showed 20.7% SPCC depletion while in the lysine reactivity assay the test item showed 15.6% SPCL depletion. The mean of the SPCC and SPCL depletion was 18.2% and as a result the test item was considered to be positive in the DPRA and classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. The test item was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, the percentages of SPCC and SPCL depletion might be underestimated..

ARE-Nrf2 luciferase test assay (KeratinoSens)

In an in vitro skin sensitisation: ARE-Nrf2 luciferase test method assay (KeratinoSens; 20243913), the test item (92.4%) was evaluated for its potential to activate the Nrf2 transcription factor in KeratinoSens cells. The test item (in 1% DMSO in DMEM culture medium) was applied to cells at concentrations from 0.06 to 125 µM (first and second experiments; top dose was the limit of solubility) for 48 h at 37°C. Luciferase production was measured by flash luminescence. Cytotoxicity was measured using an MTT assay. Two independent experiments were performed and cells were assayed for viability and luciferase activity in each experiment. The positive control was ethylene dimethacrylate glycol and the negative control was untreated cells and 1% DMSO. The controls confirmed the validity of the study. The test item showed toxicity (IC30 values of 0.9 μM and 0.6 μM and IC50 values of 1.6 μM and 0.8 μM in experiment 1 and 2, respectively). The maximum luciferase activity induction (Imax) was 1.90-fold and 1.32-fold in experiment 1 and 2 respectively. However, since positive results (>1.5-fold induction) were only observed in the first experiment at toxic test concentrations with a cell viability of <70% compared to the vehicle control, the results are considered not biologically relevant in accordance with the OECD test guideline 442D for interpretation of results. The test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5- fold induction) were observed.

U937 Cell Line Activation Test (U-SENS) Assay

In an in vitro skin sensitisation: U937 Cell Line Activation Test (U-SENS; OECD 442E/GLP), the test item (92.4%) was evaluated by the ability to induce an increase in cell surface marker expression (CD86) in U937 cells. The test item (in 0.4% DMSO in RPMI culture medium) was applied to U937 cells at concentrations of 1 -200 μg/mL (first experiment) and 10 -200 µg/mL (second experiment) for 45 ± 3 hrs. Flow cytometry analysis was carried out on IgG1-FITC and CD86-FITC antibody labelled cells. The stimulation index (S.I.) for CD86 expression for each tested concentration was then calculated. CV70 was used for viability discrimination. The positive control was 2,4,6-Trinitrobenzenesulfonic acid (TNBS) and the negative control was lactic acid.  

All acceptance criteria for controls and the test item were reached in each run; therefore, the study was considered to be valid. The test item precipitated at dose levels of 50 µg/mL and upwards in both experiments. The test item showed toxicity (CV70 of 98 µg/mL and 44 µg/mL in experiments 1 and 2, respectively). A biologically relevant induction of CD86 activity (EC150 of 37 µg/mL and 13 µg/mL in experiments 1 and 2, respectively) was measured. The test item showed colour interference at 100 µg/mL and upwards (experiment 1) and 20 and 80 µg/mL (experiment 2). However, the EC150 in both experiments was lower than the interference level, so this had no impact on the study. The test item is classified as Positive in the U-Sens assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.

This test is part of a tiered strategy for skin sensitization assessment. OECD 442C, OECD 442D and OECD 429 were also performed. The data generated with this test will be considered in the context of integrated approached such as IATA, combining the result with other complementary information from the other 3 tests.

The results of the in chemico (DPRA) and in vitro test (U-SENS) were positive, however the results did not allow discrimination between Skin sensitiser category 1A and 1B. There were no potential source candidates with relaible data found for read-across so, as a last resort, in vivo testing (OECD 429) was performed.

Skin sensitisation (in vivo):

There is one in vivo LLNA in the mouse available.

In a dermal sensitization study (OECD 429/GLP) with the test item (92.4%), young adult CBA/Ca/Ola/Hsd mice (4 females/group) were tested in the Local Lymph Node Assay. The doses tested were 0, 2, 5 and 10% (w/v) in DMF, based on the results of the vehicle compatibility and preliminary irritation/toxicity tests. The reliability of the test system was confirmed by a positive control test with 25% (w/v) α-Hexylcinnamaldehyde (HCA), formulated in DMF that was performed concurrently, using 4 animals. No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response (stimulation index value of 4.2) was noted for HCA in the main experiment. There was no mortality or clinical signs observed during the main assay. Test item residue or a minimal amount of test item residue was observed on the ears of all animals in the 10% (w/v) group from Day 1 to Day 5 and minimal amount of test item residue of the was observed in the ears of all animals in the 5% (w/v) group from Day 1 to Day 3. Marked body weight loss (>5% decrease in the body weight) was observed in only one animal in the 10% (w/v) group. However, the mean body weight was within the acceptable range. Therefore, this value was considered as individual variability. The SI values were 2.0, 1.3, 1.3 at concentrations of 10%, 5% and 2% (w/v), respectively. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item not to cause lymphoproliferation in the Local Lymph Node Assay. The resulting stimulation indices observed under these test conditions were considered to be good evidence that the test item is a  non-sensitizer. The size of lymph nodes was in good correlation with this conclusion.

Conclusion

The test item was assumed to be a skin sensitiser, based on the in vitro results (2/3 positive). However, the in vivo OECD 429 test indicated it is not a skin sensitiser. Perhaps the lack of or limited metabolic capacity of the non-animal test methods indicated the false positive result. Based on the weight of evidence, the substance is considered as a non-sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available information in the dossier, the test item is not classified for skin sensitisation when the criteria outlined in Annex I of 1272/2008/EC and Annex I of 286/2011/EC are applied.