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Description of key information

The results from the Keratinosens and h-CLAT analyses were negative for skin sensitization of the test material.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

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Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 September, 2019 - 13 March, 2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline 442E
GLP compliance:
no
Remarks:
The study was performed in accordance with UN GHS standards, OECD Test Guideline 442E
Type of study:
other: Activation of human acute monocytic leukemia cells
Justification for non-LLNA method:
The Human Cell Line Activation Test was used to assess the skin sensitization potential of the test articles by monitoring the upregulation of cell surface markers, C054 and CD86, on the surface of human acute monocytic leukemia cells (THP-1). The upregulation of CD54 and CD86 in response to a skin sensitizer is correlated to dendritic cell activation, which is the third key event of the skin sensitization pathway.
Details on the study design:
Solubility Determination:

Prior to the preliminary dose range finding assay, the test article was tested in a solubility test to determine an appropriate solvent. The following ovservations were determined during the test. The test article was found to be soluble at 500 mg/mL in DMSO with approximately 1 minute of vortexing. The test article dilution appeared to be a cloudy white non-viscous suspension. During all of the valid trials, the media tubes 1-8 used in the test substance dilution scheme appeared to contain small white particles. The tubes were vortexed immediately prior to dosing and the cloudiness remained.

Dose Range Finding Assay:

A preliminary dose range finding assay was performed to determine the viability of the THP-1 cells after 24 +/- 0.5 hour exposure to 8 test article concentrations. The CV75, which is the calculated test article concentration leading to 75% cell viability was calculated for the test article.

Definitive assays:

Based on the results from the dose range finding assay, the doses were chosen for the test article for the definitive assay. At least 2 valid definitive trials were performed.

The positive control, 2,4-Dinitrochlorobenzene, was tested in the dose rang and definitive trials.

Evaluation of test results:

The relative fluorescence intensity was calculated for the test article and control treated cell population. The EC200 and EC150 values, which are the calculated test article concentrations leading to an RFI of 200 or 150 respectively, were calcued for the test article.
Positive control results:
The positive control results were considered valid on the days that the test material assays were run. On October 1, 2019 the positive control had a cell viability (%) of 83.19 and passed the test. On October 8, 2019 the positive control had a cell viability of 77.60 and passed the test.
Key result
Run / experiment:
other: B1 Assay Date: 1 October 2019
Parameter:
other: CD54 (μg/mL), CD86 (μg/mL)
Value:
1 000
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
μg/mL
Key result
Run / experiment:
other: B2 Assay Date: 8 October 2019
Parameter:
other: CD54 (μg/mL), CD86 (μg/mL)
Value:
1 000
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
μg/mL
Key result
Run / experiment:
other: Dose Range and Definitive Assays
Parameter:
other: CV75 (μg/mL)
Value:
1 000
Remarks on result:
other: μg/mL
Other effects / acceptance of results:
The assay met acceptance criteria when:

The cell viability values of the solvent control was > 90%.

For the solvent control, RFI values of both CD86 and CD54 were less than the positive criteria (CD86 RFI < 150 and CD54 RFI < 200).

For the positive control (DNCB), RFI valus of both CD86 and CD54 were predicted to be positive (CD86 RFI >= 150 and CD54 RFI >= 200), and cell viability was > 50%.

For the medium and solvent controls, the MFI ration of both CD86 and CD54 to isotype control was >105%.

The cell viability of the test article-treated cultures was > 50% in at least four doses.

All acceptance criteria for a valid assay were met for the definitive trials.
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the h-CLAT definitive assays, the test material was considered to be a non-sensitizer.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 September - 5 October, 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
no
Remarks:
The study was performed in compliance with OECD Guideline for the Testing of Chemicals No. 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method
Type of study:
other: KeratinoSens assay (Luciferase Test Method)
Justification for non-LLNA method:
The Induction of Antioxidant-Response-Element Dependent Gene Activity in the Keratinocyte ARE-ReporterCell Line KeratinoSens skin sensitization assay is a high-throughput cell-based in vitrotest to screen for the skin sensitization potential of chemicals.
Details on the study design:
Receipt of Skin Sensitization Assay Reagents:
Reagents used for the skin sensitization bioassay were inspected upon receipt and stored according to the manufacturer’s instructions.

Cell Thawing Procedure:
A cryovial of the cryopreserved KeratinoSens cells was thawed in a water bath at approximately 37ºC (in a beaker containing 70% ethanol). Once thawed, the cryovial was immediately decontaminated with 70% ethanol and placed in a laminar flow hood. The cells were added to a sterile conical tube and diluted by slowly adding 9 mL pre-warmed Assay Medium. The cells were then centrifuged at approximately 200 x g for 5 minutes at room temperature. The supernatant was aspirated, the pellet was re-suspended in approximately 15 mL of fresh pre-warmed Assay Medium, and then transferred into a T75 tissue-culture flask. The flask was then incubated at 37 ± 1ºC, 90 ± 10% humidity, and 5.0 ± 1% CO2in air (standard culture conditions),until the KeratinoSens cells reached approximately 60% to 90% confluence.

Routine Culturing of the KeratinoSens Cells:
When the cultures reached approximately 60 to 90% confluence, they were removed from the flask by trypsinization. The medium was aspirated and the cell sheet rinsed twice with approximately 10 mL of CMF-DPBS containing 0.05% EDTA. One mL of trypsin/EDTA was added to cover the cell sheet. The flask was then placed into the incubator and incubated at standard culture conditions for 6-8 minutes, or until the cells became dislodged. When more than 50% of the cells became dislodged, the flask was rapped sharply against the palm of the hand. Then approximately 7 mL of Maintenance Medium was added to each T75 flask to obtain a single cell suspension and cells passaged at appropriate densities. The KeratinoSens cells were routinely passaged every 2-4 days.

Subculture of KeratinoSens Cells into 96-Well Plates:
The KeratinoSens cells were subcultured into transparent Costar 96-well plates or white-walled Perkin Elmer plates when the flasks were approximately 60 to 90% confluent. The flasks were rinsed and trypsinized as previously described. The cells were resuspended in 5 mL of Assay Medium per flask. The concentration of cells in suspension was determined using a Coulter Counter. A cell suspension of 1.0 x 105 cells/mL in Assay Medium was prepared. One hundred μL of the cell suspension was added to all but one well designated as a blank well (H12). The stock cell suspension was mixed often to ensure a uniform distribution of cells into each well.

Three white-walled and one transparent plate were seeded for each test article replicate set (definitive trials).The plates were incubated for approximately 24 hours at standard culture conditions. The cultures seeded into the clear plates were examined under a phase contrast microscope and evaluated for uniform seeding and confluence prior to treating the cells with the test articles or positive control.

Solubility Determination:
The solubility of the test articles were tested in DMSO on the day of the initial definitive assay (at the highest 100X concentration of 200 mM). Poor solubility in DMSO was observed for this test article, therefore it was tested as a suspension in DMSO.

MTT Direct Reduction Test:
The ability of the test articles to directly reduce MTT was assessed at the same time of test article treatment in the definitive assays. A 1.0 mg/mL MTT solution was prepared by dissolving a 10 mg/mL stock solution of MTT into warm MTT Addition Medium. Approximately 100 μL of the 100X (200 mM) test article concentration in DMSO was added to 1 mL of the MTT solution and then incubated in the dark at 37ºC for one to three hours. One hundred μL of a negative control (e.g. DMSO) was tested concurrently. If the MTT solution color turned blue/purple, the test article was presumed to have reduced the MTT. The test article did not turn the MTT solution color blue/purple. Therefore, the test article, does not directly reduce MTT. However, the test article did form a white precipitate.

Controls:
Each assay plate included a range of doses of the positive control, Cinnamic Aldehyde (Sigma). A 100X concentration of positive control was prepared by weighing an appropriate amount of Cinnamic Aldehyde into a pre-labeled conical tube and adding the necessary amount of DMSO to prepare a 64,000 μM dilution. The 64,000 μM dilution was further diluted 1:10 in DMSO to prepare a 6,400 μM 100X stock concentration. The final 1X concentrations of the positive control were 64, 32, 16, 8, and 4 μM. The solvent control for the test articles and the positive control was 1% DMSO in the dilution solvent (1% DMEM.) Each plate included a set of 6 solvent control wells.

Testing Concentrations:
The test articles had defined molecular weights provided by the Sponsor,and were diluted based on molarity. The 100 X stock dilution was prepared to a top concentration of 200 mM. The final 1X tested concentrations were 0.977, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 μM.

Definitive Assays:
The test article was tested in three independent definitive assays. Each definitive assay included a set of 4 plates (3 for gene induction, 1 for cytotoxicity assessment). Each plate tested a range of 12 dosing concentrations for the test article. Each plate also included 5 wells designated for the positive control (tested over a range of 5 dosing concentrations), 6 wells designated as the DMSO solvent control, and 1 well which was left blank. After approximately 24 hours of incubation, the Assay Medium was removed from the cells. The plates were decanted and gently blotted on sterile paper towels. One hundred and fifty microliters of fresh pre-warmed 1% DMEM were added to all wells, including the blank. The plates were returned to the incubator until the dosing was initiated. For the test article, twelve decreasing doses were selected for the assay (test articles were diluted to a final concentration of 200 mMin DMSO). For the positive control, 5 decreasing doses were prepared. For each experiment, the positive control (5 doses), and the solvent control, a 100X DMSO master plate was made, followed by a 4X Master Plate. When added to the 150μL of 1% DMEM already in each well, the addition of the 50 μL 4X dose brought the final dose on the plates to 1X.

Visual Observations:
After approximately 48 hours of post-treatment incubation, visual observations of the cultures were performed for the cytotoxicity plate and recorded. Precipitate formation precluded visual observations of cells dosed with the test article at the highest testing concentrations in all three trials.

Treatment Termination & Luciferase Induction Determination:
After 48 ±1 hours of exposure, each white-walled culture plate was removed from the incubator and allowed to equilibrate to room temperature for at least 30 minutes. Once at room temperature, the treatment medium was decanted from each plate. The cultures were rinsed with 250 μL of CMF-DPBS (room temperature), the CMF-DPBS rinsate was decanted from the wells, and the plates were gently blotted onto paper towels. Fifty microliters of CMF-DPBS was added to each well followed by fifty microliters of ONE-Glo™ Reagent. The plates remained at room temperature in the dark for at least 5 minutes before being read by the luminometer. The plates were read within 45 minutes of addition of the ONE-Glo™ Reagent. The luminescence determination of each plate was performed by a Berthold Detection Systems luminometer initiated from an IBM-PC hosting the Windows-based Simplicity™ software. The light intensity in each well was measured at 565 nm in the form of relative light units (RLUs).

Treatment Termination:Cytotoxicity Using the MTT Endpoint
A 0.59 mg/mLMTT solution was prepared in 1% DMEM and used within 2 hours. After 48 ±1 hours, the clear 96-well plates designated for the MTT endpoint were decanted and gently blotted on paper towels. No rinsing was performed. Two hundred μL of 1% DMEM containing 0.59 mg/mL MTT was added to each well. The plate was incubated with a plate seal at standard culture conditions for approximately 4 hrs. After approximately 4 hours, the MTT solution was decanted, the plate was blotted, and 200 μL of 10% SLS was added to each well. The plate was covered with a plate seal and incubated at standard culture conditions overnight. After the overnight incubation, each plate was placed on a plate shaker and shaken for at least 20 minutes at room temperature. The absorbance at 570 nm (OD570) of each well was measured with a Molecular Devices Vmax plate reader.

Data Analysis:
For each set of up to seven test articles, a copy of the standard data file, provided by Givaudan, was made. Raw data from the luminometer was transferred directly from the luminometer software into the designated Excel spreadsheet for luminescence analyses. Raw data from the Vmax was transferred into the designated Excel spreadsheet for cytotoxicity analyses.
The data file automatically calculated the gene induction and the wells with statistically significant induction over a given threshold (default value set to 1.5 = 50% enhanced gene activity). Furthermore the maximal induction (Imax), the concentration for maximal gene induction (CImax) and the EC1.5 value (concentration for induction above threshold), both with linear and log-linear extrapolation, was calculated similar to the LLNA (Local Lymph Node Assay). Relative survival (viability) was obtained by comparing the amount of MTT conversion by test article treated groups compared to the associated solvent treated group on the same plate. The Givaudan Excel file calculates an IC50 value for each test article. The IC50 is determined by averaging the viability percentage of each concentration for the 3 definitive assays and then calculating by linear interpolation the IC50 concentration which results in 50% reduced cell viability using the concentration and viability percentage below and above 50% viability.A summary of the results from the 3 definitive assays was calculated. The luciferase induction and cytotoxicity data were plotted on the graphs. For the luciferase induction, the fold gene induction (as compared to the solvent controls) was plotted over the test article concentration. For the cytotoxicity, the % viability (as compared to the solvent controls) was plotted over the test article concentration.
Positive control results:
The positive control cinnamic aldehyde ha a mean EC 1.5 (μM) of 9.27 and a Mean IC50 (μM) of >64 and was considered to be a sensitizer.
Key result
Parameter:
other: Mean CImax (μM)
Value:
250
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Mean Imax
Value:
1.15
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Mean IC50 (μM)
Value:
10.78
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Mean EC 1.5 (μM)
Value:
2 000
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Significant cytotoxicity occurred before the 2000 μM dose

Due to the standard deviation of luciferase induction in solvent control wells exceeding 20%, trial 2 was excluded from analysis for the test article.

Interpretation of results:
GHS criteria not met
Conclusions:
According to the current prediction model, the test article was predicted to be a non-sensitizer.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the restults from the two studies, the test material was not considered to be a skin sensitizer and was not classified according to GHS criteria as a skin sensitizer.