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Diss Factsheets

Administrative data

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

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
supporting 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
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 clear colorless non-viscous solution. During the B9 and B11 definitive 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 February 18, 2020 the positive control had a cell viability (%) of 76.04 and passed the test. On March 6, 2020 the positive control had a cell viability of 80.88 and passed the test. On March 13, 2020 the positive control had a cell viability of 60.13 and passed the test.
Key result
Run / experiment:
other: 18 February 2020
Parameter:
other: CD54 (μg/mL)
Value:
1 000
Positive controls validity:
valid
Key result
Run / experiment:
other: 18 February 2020
Parameter:
other: CD86 (μg/mL)
Value:
279
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: 6 March 2020
Parameter:
other: CD54 (μg/mL), CD86 (μg/mL)
Value:
1 000
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 13 March 2020
Parameter:
other: CD54 (μg/mL), CD86 (μg/mL)
Value:
1 000
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Dose Range and Definitive Assays
Parameter:
other: CV75 (μg/mL)
Value:
296.5
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.

Definitive trials 1 -8, and 10 did not meet assay acceptance criteria; therefore, they were not considered valid 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. Since the test article did not elicit RFI >= 200 at any tested concentration for CD54, and/or RFI >= 150 at any tested concentration for CD86, with cell viability >= 50% in two or at least two out of three independent runs, it was consiered to be a non-sensitizer, in accordance with the UN GHS standards.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
supporting 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
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:
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:
2 000
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Mean Imax
Value:
1.68
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Mean IC50 (μM)
Value:
2 000
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:
1 157.63
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation

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.