[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-04-20 to 2021-06-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

no guideline available

Guideline:

other: DB-Alm Protocol GARD – Genomic Allergen Rapid Detection (GARDskin)

Principles of method if other than guideline:

The Genomic Allergen Rapid Detection (GARDTM) is an in vitro assay designed to predict the ability of chemical substances to induce skin sensitization based on the analysis of the relative expression levels of a biomarker signature of 196 genes using a human myeloid leukemia cell line called SenzaCell. The GARD^TM is based on chemical stimulation of the SenzaCell line, acting as an in vitro model of human Dendritic Cells (DCs). The readout of the assay is a transcriptional quantification of the genomic predictors, collectively termed the GARD^TM Prediction Signature (GPS), using Nanostring nCounter technology.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Genomic Allergen Rapid Detection (GARD)-Assay

Justification for non-LLNA method:

The GARD^TM method mimics the immune system. It predicts the ability of chemical compounds to induce skin sensitisation. Many in vitro tests for sensitisers on the market are fairly simple to perform, but they are often not enough to give reassuring results. Genomics-based models offer reliable alternatives to animal testing. Since GARD^TM is examining the expression from more than a hundred genes that are all collectively relevant for the AOP of skin sensitisation, several key events are addressed in one single test.

Test material

Test material information

Constituent 1

Reference substance name:

N-(4-((4-(3-phenylureido)phenyl)sulfonyl)phenyl)benzenesulfonamide

Cas Number:

2437256-84-1

Molecular formula:

C25H21N3O5S2

IUPAC Name:

N-(4-((4-(3-phenylureido)phenyl)sulfonyl)phenyl)benzenesulfonamide

Specific details on test material used for the study:

- Name: N-(4-((4-(3-phenylureido)phenyl)sulfonyl)phenyl)benzenesulfonamide
- Lot/batch no.: 2019001
- Purity: 98.78%
- Appearance/physical state: white crystalline powder
- Expiry Date: 2020-10-28
- Storage: at room temperature, protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥ 99%, Lot No.: H0390). Vortex mixing was used to aid solubilisation.

In vitro test system

Details of test system:

other: human myeloid leukemia cell line Senza cells

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution : All test item solutions were freshly prepared immediately prior to use. The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥ 99%, Lot No.: H0390). Vortex mixing was used to aid solubilisation.
- Preparation of the test chemical serial dilutions : After a suitable solvent was determined, the solubility of the test item solution was tested after a 1:100 dilution in semi-complete media. The solvent was present at a constant volume ratio of 0.1% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.
- Preparation of the positive controls : p-Phenylenediamine (PPD; CAS No.: 106-50-3) at a final concentration of 50 μM was tested concurrently with the test item. PPD was dissolved in DMSO and diluted 100-fold into semi-complete medium to achieve the in-well concentration of 0.1% DMSO.
- Preparation of the solvent, vehicle and negative controls: DMSO was used as a Solvent control. The final concentration of DMSO in cell culture medium was 0.1% (v/v). Semi-complete medium was included in the test as a medium control.

DOSE RANGE FINDING ASSAY:
- Highest concentration used : 200 µM
- Solubility in solvents : All test item solutions were freshly prepared immediately prior to use. The test item was soluble in DMSO at a concentration of 500 mM. Due to precipitations of the 500 mM test item concentration in medium the test item stock solution was diluted down to 200 mM, which showed only slight turbidity in medium. 200 mM stock solution in DMSO was used for further testing. Starting from the highest soluble concentration in the most applicable solvent and medium determined
in the solvent finding experiment, a maximum of nine stock solutions (i.e. nine concentrations) down to 1 mM were prepared. (Input finder assay 1: 200 mM – 100 mM – 50 mM – 10 mM – 5 mM – 1 mM; Input finder assay 2: 200 mM – 185 mM – 170 mM – 160 mM – 150 mM – 140 mM – 125 mM – 110 mM – 100 mM). These stock solutions were further diluted 100-fold into semi-complete medium giving the stock B solutions. The stock B solutions were finally used for treatment by adding the test item to the SenzaCell suspension in complete medium in a 24-well plate to achieve a further 10-fold dilution.
- Results of selecting appropriate concentration and determination of cytotoxicity: Due to a very steep dose response between 100 µM (100.4% relative cell viability) and 200 µM (22.8% releative cell viability) observed in a first experiment, a second Dose Range Finding experiment with a narrower dose spacing was performed. An average relative viability of 90% (RV90) was detected at a test item concentration between 130 μM and 140 μM.
- Final concentration range selected on basis of: The test item that induced cytotoxicity was used for the main stimulation at the concentration that induces 90% ± 5% relative viability (RV90). The main stimulations were done with a dilution range of 120 μM to 140 μM.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates : not specified
- Number of repetitions : Three
- Test chemical concentrations : Experiment 1 and 2: 120, 130 and 140 µM. Experiment 3: 130, 135 and 140 µM.
- Application procedure : A stock solution of the positive control (50 mM) in DMSO and a stock solution of 1000 x the GARD input concentration of the test item in the solvent determined in the solvent finding pre-experiment were freshly prepared immediately before use. The stock solution of the positive control, pure DMSO as a negative control, the stock solution of the test item and the solvent of the test item were diluted 100-fold into semi-complete medium (= stock B) to achieve the in-well concentration of 0.1% DMSO. The stock B solutions of the positive control, the solvent control of the test item input concentration and the solvent of the test item were mixed 1:10 (v/v) with the cell suspension prepared in the 12-well plate.
- Exposure time : 24 ± 0.5 hours
- Description on study acceptance criteria: he criteria for the phenotypic quality control are described in section "Any other information on materials and methods incl. tables"


SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density) : For testing, SenzaCells were pre-cultured for 3 – 4 days in culture flasks at a cell density of 0.2 x 10E6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh semi-complete medium at a density of 2.22 x 10E5 cell/mL. Then 3.6 mL of cell suspension were seeded into a 12 well plate-bottom plate (8 x 10E5 cells/well). The Input Finder assay was performed using stock solutions with a concentration of 200 mM (applied concentration 200 μM) and cells harvested in passage 4 (Input finder assay 1) and 5 (Input finder assay 2). For determination of the sensitizing potential of the test substance three independent experiments
were performed using separate thawed, cultivated and harvested cells at passage 08 (main stimulation 1 and 2) and passage 9 (main stimulation 3) for all three main stimulations. For each experiment separately weighted samples and preparations were used.
- Incubation conditions : Treated plates were incubated for 24 h ± 0.5 h at 37°C ± 1 °C and 5% ± 0.5% CO2.
- Washing conditions : After 24 h ± 0.5 h of exposure, 1 mL of the cells were transferred three times into small reaction tubes and further 500 μL were transferred twice into two FACS tubes. The cells of the reaction tubes were collected by centrifugation (approx. 300 x g), re-suspended in 500 μL TRIzol® and frozen at < -20 °C. The cells of the FACS tubes were washed twice with FACS buffer. After washing, cells were resuspended in 50 μL propidium iodide (PI) solution (1 μg/mL) in FACS buffer.

MEASUREMENT OF GENE EXPRESSION
- Propidium iodide staining/cytotoxicity measurements : After washing, cells were resuspended in 50 μL propidium iodide (PI) solution (1 μg/mL) in FACS buffer. Cells were stained for approx. 10 minutes at 2-8°C in the dark. After staining, cells were washed with FACS buffer again and re-suspended in 200 μL FACS buffer. The PI uptake of the cells (as an indicator of cytotoxicity) was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10.000 living (PI negative) cells were acquired and cell viability was calculated for each sample.
- Preparation for RNA isolation: The first tube of each TRIzol® sample triplicate was thawed slowly on ice. The samples were centrifuged to remove particulate debris and the supernatant was transferred into new tubes. An equal volume of 95% - 100% ethanol was added and the whole mixture was transferred on the column of a Zymo-SpinTM IIC Columns (ZymoResearch Cat. R5052). The flow through was discarded and the column was washed twice with 400 μL with the Direct-zolTM RNA PreWash. The flow through was discarded again. After the pre-washing step the column was washed with 700 μL RNA wash buffer. The flow through was discarded again and the column was placed in a new RNAse-free tube. The RNA was eluted with 25 μL RNAse-free water. After centrifugation the collected RNA-eluate was transferred onto the column again to elute remaining RNA. The RNA was stored at -80°C.
The RNA quality and quantity was measured with an Agilent 2100 BioAnalyser. RNA samples which had passed the criteria specified in section "Any other information on materials and methods incl. tables" undergo Nanostring endpoint measurement with a codeset designed for GARD skin analysis.
- Nanostring Hybridisation: All hybridization samples used a total RNA input of 100 ng. All RNA samples were diluted to 20 ng/μL with RNA-free water: 5 μL of each diluted sample was used for hybridization. For hybridization mix 8 μL of the master mix (provided in the Nanostring master kit), 5 μL RNA sample (20 ng/μL) and 2 μL of the capture code set were mixed. The mixture was incubated in a thermocycler at 65°C for 24 h ± 0.5 h.


DATA EVALUATION
- Cytotoxicity assessment : If the cell viability passes the criteria, described in section "Any other information on mateirals and methods incl. tables", the RNA of the TRIzol® samples were collected. The cell viability was measured with FACS. The FACS data analysis was performed using the software BD FACS DIVA 6.0. The relative viability percentage calculation is based on the following equation: relative viability (Rv) = (Absolute viability of sample in %/ Absolute viability of the mean of the two unstimulated control samples in %) x 100
- Prediction model used :
Prediction for Skin Sensitisation
The prediction for skin sensitisation is defined as described below:
− If the mean decision value (DV) of biological replicate samples is ≥ 0, the substance is classified as a sensitizer
− If the mean decision value (DV) of biological replicate samples is < 0, the substance is classified as a non-sensitizer.
Prediction for Potency of Skin Sensitizer
The prediction for the potency of a skin sensitizer is defined as described below:
− If the mean decision value (DV) of biological replicate samples is ≥ 0, the substance is classified as subcategory 1A.
− If the mean decision value (DV) of biological replicate samples is < 0, the substance is classified as subcategory 1B.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

other: p-Phenylenediamine (PPD; CAS No.: 106-50-3) at a final concentration of 50 μM in DMSO

Results and discussion

Positive control results:

The mean decision value of the positive control was > 0 (9.36, individual values for the three repetitions: 9.27; 9.52; 9.29). The controls confirmed the validity of the study for all experiments for cytotoxicity (92.0; 93.0; 92.2%).

In vitro / in chemico

Results

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Decision value

Value:

7.62

Cell viability:

Mean cell viability compared to the medium control was 93.4%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for RNA samples: yes
- Acceptance criteria met for cytotoxicity: yes
- Quality control criteria met for cells used in main experiment: yes
- Range of historical values if different from the ones specified in the test guideline: See section "Any other information on results incl. tables"

Any other information on results incl. tables

Table 1: Historical Data

Criterion	mean	SD	N
Viability of DMSO control (criterion ≥ 95.5 %)	100.4	1.0	24
Viability of Positive control (criterion 84.5 % ≤ X ≤ 95.4 %)	87.0	2.2	27
RIN value (criterion ≥ 8.0)	9.4	0.4	73
RNA concentration (criterion ≥ 20 ng/μL)	119.9	48.8	73

Table 2: GARDskin - Decision Values of the Test Item and the Controls

Negative Control		Positive Control		Test item
Main Stimulation	Decision Values	Main Stimulation	Decision Values	Main Stimulation	Decision Values
1 repetition	-1.14	1	9.27	1	7.23
2	-0.687	2	9.52	2	7.09
3	-1.14	3	9.29	3	8.53
Mean	-0.990	Mean	9.36	Mean	7.62
Predicition
negative		positive		positive

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this Genomic Allergen Rapid Detection (GARD™)-Assay, the test item did change the genomic profile of the cells for sensitization in at least three independent experiment runs, resulting in a mean decision value for the test item ≥0 (7.62). Therefore, the test item the test item showed a positive result in this in vitro test for skin sensitisation.

Executive summary:

In an in vitro Genomic Allergen Rapid Detection (GARD™)-Assay for skin sensitisation, the test item N-(4-((4-(3-phenylureido)phenyl)sulfonyl)phenyl)benzenesulfonamide (purity 98.78%) was dissolved in DMSO and incubated with a human myeloid leukemia cell line (Senza Cells) at concentrations of 120 to 140 µM for 24 ± 0.5 hours at 37 °C in three independent experiments. The concentration range was chosen based on an average relative viability in a range of 90% identified in preliminary cytotoxicity tests.

The cells used for the main experiment fulfilled the quality control criteria.

After exposure, one part of the cells were stained with propidium iodide and cell viability was measured by FACS analysis and the second part of the cells were transferred into TRIzol® and stored at -80 °C.

All three main stimulations fulfilled the criteria for cytotoxicity. Cells treated with 130 μM (Experiment 1 and 2) and 135 μM (Experiment 3) of test item were used for RNA isolation because the viability was in the range of 90% relative viability. All samples fulfilled the RNA quality criteria and all single isolations were used for Nanostring measurement.

The mean decision value for the test item was ≥ 0 (7.62; individual values for the three repetitions: 7.23, 7.09, 8.53).

The mean decision value of the positive control p-Phenylenediamine was > 0 (9.36, individual values for the three repetitions: 9.27; 9.52; 9.29) and for the negative control was < 0 (-0.990; individual values for the three repetitions: -1.14; -0.687; -1.14). The controls confirmed the validity of the study for all experiments. Based on these results, the test item is considered to be a sensitizer in the in vitro GARD™ assay.