[No public or meaningful name is available]

Endpoint summary

Description of key information

The skin sensitisation potential of the test item was assessed in a weight-of-evidence approach.

In an in chemico direct peptide reactivity assay according to OECD TG 442C, a prediction could not be made due to precipitation observed in the peptide incubations.

In an in vitro KeratinoSens luciferase assay (OECD TG 442D), addressing the second molecular key event of the adverse outcome pathway (AOP) for skin sensitisation, no significant induction of the luciferase activity was observed.

In an additional in vitro Genomic Allergen Rapid Detection (GARD™)-Assay for the investigation of the third key event of the skin sensitisation AOP, changes in the genomic profile of a human myeloid leukemia cell line revealed a positive prediciton for skin sensitisation.

Due to the non-applicability of the in chemico assay and equivocal results from the in vitro assays for the second and third key event of the AOP, an in vivo local lymph node assay (LLA) according to OECD TG 429 was conducted. The test item did not show skin sensitising potential in this assay.

By assessing the results in a weight-of-evidence approach the test item is considered as non-sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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.

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.

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

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%).

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"

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

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.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-12-18 to 2021-03-16

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)

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Name: N-(4-((4-(3-phenylureido)phenyl)sulfonyl)phenyl)benzenesulfonamide
- Lot/batch number of test material: 2020003
- Purity: 99.68%
- Appearance/physical state: white cristalline powder
- Retest Date: 13 May 2021
- Storage condition of test material: refrigerated (2-8 °C), protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was weighed, and formulations prepared in DMSO daily on a weight: volume basis as % (w/v) by the Pharmacy of Charles River Laboratories Hungary Kft.

Species:

mouse

Strain:

CBA/Ca

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, The Netherlands
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF at arrival; standard housing conditions during the study
- Age at study initiation: Young adults, 8 weeks old (first experiment) and 10 weeks old (second experiment)
- Weight at study initiation: 17.1 – 19.9 grams (first experiment) and 18.1 – 21.2 grams (second experiment)
- Housing: Group caging (which is the standard procedure for small rodent studies, following AAALAC recommendations, to allow social interaction) in Type II. polypropylene/ polycarbonate cages during the observation period and individual caging in the radioactive proliferation test phase in the main experiments.
- Diet: ad libitum, ssniff® SM Rat/Mouse – "Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice" produced by ssniff Spezialdiäten GmbH (D-59494 Soest, Germany, Batch number: 71370882, Expiry date: 30 April 2021) and Geldiet Transport (Scientific Animal Food & Engineering, Route de Saint Bris, 89290 Augy, France, Batch numbers: 60200300010101 / 60200520020101, Expiry date: 29 January 2021 / 20 February 2021)
- Water: ad libitum, tap water from the municipal supply and suitable for human consumption. Routinely analysed and considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period: at least 6 days, the animals were kept under the same controlled environment conditions as during the experimental period.
- Indication of any skin lesions: The health status of the animals assigned to study were verified by the clinical Veterinarian.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.1-23.8
- Humidity (%): 22-75
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12 / 12

- IN-LIFE DATES: From: 23 December 2020 To: 02 February 2021

Vehicle:

dimethyl sulphoxide

Concentration:

25%, 10%, 5% (w/v)

No. of animals per dose:

4 females

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The solubility of the test item was examined in a Preliminary Compatibility Test. The following standard OECD No. 429 vehicles were assessed: Acetone: Olive oil 4:1 (v/v) mixture (AOO), N,N-dimethylformamide (DMF), Methyl ethyl ketone (MEK), Propylene glycol and Dimethyl sulfoxide (DMSO).
The test item did not dissolve in any vehicle at concentrations of 100 and 50% (w/v). The test item dissolved in MEK and DMSO at 25% (w/v). The formulations with MEK appeared to be an inhomogeneous suspension and DMSO appeared to be a clear solution by visual examination. The best vehicle taking into account the test item characteristics and the requirements of the relevant OECD guideline was considered to be Dimethyl sulfoxide (DMSO). The 25% (w/v) formulation was the highest concentration, which was suitable for the preliminary test, because at higher concentrations the solubility of the test item was not sufficient.
- Irritation: The Preliminary Irritation / Toxicity Test was started on CBA/CaOlaHsd mice using two doses (2 animals/dose) per test item concentrations of 25% and 10% (w/v) in DMSO. The preliminary experiment was conducted in a similar experimental manner to the Main Study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed. Besides moderate or slight test item residue on the total surface of both ears for both formulations until Day 3 for 10% (w/v) formulation and until Day 4 in case of the 25 % (w/v) formulation, no signs of irritation were observed.
- Systemic toxicity: During the Preliminary Irritation / Toxicity Test no mortality and no significant body weight loss was observed.
- Ear thickness measurements: Ear thickness of the animals was measured using a thickness gauge on Days 1, 3 and 6. No increased average ear thickness values (>25%) were detected for any of the animals in this preliminary study. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. The revealing ear punch weights were within the historical control range except in one animal in case of 10% (w/v) formulation where the data was smaller than the historical control data. It was concluded that there was no effect of test item on ear thickness.
- Erythema scores: For 25% and 10% (w/v) formulation no erythema was observed. For details on scoring scheme, please refer to Table 1 in section "Any other information on materials and methods incl. tables"

MAIN STUDY
- Mortality/Moribunity checks: Animals were inspected for signs of morbidity and mortality at least once daily.
- Clinical Observations: During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.
- Body weights: Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to ³HTdR injection) with a precision of ± 0.1g.
- Proliferation Assay: On Day 6, 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).
Five hours (±30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cutting through major cervical blood vessels.
Once removed, the lymph nodes were collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing. The nodes of each animal were processed individually. A single cell suspension (SCS) of lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10mL). LNCs was pelleted with a relative centrifugal force (RCF) of approximately 190xg for 10 minutes at 4°C. After centrifugation supernatants were discarded. Pellets were gently resuspended, and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each individual animal.
After the final washing step, supernatants were removed. Pellets were gently resuspended and 3 mL of 5% (w/v) TCA solution was added to the tubes for precipitation of macromolecules. After overnight (approximately 18 hours) incubation at 2-8°C precipitates were centrifuged (approximately 190xg for 10 minutes at 4°C), and supernatants was removed. Pellets were resuspended in 1 mL of 5% (w/v) TCA solution and dispersed by using an ultrasonic bath.
Samples were transferred into suitable sized scintillation vials filled with 10mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The β-counter expresses the ³HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1mL of 5% (w/v) TCA solution into a scintillation vial filled with 10mL of scintillation liquid.
- Euthanasia for all animals was by asphyxiation with ascending doses of carbon dioxide, followed by confirmation. Death was confirmed by cervical dislocation.
- Evaluation of Results: DPM was measured for the nodes of each individual animal. The measured DPM values were corrected with the background DPM value ("DPM"). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as "DPN" (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
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.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- 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:
- The applicable doses were based on the results of the Preliminary Irritation/Toxicity Test (see section 10.1). The test item was weighed, and formulations prepared daily on a weight: volume basis as % (w/v) by the Pharmacy of Charles River Laboratories Hungary Kft.
- 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.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

No statistical evaluation was performed in this study.

Positive control results:

The positive control substance was examined at a concentration of 25% (w/v) in the relevant vehicle (DMSO). No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. In the first assay stimulation index value was 2.8 (just below the cut-off of 3.0), therefore the main assay was repeated. In the second main assay a SI= 7.9 was noted, demonstrating the appropriate performance of the assay in the second experiment and therefore confirmed the validity of the assay.

Parameter:

SI

Value:

1

Test group / Remarks:

First main experiment: 25% test item

Parameter:

SI

Value:

0.8

Test group / Remarks:

First main experiment: 10% test item

Parameter:

SI

Value:

0.6

Test group / Remarks:

First main experiment: 5% test item

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

Second main experiment: 25% test item

Parameter:

SI

Value:

1

Test group / Remarks:

Second main experiment: 5% test item

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
- The appearance of the lymph nodes was normal in the 25%, 10% and 5% (w/v) test and negative control groups and larger and slightly larger than normal in the positive control group in both main assays (For more information on proliferation data, please refer to Table 1 and 2 in section "Any other information on results incl. tables").

DETAILS ON STIMULATION INDEX CALCULATION
- DPM was measured for the nodes of each individual animal. The measured DPM values were corrected with the background DPM value ("DPM"). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as "DPN" (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
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.

EC3 CALCULATION
- SI values after treatment with the test item were below 3. THerefore the calculation of an EC3 value was not performed.

CLINICAL OBSERVATIONS:
- There was no mortality observed during the main assays. There were no clinical signs during the studies.
In the first main assay slight or moderate amount of test item residue was observed on the ears of the 25% (w/v) group from Day 1 to Day 6, slight amount of test item residue was observed in case of the 10% (w/v) group from Day 1 to Day 3 and slight amount of test item residue was observed in case of the 5% (w/v) group on Day 3.
In the second main assay slight or moderate amount of test item residue was observed on the ears of the 25% (w/v) group from Day 1 to Day 6, slight and moderate amount of test item residue was observed in case of the 5% (w/v) group from Day 1 to Day 4.
BODY WEIGHTS
- No test item related marked decrease (>5%) of mean body weight was observed in any group. The average body weight range was within the acceptable range of all groups.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness).
- There was no indication of local irritation at the site of application (no erythema or swelling at these concentrations).

Table 1: DPM, DPN and Stimulation Index Values for all Groups (first main assay)

Test Group Name	Animal number	Measured DPM	Total DPM	No. Of Nodes	DPN	Group DPN	Stimulation Index (SI) Values
Background* (5 (w/v) % TCA)	-	36.0	-	-	-	-	-
Negative (vehicle) control DMSO	196	1770	1734.0	2	867.0	933.1	1.0
	192	1900	1864.0	2	932.0
	185	1660	1624.0	2	812.0
	183	2279	2243.0	2	1121.5
N-(4-((4-(3-phenylureido) phenyl)sulfonyl)phenyl) benzenesulfonamide (25% (w/v) in DMSO)	191	1739	1703.0	2	851.5	972.9	1.0
	197	3105	3069.0	2	1534.5
	190	1438	1402.0	2	701.0
	186	1645	1609.0	2	804.5
N-(4-((4-(3-phenylureido) phenyl)sulfonyl)phenyl) benzenesulfonamide (10% (w/v) in DMSO)	178	1251	1215.0	2	607.5	746.9	0.8
	193	2476	2440.0	2	1220.0
	180	824	788.0	2	394.0
	182	1568	1532.0	2	766.0
N-(4-((4-(3-phenylureido) phenyl)sulfonyl)phenyl) benzenesulfonamide (5% (w/v) in DMSO)	179	1376	1340.0	2	670.0	602.0	0.6
	189	1511	1475.0	2	737.5
	188	861	825.0	2	412.5
	187	1212	1176.0	2	588.0
Positive control 25 (w/v) % HCA in DMSO	184	3532	3496.0	2	1748.0	2639.9	2.8
	195	6407	6371.0	2	3185.5
	181	5251	5215.0	2	2607.5
	194	6073	6037.0	2	3018.5

Note:

1. Total DPM = Measured DPM – Background DPM.

2. *: The background values were 36 and 36.

3. DPN = (Measured DPM – Background DPM value of TCA) / Number of nodes.

4.SI = DPN value of a treated group divided by the DPN value of the negative control group.

Table 2: DPM, DPN and Stimulation Index Values for all Groups (second main assay)

Test Group Name	Animal number	Measured DPM	Total DPM	No. Of Nodes	DPN	Group DPN	Stimulation Index (SI) Values
Background (5 (w/v) % TCA)*	-	64.5	-	-	-	-	-
Negative (vehicle) control  DMSO	208	874	809.5	2	404.8	566.0	1.0
	206	860	795.5	2	397.8
	204	1375	1310.5	2	655.3
	207	1677	1612.5	2	806.3
N-(4-((4-(3-phenylureido) phenyl)sulfonyl)phenyl) benzenesulfonamide (25% (w/v) in DMSO)	213	977	912.5	2	456.3	656.0	1.2
	199	1020	955.5	2	477.8
	205	2003	1938.5	2	969.3
	210	1506	1441.5	2	720.8
N-(4-((4-(3-phenylureido) phenyl)sulfonyl)phenyl) benzenesulfonamide (5% (w/v) in DMSO)	212	823	758.5	2	379.3	547.8	1.0
	201	769	704.5	2	352.3
	202	1852	1787.5	2	893.8
	211	1196	1131.5	2	565.8
Positive control 25 (w/v) % HCA in DMSO	200	10761	10696.5	2	5348.3	4492.5	7.9
	198	6478	6413.5	2	3206.8
	203	9610	9545.5	2	4772.8
	209	9349	9284.5	2	4642.3

Note:

1. Total DPM = Measured DPM – Background DPM.

2. *: The background values were 69 and 60.

3. DPN = (Measured DPM – Background DPM value of TCA) / Number of nodes.

4.SI = DPN value of a treated group divided by the DPN value of the negative control group.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in a mouse local lymph node assay conducted according OECD TG 429, the test item is not considered to be a skin sensitizer under the test conditions of this study.

Executive summary:

In a dermal sensitization study conducted according to OECD 429, four young adult female CBA/CaOlaHsd mice per dose group were dermally exposed to N-(4-((4-(3-phenylureido)phenyl)sulfonyl)phenyl)benzenesulfonamide (purity 99.68%) in DMSO at concentrations of 5%, 10% and 25% (w/w) by topical application to the dorsum of each ear for three consecutive days and observed until day 6 in a local lymph node assay (LLNA). α-hexyl cinnamaldehyde dissolved in DMSO at a concentration of 25% was used as a positive control.

The positive control substance exceeded not the stimulation index of 3 in the first experiment (2.8). Therefore, a second main assay was performed with two dose groups (25%, 5%). The SI value for the positive control was 7.9 in the second main assay and therefore confirmed the validity of the assay.

There was no mortality, clinical signs, indication of local irritation at the application site (no erythema or swelling) or test item-related effects on the body weight.

The SI values were 1.0, 0.8 and 0.6 at test item concentrations of 25%, 10% and 5% (w/v), respectively in the first main assay. The SI values were 1.2 and 1.0 at concentrations of 25% and 5% (w/v), respectively in the second main assay. As a consequence, an EC3 value could not be calculated. In this study, the test item is not a dermal sensitizer.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-04-01 to 2020-09-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

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 freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage. The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions : 18.18 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (35.25 mL) of a 100 mM phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM. The 100 mM Phosphate buffer was prepared of 18% of a 100 mM Monobasic buffer and 82% of a 100 mM Dibasic buffer. The pH-value was adjusted by adding more or less Monobasic or Dibasic buffer.
20.13 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of 100 mM ammonium acetate buffer with pH 10.2 (37.278 mL) to reach a concentration of 0.667 mM. The pH-value was adjusted by adding ammonium hydroxide.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.
- Preparation of the test chemical solutions : The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage.
The test item was pre-weighed into a glass vial and was dissolved in dimethylformamide (DMF). Solubility was determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.
- Preparation of the positive controls, reference controls and co-elution controls :
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run.
Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run.
Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective
solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.

INCUBATION
- Incubation conditions : The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis.Reference controls, co-elution controls as well as the positive control were set up in parallel.
- Precipitation noted : Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 - 400x g) to force precipitates to the bottom of the vial.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys : A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile: 80% buffer ( v / v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions covering the range of 0.534, 0.267, 0.134, 0.067, 0.033, 0.017 an 0 mM.
- Verification of the suitability of the HPLC for test chemical and control substances : The absorbance at λ = 258 nm was also monitored for the samples of the test item and the reference controls as a co-elution control. The ratio of the peak areas (220 nm / 258 nm) was checked for consistency between reference control and test item samples. If this ratio was not consistent, a co-elution was assumed and the evaluation would be adjusted accordingly.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm

Vehicle / solvent:

other: N,N-dimethylformamide

Positive control:

cinnamic aldehyde

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.78%.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

At concentration:

100 mM

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Due to the observed precipitation the prediction model does not apply and a prediction cannot be made.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

0 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Due to the observed precipitation the prediction model does not apply and a prediction cannot be made.

Outcome of the prediction model:

other: No prediction can be made due to the observed precipitation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for reference controls A to C: yes
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): No co-elution of the test item with any of the peptide peaks was observed.
- Acceptance criteria met for variability between replicate measurements: yes

Interpretation of results:

study cannot be used for classification

Conclusions:

In this in chemico direct peptide reactivity assay (DPRA) for skin sensitisation (OECD TG 442C), the test item showed minimal reactivity towards the cysteine peptide (mean depletion ≤6.38%) while precipitation was observed after the incubation period. In the lysine peptide experiment, no HPLC analysis could be performed as a result of strong precipitation. Due to the observed precipitation in both peptide incubations, the prediction model does not apply and a prediction can not be made.

Executive summary:

In an in chemico direct peptide reactivity assay (DPRA) for skin sensitisation (OECD TG 442C), 100 mM of the test item N-(4-((4-(3-phenylureido)phenyl)sulfonyl)phenyl)benzenesulfonamide (purity 98.78%) were incubated with the cysteine and lysine peptide solutions at ratios of peptide to test item of 1:10 (cysteine peptide Ac-RFAACAA) and 1:50 (lysine peptide Ac-RFAAKAA). The reaction solutions were incubated in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.78%. The co-elution and reference controls confirmed the validity of the study for both, the cysteine and lysine run.

In the test item samples of experiments with the cysteine peptide and the lysine peptide, precipitation was observed after the incubation period. In the lysine experiment, no HPLC analysis of the precipitation supernatant could be performed  as a result of strong precipitation. In the cysteine experiment, HPLC analysis of the supernatants was performed after centrifugation. The mean depletion of the cysteine peptide was below the threshold for discrimination between sensitisers and non-sensitisers of 6.38% (0.00%).

According to the evaluation criteria in OECD TG 442C, if a precipitation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Due to the observed precipitation in the cysteine experiment no prediction can be made.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-07-17 to 2020-11-04

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 KeratinoSens™ test method)

Version / remarks:

25th June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

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
- 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%; AppliChem; Lot No.: 0001755581; 0001835436). A stock solution of 100 mM was prepared by pre-weighing the test material into a suitable tube. Vortex mixing was used to aid solubilisation. Based on the stock solution further dilutions were performed to obtain the working solutions.

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution : The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%; AppliChem; Lot No.: 0001755581; 0001835436). A stock solution of 100 mM was prepared by pre-weighing the test material into a suitable tube. Vortex mixing was used to aid solubilisation.
- Preparation of the test chemical serial dilutions : Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent.
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.
- Preparation of the positive controls : Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%; Alfa Aesar; Lot No.: 5018R23W) was used as positive control. CA was dissolved in DMSO (AppliChem; Lot No.: 0001755581; 0001835436 ) at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 μM – 64 μM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.
- Preparation of the solvent, vehicle and negative controls: A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control. DMSO (AppliChem; Lot No.: 0001755581) at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.

DOSE RANGE FINDING ASSAY:
- Highest concentration used : Based on the stock solution of 100 mM a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay: 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49 μM
- Solubility in solvents : >100 mM
- Cytotoxicity assessment performed : yes, cytotoxicity was assessed using the MTT assay

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates : Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per 96-well plate in every independent run.
- Number of repetitions : For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction.
- Test chemical concentrations : 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49 μM
- Application procedure : The assay medium was discarded and replaced by 150 μL test item exposure medium. 50 μL of the shortly before prepared 4x master test item concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
- Exposure time : Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.
- Study evaluation and decision criteria used : A KeratinoSens™ prediction is considered positive if the following conditions will be met in at least two independently prepared test repetitions:
# Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
# cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
# EC1.5 value is <1000 μM
# an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations < 1000 μM is considered as inconclusive. A negative result for test items with a log KOW > 7 has to be interpreted with care due to the applicability of the test method.
- Description on study acceptance criteria :
The test meets acceptance criteria if:
# the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
# the average induction in the three technical replicates for the positive control at a concentration of 64 μM is between 2 and 8
# the EC1.5 value of the positive control is within two standard deviations of the historical mean
# the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density) : A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 μL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel, cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2.
- Incubation conditions : Thereafter, the assay medium was discarded and replaced by 150 μL test item exposure medium. 50 μL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item. All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.
- Washing conditions : After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 μL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
- Precipitation noted : Not specified

LUCIFERASE ACTIVITY MEASUREMENTS
- Plate used : white 96-well plates (flat bottom)
- Lysate preparation : After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 μL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light. Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 μL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1 sec. before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.

DATA EVALUATION
- Cytotoxicity assessment : For the cell viability plate the medium was replaced with 200 μL test item exposure medium. 27 μL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 μL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Positive control results:

The positive control cinnamic aldehyde (CA) induced the appropriate response by showing a significant ≥2-fold induction of luciferase activity (3.10-fold at 64 µM) at a relative cell viability ≥70% (102.8%).

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

1.33

At concentration:

0.98 other: µM

Cell viability:

83%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Group:

test chemical

Run / experiment:

mean

Parameter:

IC30 [442D]

Value:

11.85 µM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

mean

Parameter:

IC50 [442D]

Value:

20.6 µM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The acceptance criteria proposed by the OECD test guideline 442D were met in this test.

- In the first experiment, no significant luciferase induction was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated.

- In the second experiment, no significant luciferase induction was found in the tested concentration range. Therefore, no EC_1.5.value could be calculated.

- No dose-response relationship for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

- Under the condition of this study the test item is therefore considered as non-sensitiser.

- The controls confirmed the validity of the study

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro skin sensitisation study (OECD 442D), the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.

Executive summary:

In an in vitro skin sensitisation assay conducted according to OECD 442D, with N-(4-((4-(3-phenylureido)phenyl)sulfonyl)phenyl)benzene-sulfonamide (99.69% purity ) in DMSO, the sensitisation potential of the test item was assessed on the basis of the activation of keratinocytes using the in vitro KeratinoSens™ cell line. Cells were incubated with the test item for 48 h at 37 °C and later checked for luciferase activity. Sensitisation was scored by measuring maximum luciferase activity induction (Imax), cell viability and EC1.5. For both experiments 1 and 2 no significant luciferase induction was observed within the tested concentration range. Therefore, no EC1.5 value could be calculated. Based on the results, the test item N-(4-((4-(3-phenylureido)phenyl)sulfonyl)phenyl)benzene-sulfonamide is considered to be a non-sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

To assess the skin sensitization potential of the target substance several studies were conducted according to current scientifically accepted guidelines.

In an in chemico direct peptide reactivity assay (DPRA) for skin sensitization (OECD TG 442C), 100 mM of the test item N-(4-((4-(3-phenylureido)phenyl)sulfonyl)phenyl)benzenesulfonamide (purity 98.78%) were incubated with the cysteine and lysine peptide solutions at ratios of peptide to test item of 1:10 (cysteine peptide Ac-RFAACAA) and 1:50 (lysine peptide Ac-RFAAKAA). The reaction solutions were incubated in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis.

In the test item samples of experiments with the cysteine peptide and the lysine peptide, precipitation was observed after the incubation period. In the lysine experiment, no HPLC analysis of the precipitation supernatant could be performed as a result of strong precipitation. In the cysteine experiment, HPLC analysis of the supernatants was performed after centrifugation. The mean depletion of the cysteine peptide was below the threshold for discrimination between sensitizers and non-sensitizers of 6.38% (0.00%). According to the OECD test guideline a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result under conditions of precipitation.

Based on the results of this study, no prediction about the sensitizing potential of the test item can be made.

In an in vitro skin sensitization assay conducted according to OECD 442D (addressing the second molecular key event of the adverse outcome pathway (AOP) for skin sensitization), the test item N-(4-((4-(3-phenylureido)phenyl)sulfonyl)phenyl)benzene-sulfonamide (purity 98.78%) was dissolved in DMSO and incubated at concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49 μM for 48 h at 37 °C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In two independent experiments, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. The controls confirmed the validity of the study. The positive control cinnamic aldehyde (CA) induced the appropriate response by showing a significant ≥2-fold induction of luciferase activity (3.10-fold at 64 µM) at a relative cell viability ≥70% (102.8%).

Under the condition of this study, the test item is therefore considered as non-sensitizer.

In an in vitro Genomic Allergen Rapid Detection (GARD™)-Assay for skin sensitization, 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 (SenzaCells) at concentrations of 120 to 140 µM for 24 ± 0.5 hours 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.

All three main stimulations fulfilled the criteria for cytotoxicity. 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 this in vitro GARD™ Test.

As the beforementioned tests, which cover all three key events on the adverse outcome pathway leading to skin sensitization, provided contradictory results and were not sufficient for the classification of the test item an in vivo dermal sensitization study was conducted according to OECD TG 429.

In this study, four young adult female CBA/CaOlaHsd mice per dose group were dermally exposed to N-(4-((4-(3-phenylureido)phenyl)sulfonyl)phenyl)benzenesulfonamide (purity 99.68%) in DMSO at concentrations of 5%, 10% and 25% (w/w) by topical application to the dorsum of each ear for three consecutive days and were observed until day 6.

The positive control substance exceeded not the stimulation index of 3 in the first experiment (2.8). Therefore, a second main assay was performed with two dose groups (25%, 5%). The SI value for the positive control was 7.9 in the second main assay and therefore confirmed the validity of the assay.

There was no mortality, clinical signs, indication of local irritation at the application site (no erythema or swelling) or test item-related effects on the body weight.

The SI values were 1.0, 0.8 and 0.6 at test item concentrations of 25%, 10% and 5% (w/v), respectively in the first main assay. The SI values were 1.2 and 1.0 at concentrations of 25% and 5% (w/v), respectively in the second main assay. Therefore, an EC3 value could not be calculated. In this study, the test item is not a dermal sensitizer.

By assessing all available results in a weight-of-evidence approach it can be concluded that the test item can be considered as not skin sensitizing.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

By assessing all available results in a weight-of-evidence approach it can be concluded that the test item can be considered as not skin sensitizing in accordance with CLP Regulation 1272/2008.