3-[(phenylcarbamoyl)amino]phenyl 4-methylbenzenesulfonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2018 - 18 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

6 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-(3-phenylureido)phenyl 4-methylbenzenesulfonate

Cas Number:

2102522-55-2

Molecular formula:

C20H18N2O4S

IUPAC Name:

3-(3-phenylureido)phenyl 4-methylbenzenesulfonate

Test material form:

solid: particulate/powder

Details on test material:

Appearance: Off-white powder

Specific details on test material used for the study:

Storage conditions: Room temperature (15-25 °C, ≤ 70 % relative humidity (RH)).

The test material was applied as supplied (although the test material was ground into a powder).

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN™ (SM) three-dimensional human epidermis model.

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ (SM) Kit
- Source: SkinEthic, France
- Tissue lot number: 18-EKIN-020
- Expiry date: 21 May 2018

TEST FOR DIRECT MTT REDUCTION
- Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test material is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test material interference with the viability measurement.
- Check-method for possible direct MTT reduction with test material: 10 mg of powdered test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded.
- If the MTT solution containing the test material turns blue/purple, the test material is presumed to have reduced the MTT.
- After three hours of incubation, yellow colour of the mixture was detected in the test tube. The test material did not react with MTT and therefore the use of additional controls was not necessary.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or extracting solution (e.g. acidified isopropanol) (simulating a tissue humid environment). As the test material had an intrinsic colour, further evaluation to detect colouring potential was necessary. Non-specific Colour % (NSCliving %) was determined in order to evaluate the ability of test material to stain the epidermis by using additional control tissues.
- Therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non-specific OD evaluation. These tissues followed the same test material application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature - 22.0 - 24.3 °C
- Temperature of post-treatment incubation: 37 °C

PRE-INCUBATION (Day [-1])
- The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37 °C in an incubator with 5 % CO2, in a > 95 % humidified atmosphere.

APPLICATION AND RINSING (Day 0)
- Test material: First an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis and then 10 mg of powdered test material was applied evenly to the epidermal surface. The test material was spread gently on the skin surface with a pipette tip without damaging the epidermis. The amount was sufficient to cover the epidermal surface.
- Negative and positive controls: 50 μL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette.
- The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature (22.0-24.3°C).
- After the 15 minutes incubation time, the EPISKIN™ (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
- After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (+ 10 minutes) at 37 °C in an incubator with 5 % CO2, in a > 95 % humidified atmosphere.

MTT TEST (Day 2)
After 42 hours incubation, all EPISKIN™ (SM) units (except the two living colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKIN™ (SM) units were incubated for 3 hours at 37°C in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere.

FORMAZAN EXTRACTION (Day 2)
- After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
- The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

CELL VIABILITY MEASUREMENTS (Day 2)
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

NUMBER OF REPLICATE TISSUES
In this assay, three replicates were used for the test material. Three negative controls and three positive controls were also run in the assay. Furthermore, as the test material was coloured, two additional test material-treated living tissues were used for the non-specific OD evaluation.

DATA EVALUATION
- The test material is considered to be corrosive to skin if the mean relative viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours post incubation is less or equal (≤) to 50 % of the mean viability of the negative controls (Category 2 or Category 1).
- The test material is considered to be non-corrosive to skin if if the mean relative viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours post incubation is more than (˃) to 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg of the test material was applied evenly to the epidermal surface. The amount was sufficient to cover the epidermal surface.

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 5 % w/v

Duration of treatment / exposure:

15 minutes.

Duration of post-treatment incubation (if applicable):

Incubated at 37 °C for 42 hours followed by 3 hours with MTT

Number of replicates:

Three replicates were used for the test material. Three negative controls and three positive controls were also run in the assay. Furthermore, as the test material was coloured, two additional test material-treated living tissues were used for the non-specific OD evaluation.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Run / experiment:

Test material

Value:

97.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ADDITIONAL CONTROLS
- As no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution, the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
- As the test material was coloured (off-white), two additional test material-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues was 0.011, Non Specific Colour % was calculated as 1.4 %. This value was below 5%, therefore additional data calculation was not necessary.

VIABILITY RESULTS
The mean OD values for the test material treated skin samples showed 97.4% relative viability compared to the negative control.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues was in the recommended range (0.753). Standard deviation of the viability results for negative control samples was 2.0 %.
- Acceptance criteria met for positive control: The positive control treated tissues showed 9.7 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 2.6 %.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of viability values of the three test material-treated tissue samples in the MTT assay was 3.2 %.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.

All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Any other information on results incl. tables

HISTORICAL CONTROL DATA

	Negative control (PBS)	Positive control (5 % (w/v) SDS solution)
Mean optical density (OD)	0.788	0.065
Standard deviation	0.129	0.041
Minimum optical density (OD)	0.573	0.019
Maximum optical density (OD)	1.362	0.354
Number of cases	251	246

PBS: Phosphate buffered saline

SDS: Sodium dodecyl sulphate

OD: Optical density (absorbance)

Note: All OD values (measured at 570 ± 30 nm) are background corrected values.

Applicant's summary and conclusion

Interpretation of results:

other: not classified according to EU criteria

Conclusions:

Under the conditions of this study the test material is non-irritant to skin.

Executive summary:

An in vitro skin irritation study was conducted in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions using the EPISKIN™ reconstructed human epidermis model.

During the study, disks of EPISKIN™ (SM) (three units) were treated with the powdered test material and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2, in a > 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2, in a > 95 % humidified atmosphere, protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.
PBS and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test material. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control, the test material is considered to be irritant to skin.
Following exposure with the test material, the mean cell viability was 97.4 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.