Benzenesulfonamide, N-(4-hydroxyphenyl)-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15/10/2019 - 11/11/2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

adopted 18 June 2019

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:

20 July 2012

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-(4-hydroxyphenyl)benzenesulfonamide

EC Number:

654-333-7

Cas Number:

5471-90-9

Molecular formula:

C12 H11 N O3 S

IUPAC Name:

N-(4-hydroxyphenyl)benzenesulfonamide

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donor

Source strain:

other:

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM
- Tissue batch number(s): 19-EKIN-043 & 19-EKIN-045
- Production date:
- Shipping date: October 22 2019 & November 5 2019
- Delivery date: October 22 2019 & November 5 2019
- Date of initiation of testing: October 22 2019 & November 5 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C (actual range 35.8 - 37.2°C)
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 35.8 - 37.2°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: /
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: /
- Filter bandwidth: /
- Linear OD range of spectrophotometer: /
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-solution (0.3 mg/ml in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 µl isopropanol (Merck, Darmstadt, Germany).
Tubes were stored refrigerated and protected from light for approximately 67 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: /
- Barrier function: /
- Morphology: /
- Contamination: /
- Reproducibility: /

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test item reacted with the MTT medium, in addition two killed tissues treated with test item and two killed untreated tissues were used for the cytotoxicity evaluation with MTT.
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item.
After rinsing, the cell culture inserts were each dried carefully and moved to a new well on
2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

yes, concurrent positive control

other: two killed tissues treated with test item

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 14.1 to 20.2 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µl PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µl 5% SDS

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item.
After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Number of replicates:

3 tissues per test item together with negative and positive controls

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 101

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

SD = 6.8%

Other effects / acceptance of results:

The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because a color change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint.
In addition to the normal procedure, two killed tissues treated with test item and two killed non treated tissues were used for the cytotoxicity evaluation with MTT.
The non-specific reduction of MTT (% NSMTT) by the test item was 7.6% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 22 ± 25 %.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically (6.8%) was less than 18%

The standard deviation of the viability of the positive control was 25% which is above the acceptability criteria (18%).
Evaluation: The high standard deviation is caused by one of three skin tissues treated with the positive control that showed a higher value. The OD value of this deviating skin tissue (0.309) is well within the historical data range for the positive control (0.023 – 0.449), and outside the range for the negative control (0.422 – 1.547). Additionally, standard deviations of the negative control (of which the OD values are used for the calculations) and test item treated tissues are all acceptable. Therefore, this deviation had no influence on the integrity of the study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, CH03951 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate CH03951 for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM). The possible skin irritation potential of the test item was tested through topical application for
15 minutes. The study procedures described in this report were based on the most recent OECD 439 (2019) and EC 440 (2008) guidelines.

Batch CH03951/ME of the test item was an off white powder. Skin tissue was moistened with 5 μL of Milli-Q water and at least 10 mg of the test item was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Therefore, in addition to the normal procedure, two killed tissues treated with test item and two killed untreated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT (% NSMTT) by the test item was 7.6% of the negative control tissues. The net OD of the treated killed tissues was then subtracted from the ODs of the test item treated viable tissues.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 101%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

The positive control had a mean cell viability of 22% after 15 ± 0.5 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 7% for the negative control and the test item. The standard deviation of the viability of the positive control was 25% (see deviation).

In conclusion, CH03951 was non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.