[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-12-20 to 2020-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted 18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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
25 mg of the test item were applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item. To ensure good contact with the skin the test item was moistened with 25 µL H2O. The test item was spread to match size of the tissue.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

Not applicable

Justification for test system used:

The EpiDerm Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm (TM) (MatTek)
- Tissue batch number(s): 30844
Epiderm Kit:
The EpiDerm tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
- 1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm²); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot No.: 30844)
- 2x 24-well plates
- 4x 6-well plates
- 1x bottle of assay medium (DMEM-based medium, Lot No.: 011620MJB)
- 1x bottle of DPBS Rinse Solution (Lot No.: 112719ISE)
- 25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)

FURTHER REAGENTS
- MTT stock solution: 5 mg/mL MTT (VWR; Lot 18I1156332) in PBS (Gibco; Lot No.: 2098592)
- MTT medium: MTT stock solution was diluted 1 + 4 with DMEM-based medium (final concentration 1 mg/mL)
- Isopropanol (AppliChem; Lot No.: 0001815947)
- Aqua dest. (Sigma Aldrich; Lot No.: RNBH8991 (pre-test), RNBG3520 (main test))

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 60 min exposure: 37 ± 1 °C; 3 min exposure: RT
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C (for 3 h, 5.0% CO2 / 95% air)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps:
At the end of exposure each tissue was rinsed about 20 times with PBS by filling and emptying the tissue insert. Excess liquid was carefully removed and transferred into new wells pre-filled with 0.3 mL/well pre-warmed MTT solution.
After 3 h MTT incubation tissues were rinsed twice in PBS and dried. Then the inserts were transferred into 12-well “extraction plates“. 2 mL of isopropanol were pipetted into each insert, thus the insert was covered from both sides. The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out either over night without shaking at room temperature or, alternatively, at least 2 h with shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts were discarded and the extraction plates were placed on a shaker for 15 min.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT stock solution: 5 mg/mL MTT (VWR; Lot 18I1156332) in PBS (Gibco; Lot No.: 2098592); MTT medium: MTT stock solution was diluted 1 + 4 with DMEM-based medium (final concentration 1 mg/mL)
- Incubation time: 3 h min at 37 ± 1 °C
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- To check the MTT-reducing capability of the test item, 25 mg of the test item were mixed per 1 mL MTT medium and incubated for 1 h at 37 ± 1 °C, 5.0% CO2 / 95% air. The mixture did not turn blue/purple. Thus, the additional test with freeze-killed tissues and the quantitative corrections were not necessary.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg + 25 μL H2O

NEGATIVE CONTROL
- Amount(s) applied: 50 μL distilled water
- Lot/batch no.: RNBG3520, Sigma

POSITIVE CONTROL
- Amount(s) applied: 50 µl KOH
- Concentration: 8N
- Lot/batch no.: B1041112511, Merck

Duration of treatment / exposure:

3 min and 60 min

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

two

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes/mean of two replicates

Value:

101.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes/mean of two replicates

Value:

76.7

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:

For detailed results please refer to box "Any other information on results incl. tables".

OTHER EFFECTS:
Pre-experiments:
The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT is determined to be 0%. The mixture of 25 mg test item per 300 µL Aqua dest. and per 90 µL isopropanol showed no colouring as compared to the solvent. Therefore, NSC is determined to be 0%. The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with Aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Table 1: Acceptance Criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nm NC (3 min Experiment)	1.708	0.8 ≤ NC ≤ 2.8	pass
Mean Absolute OD570 nm NC (60 min Experiment)	1.789	0.8 ≤ NC ≤ 2.8	pass
Mean Relative Tissue Viability [%] of PC (60 min experiment)	6.8	< 15%	pass
CV [%] (in the range of 20 – 100% viability)	0.6 – 9.8	≤ 30%	pass

NC: negative control

PC: positive control

Table 2: Results of 3 min Experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.578	1.844	0.195	0.341	1.719	1.768
	1.600	1.808	0.188	0.330	1.695	1.712
	1.598	1.818	0.190	0.336	1.724	1.755
Mean Absolute OD570	1.708****		0.263		1.729
OD570- Blank Corrected	1.530	1.796	0.147	0.293	1.671	1.720
	1.553	1.760	0.140	0.382	1.647	1.664
	1.550	1.770	0.142	0.288	1.676	1.707
Mean OD570 of 3 Aliquots (Blank Corrected)	1.544	1.775	0.143	0.288	1.665	1.697
SD OD570 of 3 Aliquots	0.012	0.018	0.004	0.005	0.016	0.029
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.660*		0.215		1.681
SD OD570 of 2 Replicate Tissues	0.163		0.102		0.023
Mean Relative Tissue Viability [%]	100.0		13.0		101.3
Coefficient Of Variation [%]***	9.8		47.5		1.4

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.

****The mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.8

Table 3: Results of 60 min Experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.786	1.789	0.168	0.163	1.475	1.317
	1.797	1.800	0.170	0.168	1.442	1.335
	1.806	1.757	0.165	0.160	1.421	1.315
Mean Absolute OD570	1.789****		0.166		1.384
OD570- Blank Corrected	1.738	1.741	0.120	0.115	1.427	1.269
	1.750	1.752	0.123	0.120	1.394	1.287
	1.759	1.709	0.117	0.112	1.373	1.268
Mean OD570 of 3 Aliquots (Blank Corrected)	1.749	1.734	0.120	0.116	1.398	1.275
SD OD570 of 3 Aliquots	0.010	0.023	0.003	0.004	0.027	0.011
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	1.741*		0.118		1.337
SD OD570 of 2 Replicate Tissues	0.010		0.003		0.087
Mean Relative Tissue Viability [%]	100.0		6.8**		76.7
Coefficient Of Variation [%]***	0.6		2.5		6.5

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

** mean relative tissue viability of the 60 min positive control < 15%

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.

**** The mean absolute OD570of the negative control is ≥ 0.8 and ≤ 2.8

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, based on the results obtained from this in vitro skin corrosion study (OECD 431), the test item is considered to be non-corrosive (UN GHS Category 1B or 1C).

Executive summary:

In an in vitro skin corrosion study conducted according to OECD guideline 431, the test item was applied topically to the EpiDerm^TM tissue for 3 min and 60 min followed by immediate determination of cytotoxic effects via MTT reduction assay.

Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after both treatment periods had been compared to the corresponding negative control tissues. The mean relative tissue viability (% negative control) was above 15% (76.7%) after 60 min treatment, and above 50% (101.3%) after 3 min treatment. The positive and negative controls confirmed the validity of the study. Based on the results, the test item can be considered as non-corrosive in conclusion with CLP Regulation 1272/2008.