[No public or meaningful name is available]

Endpoint summary

Description of key information

The potential of the test item to induce skin irritation (OECD 439, 431) and eye irritation (OECD 437) was tested in suitable in vitro test methods. Based on the results, the target substance can be considered as non-irritant to the skin and eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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)

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.

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

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

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

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

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-02-21 to 2020-07-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 31 July 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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
Firstly, 25 µL of sterile DPBS were applied to the epidermal surface in order to improve the contact between the powder and the epidermis. Afterwards, 25 mg (39 mg/cm²) of the test item were applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™-Standard Model (EPI-200-SIT, MatTek)
- Tissue batch number(s): 30849 Kit C

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.: 30849 Kit C)
- 2x 24-well plates
- 8x 6-well plates
- 1x bottle of assay medium (DMEM-based medium, Lot No.: 022020MJD)
- 1x bottle of DPBS Rinse Solution (Lot No.: 112719ISE)
- 1x 1 vial 5% SDS Solution (TC-SDS-5%)
- 25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: At RT for the first 25 ± 1 min, afterwards the plates were placed into the incubator for 35 ± 1 min.
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL DPBS
- Lot No.: 2124835

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL SDS solution
- Concentration (if solution): 5%
- Lot No.: 110519MSA

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours (The plates were post-incubated for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h.)

Number of replicates:

The test was performed on a total of 3 tissues per dose group, the tissues were treated with each dose group in triplicate.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three tissues

Value:

96.3

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 see box "Any other information on results incl. tables".

Pre-Experiments:

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%. The mixture of 25 mg of the test item per 300 µL aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 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.

Results of the main experiment:

Table 1: Results of the main experiment

Name	Negative Control****			Positive Control			Test Item
Replicate Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	2.064	1.864	2.060	0.120	0.116	0.120	1.886	1.835	2.010
	1.972	1.720	1.917	0.112	0.114	0.129	1.800	1.690	1.954
OD570 (Blank Corrected)	2.018	1.818	2.013	0.073	0.069	0.074	1.840	1.788	1.964
	1.926	1.674	1.871	0.065	0.067	0.083	1.753	1.643	1.908
Mean OD570 of the Duplicates (Blank Corrected)	1.972	1.746	1.942	0.069	0.068	0.078	1.796	1.716	1.936
Total Mean OD570 of 3 Replicate Tissues (Blank Corrected)	1.887*			0.072			1.816
SD of Mean OD570 of 3 Replicate Tissues (Blank Corrected)	0.123			0.005			0.111
Relative Tissue Viability [%]	104.5	92.5	102.9	3.7	3.6	4.1	95.2	90.9	102.6
Mean Relative Tissue Viability [%]	100.0			3.8**			96.3
SD of Relative Tissue Viability [%]***	6.5			0.3			5.9
CV of Relative Tissue Viability [%]	6.5			7.5			6.1

* Blank-corrected mean OD_{570 nm} of the negative control corresponds to 100% absolute tissue viability.

**Mean relative tissue viability of the three positive control tissues is ≤ 20%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

****The mean absolute OD_{570 nm} of the negative control is ≥ 0.8 and ≤ 2.8).

Table 2: Quality criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nm NC	1.933	0.8 ≤ NC ≤ 2.8	pass
Relative Viability [%] PC	3.8	≤ 20%	pass
SD of Relative Viability [%] (min-max)	0.3 - 6.6	≤ 18%	pass

NC: Negative Control

PC: Positive Control

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro skin irritation study (OECD 439), the test item showed no irritant effects. Therefore, classification as irritant to the skin is not warranted.

Executive summary:

In an in vitro dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT) was topically exposed to to the test item for 60 min and 42 h post incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability of the test item (% negative control) was greater than 50% (96.3%). Based on this result, the test item is considered to be non-irritating to the skin and no classification according to CLP Regulation 1272/2008 is warranted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-03-17 to 2020-05-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 suspended with physiological saline 0.9% NaCl (B. Braun Melsungen, lot no. 19393414, expiry date: 08/2022) to give a 20% concentration.

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories.
- Time interval prior to initiating testing: At day of testing, immediately after arrival of the eyes, cornea preparation was initiated.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.
- Indication of any antibiotics used: Hanks’ balanced salt solution (HBSS) with Ca++ and Mg++ containing penicillin/ streptomycin
- Selection and preparation of corneas: The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. The corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI 1640 medium (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI 1640 medium). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.
- Quality check of the isolated corneas: Before the corneas were mounted in corneal holders, they had been visually examined for defects and any defective cornea had been discarded.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 μL
- Concentration: 20%

VEHICLE
- Amount(s) applied: 750 µL
- Concentration: 0.9% NaCl
- Lot/batch no.: B. Braun Melsungen, lot no. 19393414, expiry date: 08/2022

Duration of treatment / exposure:

4 hours ± 5 minutes

Duration of post- treatment incubation (in vitro):

The optical density at 490 nm was measured upon 90 minutes of incubation with fluorescein after exposure to the test item by using a spectrophotometer.

Number of animals or in vitro replicates:

3 corneas each for the test item, solvent control and positive control

Details on study design:

NUMBER OF REPLICATES : 3 corneas per treatment group

NEGATIVE CONTROL USED : yes, 0.9% NaCl

POSITIVE CONTROL USED : 20% imidazole

APPLICATION DOSE AND EXPOSURE TIME : 750 µL of 20% test item in 0.9% physiological saline for 4 hours ± 5 minutes

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI 1640 medium (without phenol red). The anterior chamber was refilled with complete RPMI 1640 medium and an illuminance measurement was performed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer (BASF-OP3.0, Duratec GmbH). Calibration was performed before the test and is documented in the raw data. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout lay in the range between 540-560 lux.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)
- Others: Following treatment and washing steps, each cornea was observed visually and pertinent observations were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15x mean permeability OD490 value)

DECISION CRITERIA: As indicated in the OECD TG 437:
- IVIS ≤ 3: No Category
- 3 >IVIS≤: No prediciton can be made
- >55: Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of triplicates

Value:

0.57

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None of the corneas treated with N-(4-((4-(3-phenylureido)phenyl)sulfonyl) phenyl)benzene-sulfonamide showed any opacity of the tissue.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.
- Acceptance criteria met for positive control: The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, based on the mean in vitro irritation score of 0.57 obtained in the bovine corneal opacity and permeability assay (BCOP, OECD 437), the test item can be considered to be non-irritant and no classification as eye-irritant is warranted.

Executive summary:

The eye irritation potential of the test item was investigated in the bovine corneal opacity and permeability assay (BCOP, OECD 437). The test item was suspended in 0.9% NaCl to gain a 20% concentration. A mean in vitro irritation score of 0.57 was determined. The positive and negative controls induced the appropriate responses, indicating the validity of the assay. Based on the results obtained, no classification as eye-irritant is warranted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

To assess the skin corrosion potential of the target substance, an in vitro skin corrosion study was conducted according to OECD guideline 431. The test item was applied topically to the EpiDerm 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. Based on the results, the test item can be considered as non-corrosive in conclusion with CLP Regulation 1272/2008. To assess the skin irritation potential of the target substance an in vitro skin irritation study was conducted according to OECD TG 439. The test item showed no irritant effects and the mean relative tissue viability (% negative control) was > 50% (96.3%) after 60 min treatment and 42 h post-incubation.

By assessing the results from both in vitro studies, it can be concluded that no classifcation for skin irritation is warranted.

For the investigation of the eye damaging potential of the test item, an in vitro Bovine Corneal Opacity and Permeability (BCOP) Test (OECD TG 437, closed-chamber method) was performed. Treatment with the test item did not cause opacity in any of the treated corneas. The mean in vitro irritation score was calculated as 0.57, which suggests that the test substance does not require classification for eye irritation or serious eye damage.

Justification for classification or non-classification

Based on the results obtained from suitable in vitro studies conducted according to OECD test guidelines, no classification for skin and eye irritation is warranted according to CLP Regulation 1272/2008.