[No public or meaningful name is available]

Eye irritation

Administrative data

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

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 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)

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
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.

Test animals / tissue source

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.

Test system

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

Results and discussion

In vitro

Results

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.

Applicant's summary and conclusion

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.