4-Methyl-N-[[[3-(trifluoromethyl)phenyl]amino]carbonyl] benzenesulfonamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28-January-2022 to 3-June-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

2010

Deviations:

no

Test guideline 2

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:

2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-Methyl-N-[[[3-(trifluoromethyl)phenyl]amino]carbonyl] benzenesulfonamide

EC Number:

850-929-8

Cas Number:

1584-79-8

Molecular formula:

C15H13F3N2O3S

IUPAC Name:

4-Methyl-N-[[[3-(trifluoromethyl)phenyl]amino]carbonyl] benzenesulfonamide

Test material form:

solid

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Freshly isolated bovine cornea (12 - 24 months old donor cattle)
The isolated eyes were stored in HBSS (Hank’s Balanced Salt Solution) containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughterhouse and during transportation on the same morning to the laboratory using a Styrofoam box. The corneas were isolated on the same day after delivery of the eyes.

Test system

Vehicle:

physiological saline

Remarks:

The test item was tested as a 20% suspension (w/v) in saline using sonication for 10 minutes.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The anterior compartment received the test item suspension or the negative or positive controls at a volume of 0.75 mL each on the surface of the corneas via open chamber method, respectively. The corneas were incubated in a horizontal position at 32 ± 1 °C in the water-bath.

Duration of treatment / exposure:

The incubation period is 240 minutes.

Duration of post- treatment incubation (in vitro):

After exposure, the test item or the control items, respectively, were each rinsed off from the according application sides with EMEM containing phenol red for at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible. Since the test item proved difficult to remove by the rinsing method, the front cover of the holder was opened and the cornea was carefully washed using a gentle stream of incubation medium. Once the medium was free of the test item the corneas were given a final rinse with cMEM without phenol red. Fresh cMEM was added into both compartments and opacity was measured (t240).

Number of animals or in vitro replicates:

Negative Control 1: Saline (0.9% NaCl in deionised water) : 3 Replicates
Negative Control 2: Deionized water: 3 Replicates
Positive Control 1: 10% (w/v) Benzalkonium chloride in saline using sonication: 3 Replicates
Positive Control 2: 20 % (w/v) Imidazole in saline: 3 Replicates

Details on study design:

All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. The corneas were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437.
For equilibration, the corneas in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed before basal opacity measurement (t0).
Only corneas with a value of the basal opacity < 7 were used. Sets of three corneas were used for treatment with the test item and for the negative and positive controls, respectively.
The opacitometer determines changes in the light transmission passing through the corneas and displays a numerical opacity value. This value was recorded in a table. The opacitometer was calibrated and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
Following to the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneas were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate.
The optical density was measured with a microplate reader at 490 nm (OD490). The absorbance values were determined using software.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Permeability

Run / experiment:

run 1

Value:

0.261

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: Permeability

Run / experiment:

run 2

Value:

0.375

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

other: Permeability

Run / experiment:

run 3

Value:

0.563

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

run 1

Value:

9.59

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Results 5

Irritation parameter:

in vitro irritation score

Run / experiment:

run 2

Value:

9.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Results 6

Irritation parameter:

in vitro irritation score

Run / experiment:

run 3

Value:

12.12

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Results 7

Irritation parameter:

cornea opacity score

Run / experiment:

run 1

Value:

3.67

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Results 8

Irritation parameter:

cornea opacity score

Run / experiment:

run 2

Value:

5.67

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Results 9

Irritation parameter:

cornea opacity score

Run / experiment:

run 3

Value:

3.67

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction can be made on GHS classification as the mean IVIS was determined to be 10.33.

Executive summary:

No prediction can be made on GHS classification as the mean IVIS was determined to be 10.33.