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

Endpoint summary

Description of key information

The in-vitro BCOP study (OECD 437) proved that the test item is not classified according to Category 1 for eye irritation/corrosion. The result for this study is, that no prediction on the classification can be made.

The in-vitro RhCE study (OECD 492) gives clear result in case no eye irritation/corrosion is expected. In the present study, the result was that the test item needs to be classified.

Based on QSAR (Nexus DEREK and Toxtree) no alerts on eye irritation/corrosion were identified. At the same time CLP guidance states in section 3.3.2.1.5. “(Q)SARs are in general not very specific for eye irritancy.” And “In the absence of any other existing data, conclusions on the presence or absence of an effect can be made if the (Q)SAR or expert system has been shown to make an adequate prediction. The suitability of the model (reliability, relevance) should be very carefully checked to make sure that the prediction is fit for purpose, and the applicability of the model to the substance should also be justified. The predicted endpoint should be adequate for classification and labelling. In case of negative QSAR data the need for classification cannot be excluded.”

Because of the fact, that OECD 437 does not support Category 1 classification of the test item, while the results of the OECD 492 indicate that the test item needs classification, the subsequent classification for eye irritation must be GHS Category 2.

For skin irritation/corrosion the test item was "Non-irritant"

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 February 2020 to 14 April 2020

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

(2019)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: The EPSKIN TM model is a three dimensional reconstructed human epidermis model consisting of adult human derived epdermal keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDermTm SIT kit:
Manufacturer MatTek Corporation
receipt date: 2 March 2020
Components: EpiDerm tissue (Lot Number 32183), assay medium (Lot Number: 022620TVKD), Nylon mesh (Lot Number: 0551428-00), Phosphate buffered saline without Ca2+ and MG2+, 5% SDS solution
The tissue insert and the medium were stored in a cold place. Nylon mesh and phosphate buffered saline without Ca2+ and Mg2+ were stored at room temperature.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg test substance or 30µL of the control substances respectively per tissue

Duration of treatment / exposure:

Incubation of all plates for 35 +/- 1 minute and until 60 minutes at room temperature afterwards.

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 per control group (Test substance, negative control and positive control)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

positive control

Value:

2.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean Negative Control

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean test item

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Acceptable criteria of the test were met:

Mean OD in the negative control substance group is >= 0.8 and =< 2.8

Mean cell viability in the positive control substance group is <10%

SDs of cell viablities in each treatment group used three tissue inserts are 0< 18%.

 

In a preliminary test it was confirmed that the test item has no reactivity with MTT. Therefore the interference of the test substance with MTT was not evaluated in the skin irritation test.

As a result of tissue-binding test, the staining ratio was 02% whic is く 5%. Therefore, correction of OD was not conducted.
The mean OD in the negative control was l.809. The mean cell viability in the
 positive control substance was 2.2%. The SDs of cell viabilities in the negative and the positive control substances, and the test substances were 4.9% , 0.1% and 3.4 %, respectively. These results indicated thatt the present study was appropriately performed. As a result of skin irritation test, the cell viability treated by the test item was 100.0%, exceeding 50%, which is the judgement criteria of skin irritation.

 

Interpretation of results:

GHS criteria not met

Conclusions:

It was concluded that the test item was "Non-irritant" (UN GHS Category: not classified (including UN GHS Category 3).

Executive summary:

The ability of the test item to induce skin irritation was investigated using EpiDerm^TM SIT (EPI-200). As a result of the skin irritation test, the cell viability treated with the test item was 100.0% exceeding 50% which is the judgement criteria of skin irritation. Consequently, it was concluded that the test item was "Non-irritant" (UN GHS Category: not classified (including UN GHS Category 3).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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

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)

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.

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.

Irritation parameter:

other: Permeability

Run / experiment:

run 1

Value:

0.261

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Permeability

Run / experiment:

run 2

Value:

0.375

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Permeability

Run / experiment:

run 3

Value:

0.563

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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

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

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

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

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

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

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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 February 2020 to 2 March 2020

Reliability:

2 (reliable with restrictions)

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

(2019)

Principles of method if other than guideline:

The test method is only recommended in case non classification is expected.

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

TTEST KIT
Name: Epiocular™ EIT kit

Manufacturer: MatTek Corporation

Receipt date: March 2, 2020

Components: Epiocular tissue(tissue insert, Lot number: 28052, manufactured on February 27, 2020)
Assay medium (medium, Lot number: 022420ALC)
Methyl acetate
Phosphate buffered saline without Ca2+ and Mg2+ (PBS(-), Lot number: 111219RAHA)

Reason for selection:
Epiocular™ EIT kit is recommended in the test method.
Storage condition:
The tissue insert and the medium were stored in a cold place in the cell experimental
room No. 1 (permissible range: from 1 °C to 10°C). PBS(-) was stored at room
temperature in the cell experimental room No. 1.

Culture Condition (setting value):

Incubator: C02 incubator (MCO-18AIC, SANYO Electric)
Temperature: 37°C
Humidity: Under humid condition
C02 concentration 5%

Buffer Solution, Medium Containing MTT Solution and MTT Extraction Solvent

Buffer solution: PBS(-)

Medium containing MTT solution:

Preparation method:
3-( 4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Lot
number: NW129, for research, DOJINDO Laboratories) was dissolved in PBS(-) to
prepare 5 mg/mL MTT solution. This solution was diluted with the medium to
prepare medium containing 1 mg/mL MTT solution (MTT medium).

Timing of preparation and storage condition:
MTT medium was prepared just before use. MTT medium was stored at room
temperature under light shielding until use.

MTT extraction solvent:
2-Propanol (Lot number: KCG0694, special grade, FUJIFILM Wako Pure Chemical)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

20 µl of PBS (-) was added onto each tissue
50 µl control substance or 50 mg test substance

Duration of treatment / exposure:

6 hours +/- 15 min (incubation)

Duration of post- treatment incubation (in vitro):

after incubation each tissue was rinsed with PBS(-). After rinsing, PBS(-) was removed from the tissue surface. Afterwards the tissue inserts were transferred into 6-well plates filled with 1 mL/well of the frest medium and incubated for 18 hours. +/-15 min.

Number of animals or in vitro replicates:

2 replicates per negative control, positive control and test substance.

Details on study design:

PRELIMINARY TEST
a) Test for reactivity with MTT
Fifty milligrams of the test substance and 1 mL of MTT medium were mixed, the
mixture was incubated for 180 minutes. After the incubation, the change in color of the MTT medium was evaluated. As a result, the change in color was not observed and it was judged that the test substance had no reactivity with MTT. Therefore, interference of the test substance with MTT (interference test) was not evaluated in the eye irritation test.
EYE IRRITATION TEST
Duplicate tissue inserts were used for the test substance, negative control substance and
positive control substance, respectively. Duplicate tissue inserts were used to check the
tissue-binding of the test substance (tissue-binding test).
a) Pre-incubation
1) Tissue inserts were placed in 6-well plates (AGC TECHNO GLASS) filled with 1
mL/well of the medium and incubated for 60 ± 5 minutes.
2) The medium was aspirated, and 1 mL of the fresh medium was added to each well.
Then, the tissue inserts were incubated for 16 hours to 24 hours.
b) Exposure of the test substance
1) Twenty microliters of PBS(-) was added onto each tissue surface at 1 minute interval.
Two tissue inserts were treated at once. Each plate was incubated until 3 0 ± 2
minutes was completed for the first added tissue insert in each plate.
2) Fifty microliters of the control substances or 50 mg of the test substance were applied
onto each tissue surface at 1-minute interval. Two tissue inserts were treated at once.
Each plate was incubated until 6 hours ± 15 minutes was completed for the first
exposed tissue inserts in each plate.
c) Rinsing and post-incubation
1) After the incubation, each tissue insert was completely submerged three times per
beaker into three beakers filled with about 100 mL/beaker of PBS(-) for rinsing.
After rinsing, PBS(-) was removed from the tissue surface.
2) The tissue inserts were transferred into a 12-well plate (Coming) filled with 5
mL/well of the fresh medium and soaked for 25 ± 2 minutes. After the soak, the
medium was removed from the tissue surface.
Microplate Reader (FLUOstar Omega, BMG LABTECH) at 570 nm.

Irritation parameter:

percent tissue viability 

Run / experiment:

1

Value:

2.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

study cannot be used for classification

Remarks:

The study is only recommended to identify substances not requiring classification and labelling for eye irritation or serious eye damage. It is not recommended for the identification of chemicals which should be classified for eye irritation or serious eye damage.

Conclusions:

It was concluded that the test item was eye irritant under the present test conditions. However it cannot be excluded that it is a false positive result, as the study design is only recommended for substances where no classification for eyer irritation or serious eye damage is required.

Executive summary:

As a result of the tissue-binding test, the staining ratio was 0.5% that was <= 60%. However, the mean cell viability in the test substance group was <= 60%. Therefore, correction of the cell viability was not conducted.

The mean 0D in the negative control substance group was 1.495. The mean cell viability in the positive control substance group was 24.3%. The differences of two cell viabilities in the negative and the positive control substances, and the test substance were 3.8%, 6.7% and 0.0%, respectively. These results indicated that the present study was appropriately performed.

As a result of the eye irritation test, the cell viability treated with the test item was 2.5%, falling equal or below 60% which was the judgement criteria of eye irritation.

Reference 3

Endpoint:

eye irritation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

Three QSAR models; Toxtree , Nexus Derek (and Consensus Prediction) did not indicate any alerts on eye irritation/corrosion.

Qualifier:

no guideline followed

Principles of method if other than guideline:

QSAR prediction

Specific details on test material used for the study:

CC1=CC=C(C=C1)S(=O)(=O)NC(=O)NC2=CC=CC(=C2)C(F)(F)F

Run / experiment:

other QSAR

Remarks on result:

other: No indication of eye irritation/corrosion based on QSAR prediction

Interpretation of results:

study cannot be used for classification

Conclusions:

QSAR did not indicate eye irritation/corrosion. However QSARs are in general not very specific for eye irritancy.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)