4-[(2E)-3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-[(4R)-2-ethyl-3-oxo-1,2-oxazolidin-4-yl]-2-methylbenzamide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

Juyl 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-[3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Molecular formula:

C23H18Cl2F4N2O4

IUPAC Name:

4-[3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Test material form:

solid

Details on test material:

Off-white

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Details on animal used as source of test system:

EpiSkinTM (Manufacturer: SkinEthic, France, Batch No.: 18-EKIN-008, Expiry Date: 26 February 2018) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum

Justification for test system used:

EpiSkin model has been validated for corrosivity testing in an international validation study and its use is recommended by the relevant OECD guideline 431.

Vehicle:

physiological saline

Details on test system:

Preparation: adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Units: EpiSkinTM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EpiSkinTM biopsy punch for easy sampling of epidermis
A flask of sterile “Maintenance Medium” (Batch No.: 18 MAIN3 008; Exp. Date: 28 February 2018)
A flask of sterile “Assay Medium” (Batch No.: 18 ESSC 007; Exp. Date: 28 February 2018)

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg , then 100 µL physiological salline was added

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

Duration of treatment / exposure:

Incubation for 4 hours

Duration of post-treatment incubation (if applicable):

3 hours after MTT solution was added to each well below the skin units

Number of replicates:

Two

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean cell viability of two discs

Value:

64.6

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results of the in vitro EpiSkin corrosivity assay show that the substance is non-corrosive to skin.

Executive summary:

An in vitro skin corrosivity test was conducted under GLP to OECD TG 431, using a reconstructed human epidermis model. Disks of the EpiSkin model (two units) were treated with powdered test material (20 mg per unit) and 100 µL of physiological saline and then incubated for 4 hours at room temperature. Exposure was terminated by rinsing the units with phosphate buffered saline solution. The cell viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically at 570 nm. The two negative control epidermis units were treated with physiological saline (0.9% w/v NaCl solution), and the two positive control units were treated with glacial acetic acid. Two additional disks were used to provide an estimate of colour contribution from the test item. For each treated tissue the cell viability was expressed as the percentage relative to the negative control. The cell viability in the units treated with the test substance was 64.6%, which is above the threshold of 35%. The substance wsa considered to be non-corrosive to skin.