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

Endpoint summary

Description of key information

Skin corrosion: no adverse effects in vitro, OECD TG 431, Orovecz 2018

Skin irritation: no adverse effects in vitro, OECD TG 439, Orovecz 2019

Eye irritation: no adverse effects in vitro, OECD TG 438, Orovecz 2019

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:

key study

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:

Juyl 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

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

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.

Reference 2

Endpoint:

skin irritation: 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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:

EpiSkin (Manufacturer: SkinEthic, France, Batch No.: 19-EKIN-029, Expiry Date: 22 July 2019) 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 (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Justification for test system used:

The EpiSkin has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

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
Medium: A flask of sterile “Maintenance Medium” (Batch No.: 19 MAIN3 032; Exp. Date: 24 July 2019)
A flask of sterile “Assay Medium” (Batch No.: 19 ESSC 030; Exp. Date: 24 July 2019)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

A volume of 10 μL of distilled water was applied to the epidermal surface to improve the contact of the test substance with the epidermis. Then, an aliquot of 10 mg test substance was applied evenly to the skin surface. Aliquots of 20 μL of negative control (phosphate buffered saline) and positive control (5% w/v sodium dodecyl sulphate) were applied evenly to the skin surfaces.

Duration of treatment / exposure:

15 minutes ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours ± 1 hour

Number of replicates:

Three

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Results of three replicates

Value:

>= 88.8 - <= 97.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Mean cell viability was 93.2%

Other effects / acceptance of results:

After receipt, the two indicators included in the package of the delivered kits (reflecting the storage temperature history and the pH) were checked. Based on the observed colours, the epidermis units were in a suitable condition for use in the assay.
The mean OD value of the three negative control tissues was in the recommended range (0.848). Standard deviation of the viability results for negative control samples was 4.1%.
The positive control treated tissues showed 3.7% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.6%.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 4.4%.
The mean OD value of the blank samples (acidified isopropanol) was 0.048.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

The results of this in vitro study indicated that the substance was not irritating to skin under the conditions of the test.

Executive summary:

An in vitro skin irritation study was conducted under GLP to OECD TG 439 in a reconstructed human epidermis model. Three disks each of the EpiSkin model were treated with the test substance, the negative control phosphate buffered saline or the positive control substance sodium dodecyl sulphate (applied as 5% w/v solution). The disks were incubated at room temperature for a duration of 15 minutes. Exposure was stopped by rinsing the units with phosphate buffered saline. The cleaned epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5% CO2 in a >95% relative humified atmosphere. After this incubation, MTT ("Thiazolyl blue") solution was added to the units, which were then incubated for a further 3 hours to determine cell viability. The precipiated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically at 570 nm.

Two additional disks were used to provide an estimate of colour contribution from the test item. For each treated tissue, the viability was expressed as a percentage relative to the negative control. The mean cell viability of the disks treated with the substance was 93.2% compared to the cell viability of the negative control disks. This is clearly above the threshold of 50% viability, indicating that the test substance was not irritating to the skin under the conditions of the test. The mean cell viability of the plates treated with the positive control substance was 3.7%, showing the suitability of the applied test system.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: COBB 500 and ROSS 308

Details on test animals or tissues and environmental conditions:

Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni u 129., Hungary
Collection: heads were collected by a technician from a commercial abattoir after approximately 7 weeks old chicken were slaughtered for human consumption. Heads were inspected for appropriate quality and wrapped with paper moistened with saline and then placed in a sealed plastic box. The heads were then immediately transported to the laboratory at ambient temperature and processed within 2 hours of collection in each experiment.
Eye selection: delivered heads were removed from the plastic box and placed on soft paper. The eyelids were carefully cut away with scissors, avoiding damage of the cornea. One small drop of 2% w/v fluorescin solution was applied onto the cornea surface for a few seconds. The eye was subsequently rinsed with 20 mL of physiological saline. The treated cornea was then examined with a hand-held slit lamp or microscope to ensure that it was undamaged. If the cornea was in a good condition, the eyeball was carefully removed from the orbit.
Preparation of the eye: the eyeball was removed without cutting off the optical nerve too short avoiding pressure on the eye to prevent distortion of the cornea and subsequent corneal opacity. The removed eyeball was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eye was kept on the wet papers in a closed box so that the appropriate humidity was maintained.
Eyes examination and acclimatisation: the prepared eye was placed in a steel retainer. The cornea was positioned vertically with the eye in the correct relative position, by avoiding too much pressure on the eye. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The retainer holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline dripping from a stainless steel tube, at a rate of approximately 3-4 drops per minute or 0.1 to 0.15 mL/minute. The chamber was closed except for manipulations and examinations, to maintain temperature and humidity. The appropriate number of eyes (nine to twelve) were selected, placed in the superfusion apparatus, and examined again with the slit lamp microscope. The focus was adjusted to clearly see the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescine staining or corneal opacity score were rejected. The cornea thickness was measured with an optical pachymeter on a slit-lamp microscope, which was set at a 0.095 mm slit-width. Any eye with cornea thickness deviating by more than 10% from the mean value of all eyes, or eyes showing any other signs of damage were rejected and replaced. Once the selected eyes were appropriate for the test, acclimatisation was started for a period of about 45 to 60 minutes. The chambers of the superfusion apparatus were held at a controlled temperature of 32 ± 1.5 °C.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

An aliquot of 30 mg powdered test substance was applied onto the entire surface of the cornea. The positive control eyes were treated in a similar way with 30 mg powdered imidazole. The negative control eye was treated with 30 μL of physiological saline.

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

The negative and positive control eyes as well as all test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.

Number of animals or in vitro replicates:

Three for test substance, one for negative and three for positive control

Details on study design:

At the end of the acclimatisation period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a base line (t = 0) for each individual eye. The corneal thickness of the eyes should not change by more than 5% between the -45 min and the zero time. No changes in thickness (0.0%) were observed in any eye in each experiment. Base line values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment I

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment II

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment I

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment II

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment I

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment II

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment I and II

Value:

>= 12.2 - <= 29.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Positive control

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment I and II

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Positive control

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment I and II

Value:

>= 2.5 - <= 3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Positive control

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment I and II

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative control

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment I and II

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Netative control

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment I and II

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative control

Other effects / acceptance of results:

The results from all eyes used met the quality control standards. The negative control and positive control results were in good correlation with historic data. This experiment was considered to be valid. Historical data for negative control (n = 497) were -3.2 to 3.4% after 75 minutes and -4.8 to 3.4% after 240 minutes for corneal swelling, maximum 0.5 for opacity change and 0.5 for fluorescine retention change. Historical data for positive control (n = 221) were -6.6 to 25% after 75 minutes and -15.9 to 36.7% after 240 minutes for corneal swelling, 3.5 to 4.0 for opacity change and 2.0 to 3.0 for fluorescine retention change.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the in vitro isolated chicken eye assy, the substance is non-irritating to the eye.

Executive summary:

An in vitro eye irritation study was conducted under GLP to OECD TG 438, using the isolated chicken eye test. The study used three eyes for the treatment with test substance, alongside with one negative and three positive control eyes in each of two experiments. After the zero reference (baseline) measurement, each eye in the treatment group was held in a horizontal position and 30 mg of powdered test material was applied onto the centre of teh cornea such that the entire surface was covered. After 10 seconds of exposure, the surface was rinsed with physiological saline. In both experiments, the positive control eyes were treated in a similar way with 30 mg of powdered imidazole and the negative control eye was treated with 30 μL of physiological saline (i.e. 0.9% NaCl solution). Corneal thickness, corneal opacity and fluorescein retention changes were measured and any morphological effects (such as pitting or loosening of the epithelium) were evaluated over a four-hour period. In experiment I, no corneal swelling, no cornea opacity, and no fluorescein retention change was observed during the observation period in any of the three treated eyes. Test substance was stuck on all corneal surfaces after the rinsing, and the surfaces were cleared at 30 minutes after the rinsing. The negative and positive control group results demonstrated the validity of the study and the sensitivity of the test system. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

On the basis of the results of the available reliale and valid in vitro studies on skin corrosion and irritation and eye irritation, the substance does not need to be classified under Regulation (EC) No. 1278/2008.