[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09 to 15 Feb 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(S)-tert-butyl 5'-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-3'H-spiro[azetidine-3,1'-isobenzofuran]-1-carboxylate

Cas Number:

1398610-06-4

Molecular formula:

C25H22Cl2F4N2O4

IUPAC Name:

(S)-tert-butyl 5'-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-3'H-spiro[azetidine-3,1'-isobenzofuran]-1-carboxylate

Specific details on test material used for the study:

Batch No.: 465884
Purity: 100%

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: human epidermis model

Justification for test system used:

recommended in international guidelines (e.g. OECD and EC).

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM
- Tissue batch number(s): 21-EKIN-006

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with phosphate buffered saline to remove residual test item.
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 ± 1 hours at 37°C.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS
- Incubation time: 3h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test item is considered an irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to
the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative
control.
A test item is considered a non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to
the test item and 42 hours of post incubation is > 50% of the mean viability of the negative
control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 μL

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Because the solutions did not turn blue / purple, nor was a blue / purple precipitate observed and the OD for the test item solution was 0.08, it was concluded that the test item did not interfere with the MTT endpoint.
The positive control had a mean cell viability of 5.4% after 15 ± 0.5 minutes exposure. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 6.8%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is a non-irritant in the in vitro skin irritation test.

Executive summary:

The objective of this study was to evaluate test item for its ability to induce skin irritation. For this purpose the test item was topically applied on a human three dimensional epidermal model, according to OECD Guideline 439.

The test item is a non-irritant in the in vitro skin irritation test.