isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29-1-2019 to 4-02-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

EC Number:

950-484-0

Molecular formula:

C9H17NO2*C4H6O4

IUPAC Name:

isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

Test material form:

solid

Details on test material:

Batch (Lot) Number: GR13335
Expiry date: 31 January 2020 (expiry date)
Physical Description: White solid
Purity/Composition: 97.1%
Storage Conditions: At room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Source strain:

not specified

Details on animal used as source of test system:

Adult human donors

Justification for test system used:

The test is based on the experience that irritant chemicals show cytotoxic effects following
short term exposure to the stratum corneum of the epidermis. The test is designed to predict
and classify the skin irritation potential of a test item by assessment of its effect on a three
dimensional human epidermis model

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model
- Tissue batch number(s): 19-EKIN-005
- Production date: 29-01-2019
- Shipping date: 29-01-2019
- Delivery date: 30-01-2019
- Date of initiation of testing: 31-01-2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 35.9 - 36.8°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: not given
- Observable damage in the tissue due to washing: not noted
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS
- Incubation time: 3 h at 37°C
- Spectrophotometer: the TECAN Infinite® M200 Pro Plate Reader
- Filter: 570nm
- Filter bandwidth: not given
- Linear OD range of spectrophotometer: not given

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: IC50 for SDS = 1.9mg/mL (acceptance criteria between 1.5 and 3 mg/mL)
- Barrier function: not measured
- Morphology: HIstology scoring >19.5, satisfactory morphology following qualitative assessment
- Contamination: not present
- Reproducibility:

NUMBER OF REPLICATE TISSUES: triplicate (3 tisues per treatment)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
-conducted in Skin corrosion study

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
-A test item is considered 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
controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

15.4 to 29.8 mg per tissue

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 (three

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

experiment 1

Value:

95

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: standard deviation 7.4

Other effects / acceptance of results:

PF-07097547-24 was checked for possible direct MTT reduction and color interference in the
Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20171510).
Because no color changes were observed it was concluded that PF-07097547-24 did not
interact with the MTT endpoint.
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 5.4%. 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 < 8%, indicating that the test system functioned
properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, PF-07097547-24 is non-irritant in the in vitro skin irritation test under the
experimental conditions described in this report and should not be classified according to the
Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the
United Nations.

Executive summary:

The objective of this study was to evaluate  PF-07097547-24 for its ability to induce skin

irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKINSM

TM)). The possible skin irritation potential of  PF-07097547-24 was tested through topical

application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC

guidelines.

Batch GR13335 of  PF-07097547-24 was a white solid with a purity of 97.1%. Skin tissue

was moistened with 5 µL of Milli-Q water and at least 10 mg of  PF-07097547-24 was applied

directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period,

determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as

the reduction of mitochondrial dehydrogenase activity measured by formazan production

from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The

relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with  PF-07097547-24

compared to the negative control tissues was 95%. Since the mean relative tissue viability for

PF-07097547-24 was above 50% after 15 ± 0.5 minutes treatment  PF-07097547-24 is

considered to be non-irritant.

The positive control had a mean cell viability of 5.4% after 15 ± 0.5 minutes exposure. The

absolute mean OD570 (optical density at 570  nm)  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 < 8%, indicating that the test system

functioned properly.

In conclusion,  PF-07097547-24 is non-irritant in the in vitro skin irritation test under the

experimental conditions described in this report and should not be classified according to the

Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the

United Nations.