Reaction mass of (1R,2S,5S)-N-{(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide and tertbutyl methyl ether

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30th July 2021 to 6th August 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142, 31 May 2008.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(1R,2S,5S)-N-[(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl]-3-[(2S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide; 2-methoxy-2-methylpropane

EC Number:

955-078-7

Cas Number:

2755812-44-1

Molecular formula:

C28H44F3N5O5

IUPAC Name:

(1R,2S,5S)-N-[(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl]-3-[(2S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide; 2-methoxy-2-methylpropane

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm Skin Model (EPI-200, Lot no.: 35631 kit C&D,
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm2) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
Test for Color Interference by the Test Item
To assess the color interference, at least 50 mg of the test item or 50 uL Milli-Q water as a negative control were added to 1 mL Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 +/- 1.0°C in the dark. Furthermore, at least 50 mg of the test item or 50 uL Milli-Q water as a negative control were added to 2.0 mL isopropanol. The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking. At the end of the exposure time, the mixtures were centrifuged for 30 seconds at 16000 g if needed and the absorbance of the solutions were determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. If after subtraction of the negative control, the OD for the test item solution was >0.08, the test item was considered as possibly interacting with the MTT measurement.
Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of -0.0032 and 0.0017. Therefore it was concluded that the test item did not induce color interference.

Test for Reduction of MTT by the Test Item
To assess the ability of the test item to reduce MTT, at least 50 mg of the test item or 50 uL Milli-Q water as a negative control were added to 1mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 3 hours at 37.0 +/- 1.0°C in the dark. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.
No color change was observed in the presence of MTT therefore it was concluded that the test item did not interact with the MTT endpoint.

Application/Treatment of the Test Item
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 3 hours at 37.0 +/- 1.0°C. The medium was replaced with fresh DMEM just before the test item was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. The skin was moistened with 25 uL Milli-Q water to ensure close contact of the test item to the tissue and 26.4 to 40 mg of the solid test item was added into the 6-well plates on top of the skin tissues.
For the negative and positive controls, 2 tissues were treated with 50 uL Milli-Q water (negative control) and 2 tissues were treated with 50 uL 8N KOH (positive control) for both the 3-minute and 1-hour time point. Negative and positive controls were shared with parallel studies. After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 uL DMEM until 6 tissues (= one application time) were dosed and rinsed.

Cell Viability Measurement
The DMEM was replaced by 300 uL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

26.4 to 40.0 mg of the solid test

Duration of treatment / exposure:

The method utilizes a 3-minute exposure for a corrosive classification and a 60- minute confirmatory exposure for materials found to be non-corrosive at the 3-minute exposure.

Number of replicates:

4 tissues per test item

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean

Run / experiment:

60 minutes

Value:

92

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean

Run / experiment:

3 minutes

Value:

94

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8).
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability > 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

The absolute mean OD570 of the negative control tissues of the 1-hour treatment was just above the laboratory historical control data range. Since the absolute mean OD570 values were the within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8), this minor deviation has no impact on the study results.
The mean relative tissue viability following the 1-hour exposure to the positive control was 8.3%.
In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 7.2%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, PF-07321332 MTBE Solvate is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate PF-07321332 MTBE Solvate for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)).
The possible corrosive potential of the test item was tested through topical application for 3 minutes and | hour.
The study procedures described in this report were based on the most recent OECD and EC guidelines.
Batch 902/7321332/B/1/1 of the test item was a white to off-white solid. Skin tissue was moistened with 25 uL of Milli-Q water and at least 25 mg of the test item was applied directly on top of the skin tissue.
The positive control had a mean relative tissue viability of 8.3% after the 1-hour exposure. The absolute mean OD570 of the negative control tissues of the 1-hour treatment was just above the laboratory historical control data range. Since the absolute mean OD570 values were the within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8), this minor deviation has no impact on the study results. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 7.2%, indicating that the test system functioned properly.
Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 94% and 92%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.
In conclusion, PF-07321332 MTBE Solvate is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.