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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30th July 2021 to 3rd 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:

Used updated guideline 437 (26 June 2020)

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

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.

Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and tested the day of arrival in the laboratory.
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

The isolated corneas were stored in a petri dish with CMEM (Eagle’s Minimum Essential Medium) containing 1% (v/v) L-glutamine and 1% (v/v) Fetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of Duratec Analysentechnik GmbH with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with CMEM of 32 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

The test item was used as delivered and added pure on top of the corneas.
(306.0 to 351.6 mg).

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

Three corneas were incubated in the presence of the test article and each control.

Details on study design:

After the incubation period, the medium was removed from both compartments and replaced with fresh CMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a CMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

The medium from the anterior compartment was removed and 750 uL of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (306.0 to 351.6 mg). The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 + 10 minutes at 32 + 1°C. After the incubation the solutions and the test item were removed and the epithelium was washed at least three times with MEM with phenol red. Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh CMEM and the opacity determinations were performed.

Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 + 5 minutes at 32 + 1°C.

After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 uL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader. Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using CMEM corrected OD590 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

2

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

-1.1

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The assay is considered acceptable if:
a) The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
b) The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 143 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since PF-07321332 MTBE solvate induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of PF-7321332 MTBE Solvate as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).
This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage potential of the test item was tested through topical application for approximately 240 minutes.
The study procedures described in this report were based on the most recent OECD guideline. 

Batch 902/7321332/B/1/1 of the test item was a white to off-white solid. Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure on top of the corneas.
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 143 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.1 after 240 minutes of treatment. 

In conclusion, since PF-07321332 MTBE Solvate induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.