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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21st July 2021 - 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)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
(1R,2S,5S)-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxylic acid
EC Number:
955-024-2
Cas Number:
2755812-45-2
Molecular formula:
C16 H23 F3 N2 O4
IUPAC Name:
(1R,2S,5S)-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxylic acid
Test material form:
solid

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
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:
water
Controls:
yes, concurrent positive control
Amount / concentration applied:
The test item was used as delivered and added pure on top of the corneas.
(301.6 to 353.6 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:
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 (301.6 to 353.6 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 (Eagle’s Minimum Essential Medium). 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 OD490 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

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
26
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
25
Negative 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-07320267 induced an IVIS > 3 < 55, no prediction on the classification can be made.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of PF-07320267 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 JR-C200917012-D21001 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 induced ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 26 after 240 minutes of treatment.
In conclusion, since PF-07320267 induced an IVIS > 3 < 55, no prediction on the classification can be made.