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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-11-2018 to 13-11-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

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

Test animals / tissue source

Species:

cattle

Strain:

not specified

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

A 20% (w/v) solution of PF-07097547-24 was prepared in physiological saline.

Duration of treatment / exposure:

240 minutes +/- 10 minutes

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

90 minutes with Na-fluorescein

Number of animals or in vitro replicates:

3 (three)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Source: 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.
Transport: Eyes were collected and transported in physiological saline in a
suitable container under cooled conditions.
Preparation of Corneas
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 (Earle’s Minimum Essential
Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine
(Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated
corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen,
Germany) 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 wereincubated for the minimum of 1 hour at 32 ± 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS
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 (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined wasrecorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
Physiological saline

SOLVENT CONTROL USED (if applicable)

POSITIVE CONTROL USED
20% imidazole in physiological saline

APPLICATION DOSE AND EXPOSURE TIME
The medium from the anterior compartment was removed and 750 µl of either the negative
control, positive control (20% (w/v) Imidazole solution) or 20% (w/v) solution of the test
item was introduced onto the epithelium of the cornea. The medium from the anterior
compartment was removed and 750 µl of the negative control and 20% (w/v) Imidazole
solution (positive control) were introduced onto the epithelium of the cornea. 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.

TREATMENT METHOD:closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea.
The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490) : 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 µl 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 (TECAN Infinite® M200 Pro Plate 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 verifiedbefore the start of the experiment). OD490
values of less than 1.500 were used in thepermeability calculation.
- Others (e.g, pertinent visual observations, histopathology): (please specify)
Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

16

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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

Any other information on results incl. tables

The individual in vitro irritancy scores for the negative controls ranged from 4.1 to 6.3. The

corneas treated with the negative control item were clear after the 240 minutes of treatment.

The individual positive control in vitro irritancy scores ranged from 116 to 168.

The corneas treated with the positive control were turbid after the 240 minutes of

treatment.

The corneas treated with  PF-07097547-24 showed opacity values ranging from 4.6 to 26 and

permeability values ranging from -0.008 to 0.460. The corneas were turbid and translucent

after the 240 minutes of treatment with  PF-07097547-24. A pH effect of the test item was

observed on the rinsing medium, the corneas were rinsed until no colour change of the

medium was observed. Hence, the in vitro irritancy scores ranged from 4.5 to 33 after

240 minutes of treatment with  PF-07097547-24.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, since PF-07097547-24 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-07097547-24 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 of  PF-07097547-24 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 GR13335 of  PF-07097547-24 was a white solid with a purity of 97.1%. The test item

was applied as a 20% (w/v) solution (750 µL) directly 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 140 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.  

PF-07097547-24 induced ocular irritation through both endpoints, resulting in a mean in vitro

irritancy score of 16 after 4 hours of treatment.

In conclusion, since  PF-07097547-24 induced an IVIS > 3 ≤ 55, no prediction on the

classification can be made.