Propan-2-yl-oxocyclobutane carboxylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

06-11-2018

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 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

1

Reference substance name:

propan-2-yl 3-oxocyclobutane-1-carboxylate

EC Number:

834-970-9

Cas Number:

130111-95-4

Molecular formula:

C8H12O3

IUPAC Name:

propan-2-yl 3-oxocyclobutane-1-carboxylate

Test material form:

liquid

Details on test material:

Batch (Lot) Number: GR13224
Expiry date: 31 January 2020 (expiry date)
Physical Description: Colourless to pale yellow liquid
Purity/Composition: 99.6%
Storage Conditions: At room temperature

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System
Test System: Bovine eyes were used as soon as possible after slaughter.
Rationale: In the interest of sound science and animal welfare, a sequential
testing strategy is recommended to minimize the need of in vivo
testing (1-6). As a consequence a validated and accepted in
vitro test for eye irritation should be performed before in vivo
tests are conducted. One of the proposed validated in vitro eye
irritation tests is the Bovine Corneal Opacity and Permeability
(BCOP) test.
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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750µl

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

triplicate (three)

Details on study design:

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) Foetal 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 were
incubated for the minimum of 1 hour at 32 ± 1°C.

Cornea Selection and Opacity Reading
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 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.

Test Item Preparation
No correction was made for the purity/composition of the test item.
The test item was tested neat.

Treatment of Corneas and Opacity Measurements
The medium from the anterior compartment was removed and 750 µl of either the negative
control, positive control (Ethanol) or test item was introduced onto the epithelium of the
cornea. The holders were slightly rotated, with the corneas maintained in a horizontal
position, to ensure uniform distribution of the control or the test item over the entire cornea.
Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. After the
incubation the solutions were removed and the epithelium was washed with MEM with
phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with
cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in
the posterior compartment was removed and both compartments were refilled with fresh
cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After
the completion of the incubation period opacity determination was performed. Each cornea
was inspected visually for dissimilar opacity patterns.
4.6.5. Opacity Measurement
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.
The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity = (Io/I - 0.9894)/0.0251

With I0 the empirically determined illuminance through a cornea holder but with windows
and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was
calculated by subtracting the initial opacity reading from the final post-treatment reading.
The corrected opacity for each treated cornea with the test item or positive control was
calculated by subtracting the average change in opacity of the negative control corneas from
the change in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected
opacity values of the treated corneas for each treatment group.

Application of Sodium Fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein
(Sigma-Aldrich, Germany) 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 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/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.

Permeability Determinations
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 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.

ACCEPTABILITY CRITERIA
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.
All results presented in the tables of the report are calculated using values as per the raw data
rounding procedure and may not be exactly reproduced from the individual data presented.

INTERPRETATION
The mean opacity and mean permeability values (OD490) were used for each treatment group
to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine
whether the test item induced irritation through only one of the two endpoints.
The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN
GHS Category 1) and test items not requiring classification for eye irritation or serious eye
damage (UN GHS No Category) are given hereafter:

In vitro score range UN GHS
< 3 No Category
> 3; < 55 No prediction can be made
>55 Category 1

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Range

Value:

>= 6.9 - <= 7.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 1

Value:

7.7

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 2

Value:

6.9

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 3

Value:

6.9

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The individual in vitro irritancy scores for the negative controls ranged from -0.7 to 1.3. The
corneas treated with the negative control item were clear after the 10 minutes of treatment.
The individual positive control in vitro irritancy scores ranged from 30 to 52. The corneas treated with the positive control item were turbid after the 10 minutes
of treatment.

Applicant's summary and conclusion

Interpretation of results:

other: further testing required

Conclusions:

In conclusion, since PF-06238566 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-06238566 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-06238566 was tested through topical application for

10 minutes.

The study procedures described in this report were based on the most recent OECD guideline.

Batch GR13224 of  PF-06238566 was a colourless to pale yellow liquid. The test item was

applied as it is (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 (Ethanol) was 44 and

was 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-06238566 induced ocular irritation through one endpoint (opacity), resulting in a mean in

vitro irritancy score of 7.1 after 10 minutes of treatment.

In conclusion, since  PF-06238566 induced an IVIS > 3 ≤ 55, no prediction on the

classification can be made.