4-chloro-7H-pyrrolo[2,3-d]pyrimidine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21-11-2018 to 2712-2018

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:

4-chloro-7H-pyrrolo[2,3-d]pyrimidine

EC Number:

628-079-2

Cas Number:

3680-69-1

Molecular formula:

C6H4ClN3

IUPAC Name:

4-chloro-7H-pyrrolo[2,3-d]pyrimidine

Test material form:

solid

Details on test material:

Batch (Lot) Number: GR13290
Expiry date: 31 January 2020 (expiry date)
Physical Description: White to off-white solid
Purity/Composition: 99.1%
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: 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:

303 to 419 mg

Duration of treatment / exposure:

240 ± 10 minutes at 32 ± 1°C

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

N/A

Number of animals or in vitro replicates:

34 (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) 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 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 will be made for the purity/composition of the test compound.
Since no workable suspension of PF-01323624 in physiological saline could be obtained, the
test item was used as delivered by the sponsor and added pure on top of the corneas.

Treatment of Corneas and Opacity Measurements
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. PF-01323624 was weighed in a bottle and applied directly on the corneas in such
a way that the cornea was completely covered (303 to 419 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 compound were
removed and the epithelium was washed at least three times with MEM with phenol red
(Earle’s Minimum Essential Medium Life Technologies). 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.

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 was measured with the device OP-KIT)
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 5 mg Na-fluorescein/mL cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). 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
valuesof 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

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

experiment1

Value:

1.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The individual in vitro irritancy scores for the negative controls ranged from 0.4 to 2.4. 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 115 to 130
The corneas treated with the positive control were turbid after the 240 minutes of
treatment.

Any other information on results incl. tables

The corneas treated with  PF-01323624 showed opacity values ranging from 1.3 to 2.1 and

permeability values ranging from 0.004 to 0.012. The corneas were translucent after the

240 minutes of treatment with  PF-01323624. No pH effect of the test item was observed on

the rinsing medium. Hence, the in vitro irritancy scores ranged from 1.4 to 2.3 after

240 minutes of treatment with  PF-01323624.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since PF-01323624 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-01323624 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-01323624 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 GR13290 of  PF-01323624 was a white to off-white solid with a purity of 99.1%. 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 124 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-01323624 did not induce ocular irritation through both endpoints, resulting in a mean in

vitro irritancy score of 1.7 after 4 hours of treatment.

In conclusion, since  PF-01323624 induced an IVIS ≤ 3, no classification is required for eye

irritation or serious eye damage.