Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Feb 2018 to 13 Mar 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:
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:
yes
Remarks:
None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Off-white powder
Specific details on test material used for the study:
Purity/Composition: 98.7% Test item storage: At room temperature protected from light Stable under storage conditions until: 20 November 2019 (retest date) (taken from label)

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.

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 +/- 1oC. The corneas were incubated for the minimum of 1 hour at 32  1C.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
PF-06932437 was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (317.31 to 463.7 mg over 2 experiments).
Duration of treatment / exposure:
240 +/- 10 minutes
Number of animals or in vitro replicates:
3
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 +/- 1oC. The corneas were incubated for the minimum of 1 hour at 32 +/- 1oC.

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-06932437 in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas. To protect the test item from light, tubes wrapped in tin-foil were used.

Treatment of Corneas and Opacity Measurements
Initially, the second experiment was rejected since some of the acceptability criteria were not met. This part of the study was repeated. The medium from the anterior compartment was removed and 750 l of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. PF-06932437 was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (317.31 to 463.7 mg over 2 experiments). 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 +/- 1oC. 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 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 +/- 1oC.

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 l 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.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
run 1- mean after 4 hours treatment with the test item
Value:
ca. 0.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
run 2- mean after 4 hours
Value:
ca. -0.8
Vehicle controls validity:
valid
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: 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.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, since the test item induced an IVIS ≤ 3 in 5 out of 6 cornea’s treated with the test item, 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-06932437 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-06932437 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 GR12302 of PF-06932437 was an off-white powder with a purity of 98.7%.  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. In the first experiment, 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 144 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 mean in vitro irritancy score was 0.3 after 4 hours of treatment with the test item. Since the results were spread over 2 categories (-1.8, -1.2 and 4.0, respectively), the test was inconclusive and a repeat experiment was performed. In the second experiment, 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 164 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 -0.8 (0.8, -1.3 and -1.9, respectively) after 4 hours of treatment.   In conclusion, since the test item induced an IVIS ≤ 3 in 5 out of 6 cornea’s treated with the test item, no classification is required for eye irritation or serious eye damage.