Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Jul 2017 to 13 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Lot number: 2000850599
Analysis date: 19 August 2016
Specific details on test material used for the study:
Appearance: Off-white crystalline powder (determined by Charles River Den Bosch)
Batch: B64002 (Pfizer Lot no. 2000850599)
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature
Stable under storage conditions until: Unknown

Since no workable suspension of Tri-o-tolylphosphine in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- 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)
Remarks:
Since no workable suspension of the test item in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.
Controls:
yes, concurrent positive control
Amount / concentration applied:
The medium from the anterior compartment will be removed and 750 µl of the negative or positive control or test item solution will be introduced onto the epithelium of the cornea.
Duration of treatment / exposure:
4 hours
Duration of post- treatment incubation (in vitro):
240 ± 10 minutes at 32 ± 1oC.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS : 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.

QUALITY CHECK OF THE ISOLATED CORNEAS : 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.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : A negative control, physiological saline (Eurovet, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

POSITIVE CONTROL USED : 20% (w/v) Imidazole (Merck Schuchardt OHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.

APPLICATION DOSE AND EXPOSURE TIME : Tri-o-tolylphosphine was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (329 to 363 mg). Exposure time: 240 ± 10 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions will be removed and the epithelium will be rinsed with MEM with phenol red (Eagle's Minimum Essential Medium) and thereafter with cMEM.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea will be measured by the diminution of light passing through the cornea. The light will be measured as illuminance (I
= luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) will be calculated according to:

Opacity = [(Io/I)-0.9894] / 0.0251

With Io 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) will be calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity of each cornea treated with the test item or positive control will be 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 is then calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Corneal permeability: Following the final opacity measurement, permeability of the cornea to Na-fluorescein will be evaluated. The medium of both compartments (anterior compartment first) will be removed. The
posterior compartment will be refilled with fresh cMEM. The anterior compartment will be filled with 1 ml of 5 mg sodium-fluorescein/ml cMEM solution. The holder will be slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas will be 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 will be removed and placed into a sampling tube relabeled according to holder number. 360 µl of the medium from each sampling tube will be transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube will be measured in triplicate using a microplate reader (TECAN Infinite® 1200 Pro Plate Reader). Any OD490 that is 1.500 or higher must be diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 will be verified before the start of the experiment). OD490 values of less than 1.500 will be used in the permeability calculation. The mean OD490 for each treatment will be calculated using смЕм corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item will be corrected for the mean negative control OD490 before the dilution factor will be applied to the reading.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) : 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
Irritation parameter:
in vitro irritation score
Value:
<= 3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
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.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, since Tri-o-tolylphosphine 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 Tri-o-tolylphosphine 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 Tri-o-tolylphosphine 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 B64002 (Pfizer Lot no. 2000850599) of Tri-o-tolylphosphine was an off-white crystalline powder. 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 147 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. Tri-o-tolylphosphine 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 Tri-o-tolylphosphine induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.