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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 to 15 Jul 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

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

Test material

Constituent 1
Chemical structure
Reference substance name:
(1S)-1-(2,6-dichloro-3-fluorophenyl)ethan-1-ol
EC Number:
700-699-9
Cas Number:
877397-65-4
Molecular formula:
C8H7Cl2FO
IUPAC Name:
(1S)-1-(2,6-dichloro-3-fluorophenyl)ethan-1-ol
Specific details on test material used for the study:
- Purity: 100.0 % area (HPLC)
- Batch No.: E010010678 (PFI-09-01-097)

Test animals / tissue source

Species:
other: cattle
Strain:
other: an isolated bovine cornea
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Age at study initiation:
- Weight at study initiation:
- Housing:
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period: minimium of 1 hour

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 301.5 to 302.0 mg
Duration of treatment / exposure:
240±10 minutes
Observation period (in vivo):
Immediate opacity measurement and permeability evaluation of the cornea.
Number of animals or in vitro replicates:
Three corneas for each treatment group (total 9 corneas).
Details on study design:
Negative control: Physiological saline
Positive control: 20% (w/v) Imidazole in physiological saline

Treatment of corneas and opacity measurements:
The medium from the anterior compartment was removed and 750 l of the negative control and 20% (w/w) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. Three corneas were covered with 301.5 to 302.0 mg the test substance. 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℃. 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. Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.

Opacity measurement:
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final posttreatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance 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 (Merck) 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. 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℃.

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 (Multiskan spectrum, Thermo labsystems, Breda, The Netherlands). 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 was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Remarks:
Mean
Value:
ca. 143
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
other:
Remarks:
Permeabillity Mean
Value:
ca. 9.152
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
cornea opacity score
Remarks:
Mean
Value:
ca. 6
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Any other information on results incl. tables

Summary of opacity, permeability and in vitro scores

Treatment

Mean Opacity

Mean Permeability

Mean In vitro Irritation Score1,2

Negative control

0

0.000

0

Positive control

79

3.122

126

Test substance

6

9.152

143

1 Calculated using the negative control mean opacity and mean permeability values.

2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye)
Conclusions:
It is concluded that the test substance is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.
Executive summary:

Screening for the eye irritancy potential of PF-00968603 using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the ocular irritation properties of PF-00968603 on an isolated bovine cornea. The possible ocular irritancy of PF-00968603 was tested through topical application for 240 ± 10 minutes.  

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

Batch E010010678 (PFI-09-01-097) of PF-00968603 was a white powder with lumps with a purity of 100.0 % area (HPLC). Since no workable suspension of PF-00968603 in physiological saline could be obtained, the test substance was added pure on top of the corneas (301.5 to 302.0 mg).

The negative control responses of the opacity and permeability values 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 126 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.  

PF-00968603 induced severe ocular irritation at one endpoint mainly (permeability), resulting in a mean in vitro irritancy score of 143 after 240 minutes of treatment.

Since PF-00968603 induced an IVIS ≥ 55.1, it is concluded that PF-00968603 is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.  

Based on these results: According to the test guideline, PF-00968603 should be regarded as severe eye irritant.  According to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007), PF-00968603 would be classified as:  having irreversible effects on the eyes (Category 1). According to the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures, PF-00968603 would be classified as: Irreversible effects on the eye (Category 1) and labeled as H318: Causes serious eye damage.

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