3-chloro-4-fluoroaniline

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25th August 2011 to 15th November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review D

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-chloro-4-fluoroaniline

EC Number:

206-682-8

EC Name:

3-chloro-4-fluoroaniline

Cas Number:

367-21-5

Molecular formula:

C6H5ClFN

IUPAC Name:

3-chloro-4-fluoroaniline

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands),
- Characteristics of donor animals: Bovine eyes from young cattle
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container as soon as possible after slaughter. .
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.
- Selection and preparation of corneas: The isolated corneas were stored at 32 +/- 1 C in a petri dish with cMEM (Eagle’s Minimum Essential Medium, containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 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.
- Quality check of the isolated corneas: 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. The opacity of each cornea was read against an air 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 system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test substance was used as delivered and added pure on top of the corneas (309 to 345 mg).

Duration of treatment / exposure:

The corneas were incubated for 240 minutes +/- 10 minutes at 32 +/-1 C.

Details on study design:

Treatment of corneas and opacity measurements
The medium from the anterior compartment was removed and 750 ul of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. PF-01458762 was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (309 to 345 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 minutes +/- 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 (Eagle’s Minimum Essential Medium. 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 post-treatment 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 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 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 ul 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. 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

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

mean, three replicates

Value:

25

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

mean, three replicates

Value:

98

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

PF-01458762 induced severe ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 98 after 240 minutes of treatment.
Since PF-01458762 induced an IVIS ≥ 55.1, it is concluded that PF-01458762 is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Executive summary:

This report describes the ocular irritation properties of PF-01458762 on an isolated bovine cornea. The possible ocular irritancy of PF-01458762 was tested through topical application for approximately 240 ± 10 minutes.
The study procedures described in this report were based on the most recent OECD and EC guideline.
Batch E010012175 of PF-01458762 contained white crystal lumps with a purity of 99.5%. Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas (309 to 345 mg).
The mean 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 106 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-01458762 induced severe ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 98 after 240 minutes of treatment.
Since PF-01458762 induced an IVIS ≥ 55.1, it is concluded that PF-01458762 is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.