4-(1-piperidinyl)-2-(E)-butenoicacid hydrochloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18th October 2011 to 18th October 2011

Reliability:

1 (reliable without restriction)

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:

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 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: 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.

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:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-[(2E)-3-carboxyprop-2-en-1-yl]piperidin-1-ium chloride

EC Number:

690-933-5

Cas Number:

197892-69-6

Molecular formula:

C9H16ClNO2

IUPAC Name:

1-[(2E)-3-carboxyprop-2-en-1-yl]piperidin-1-ium chloride

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)
The eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container.
- 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 +/- 1oC 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 +/- 1o C.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

A 20% (w/w) solution of PF-00818977-01 was prepared in physiological saline (Merck, Darmstadt, Germany).
750 ul of 20% (w/w) test substance solution was introduced onto the epithelium of the cornea.

Duration of treatment / exposure:

240 +/- 10 minutes

Duration of post- treatment incubation (in vitro):

None

Number of animals or in vitro replicates:

Three

Details on study design:

Preparation of corneas
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.
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 (Invitrogen Corporation) 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.

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. 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 3 were not used. Three corneas were selected at random for each treatment group.

Treatment of corneas and opacity measurements
The medium from the anterior compartment was removed and 750 ul of the negative control, 20% (w/v) Imidazole solution (positive control) or 20% (w/w) test substance solution were introduced onto the epithelium of the cornea. 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 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 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:

other: Mean Permeability

Run / experiment:

mean

Value:

0.004

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

87

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

87

Vehicle 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:

Since PF-00818977-01 induced an IVIS ≥ 55.1, it is concluded that PF-00818977-01 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-00818977-01 on an isolated bovine cornea.
The possible ocular irritancy of PF-00818977-01 was tested through topical application for approximately 240 minutes.
The study procedures described in this report were based on the most recent OECD and EC guideline.
Batch E010012221 of PF-00818977-01 was a white powder with a purity of 99.2%. The test substance was applied as a 20% (w/w) solution (750 μl) directly 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 (IVIS) of the positive control (20% (w/v) Imidazole) was 105 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-00818977-01 induced severe ocular irritation at one endpoint only (opacity), resulting in a mean in vitro irritancy score of 87 after 240 minutes of treatment.
Since PF-00818977-01 induced an IVIS ≥ 55.1, it is concluded that PF-00818977-01 is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.