N-(1-benzylpiperidin-4-yl)-N-phenylpropionamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-06-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Study performed according to INVITTOX Protocol 98 (Bovine Corneal Opacity and Permeability Assay, February 1994) and Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK, April 1997 and similar to OECD guideline 437.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol 98 (Bovine Corneal Opacity and Permeability Assay, Feb. 1994, SOP of Microbiological Associates Ltd., UK, April 1997.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

N-(1-benzylpiperidin-4-yl)-N-phenylpropionamide

EC Number:

216-014-7

EC Name:

N-(1-benzylpiperidin-4-yl)-N-phenylpropionamide

Cas Number:

1474-02-8

Molecular formula:

C21H26N2O

IUPAC Name:

N-(1-benzylpiperidin-4-yl)-N-phenylpropanamide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-1594164-AAA (T000424)
- Physical state: solid (powder)
- Appearance: White powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RT000424G1A251
- Expiration date of the lot/batch: 2005-12-31
- Purity test date: no data
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (range of 20 +/- 5°C), light protected
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was tested at a concentration of 20% in saline. Strong stirring with a magnetic stirrer resulted in a suspension. Until administration, the suspension was stirred with a magentic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material): th test item was tested at a concentration of 20% in saline.

Test animals / tissue source

Species:

other: bovine eyes

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Freshly isolated bovine eyes were collected from Abattoir Basel, Schlachthofstrasse 55, CH-4055 Basel, Switzerland. After excess tissue was removed from the excised eyes, the were stored at room temperature in Hank's balanced salt solution containing penicillin/streptomycin and then transported for further preparations. The eyes were delivered the before treatment and the isolated corneas were stored over night in a preservation medium in a regriferator.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL
- Concentration: 20% in saline


VEHICLE/NEGATIVE CONTROL
- Amount(s) applied: 0.75 mL
- Concentration: 0.9%

POSITIVE CONTROL:
- Amount(s) applied: 0.75 mL
- Concentration: 20% in saline
- Lot/batch no.: 054063/2
- Purity: >99.5% (assay)

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

After 240 minutes of treatment, opacity was measured with an opacitometer. The permeability measurement of the corneas was performed after the incubation period of about 90 minutes following the opacity measurement.

Number of animals or in vitro replicates:

9 (3 per treatment group)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
- All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. Each cornea was dissected from the eye using scalpel and rounded scissors. A rim of about 2-3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the expreiment were collected in complete minimum essential medium (cMEM) and were checked finally with a view box for defects listed above.
- Since the boven eyes were delivered in the afternoon, corneas were stored in a preservation medium over night in a refrigerator at about 4 °C. The preservation medium was composed of Medium 199 supplemented with L-glutamine, Na-bicarbonate and Taurine. Shortly before use, Dextran was added.
- Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching had to be avoided. After the anterior part of the holder was positioned on the top of the cornea and fixed in place with screws, both compartments of the holder were filled with cMEM. The posteriar compartment had to be filled first to return the cornea to its natural convex position. Care had to be taken to assure no air bubbles were present within the compartments.
- For equilibration, the corneas in the holder were incubated for about one hour at 32°C +/- 2°C in a water-bath. At the end of the incubation periode, the medium was removed from both compartments and replaced by fresh cMEM.

TREATMENT METHOD:
- Fresh cMEM was filled into the posterior compartment, while the anterior compartment has received 0.75 mL-aliquots of the test item, or negative of positive control, respectively and were evenly distributed on the surface of the corneas.
- The incubation lasted 240 minutes. During the whole experiment, cornea holders and medium were maintained in a water-bath at 32°C +/- 2°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: the test item was rinsed off from the application side by changing sMEM several times untill precipitates of the test item could be obbserved no longer.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
The opacitometer determined changes in the light transmission passing throught the corneas, and displayed a numerical opacity value.
The opacitometer was calibrated with a standardized opaque polyester sheet as described in the manual and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
The change of opacity value of each treated cornea or positive or negative control corneas was calculated by subtracting the initial opacity reading from the post-treatment reading, for each individual cornea. The average change in opacity of the negative control corneas was calculated and this value was subtracted from from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
The mean corrected opacity value of each treatment group was then calculated from the individual corrected opacity values of the treated corneas for each treatment condition.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometry (OD490)
After the final opacity measurement was performed, the medium was removed from the anterior compartment and 1 mL of the fluorescein dye solution, 0.5% dissolved in Dumlbecco's phosphate-buffered saline, was placed in the anterior compartment. Corneas were incubated again in a horizontal position for about 90 minutes an a water-bath at 32°C +/- 2°C. Medium from the posterior compartment was removed with a 5 mL-syringe, well mixed and trasferred to a cuvette of 10 mm path length and the optical density was measured by photometry at 490 nm.
The corrected OD490 value of each treated cornea or positive control cornea was calculated by subtracting the average negative control value from the original permeability value for each cornea.
The mean corrected permeability values of each treatment group was calculated from the individual corrected permeability values of the treated corneas for each treatment condition.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- The following formula was used to determine the in vitro score:
In vitro score = opacity value + (15 x OD490 value)

DECISION CRITERIA
- Depending on the score, the test substance was classified into one of the following categories:
0-3 non eye irritant
3.1-25 mild eye irritant
25.1-55 moderate eye irritant
55.1-80 severe eye irritant
> 80.1 very severe eye irritant

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

1

Value:

0.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

SD +/- 1.2; range: -0.2 to 1.9

Results 2

Irritation parameter:

cornea opacity score

Remarks:

mean

Run / experiment:

1

Value:

0.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

SD +/- 1.2; range: 0 to 2

Results 3

Irritation parameter:

other: permeability

Remarks:

mean

Run / experiment:

1

Value:

-0.008

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

SD +/- 0.003; range: -0.012 to -0.007

Other effects / acceptance of results:

mean in vitro irritancy score (range):
negative control: 1.3 +/- 1.2 (0.1 to 2.5)
positive control: 86.1 +/- 4.9 (82.7 to 91.7)

mean opacity scores (range):
negative control: 1.0 +/- 1.0 (0 to 2)
positive control: 58.0 +/- 2.0 (56.0 to 60.0)

mean permeability scores (range):
negative control: 0.018 +/- 0.015 (0.009 to 0.035)
positive control: 1.870 +/- 0.338 (1.586 to 2.244)

The In vitro score of the positive control (imidazole, 20%, dissolved in saline) was 86.1 +/- 4.9, proving the vailidty of the study.

Any other information on results incl. tables

Before starting the permeability test, the dye solution sodium fluorescein was checked for its quality. The dye solution is valid for use, if a dilution of the stock solution containing 10 µg/mL showed an optical density (OD490) of 1.610 to 1.910. The value found by spectroscopy was 1.693.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

As the mean in vitro irritation score was within the range 0-3, T000424 is considered to be non eye irritant.