1-benzyl-N-phenylpiperidin-4-amine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-03-23 to 2006-03-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Non-GLP study performed according to the protocols on which the OECD guideline was based (INVITTOX Protocol no. 98 "Bovine Corneal Opacity and Permeability Assay", dated February 1994 and Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997). No deviations were recorded.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol no. 98 "Bovine Corneal Opacity and Permeability Assay", dated February 1994.

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

Principles of method if other than guideline:

Study was performed equivalent to the OECD437 guideline

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

1-benzyl-N-phenylpiperidin-4-amine

EC Number:

214-583-6

EC Name:

1-benzyl-N-phenylpiperidin-4-amine

Cas Number:

1155-56-2

Molecular formula:

C18H22N2

IUPAC Name:

1-benzyl-N-phenylpiperidin-4-amine

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-1594008-AAA (T000293)
- 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: 00479757RT000293G1A401
- Expiration date of the lot/batch: 2006-06-30
- Purity: 100%
- Purity test date: no data

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: the test item was tested at a concentration of 20% in saline. Stron stirring with a magnetic stirrer resuted in a suspension. Until administration, the suspension was stirred with a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspension in saline

Test animals / tissue source

Species:

other: bovine eyes

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Abattoir Basel, Schlachthofstrasse 55, CH-4055 Basel, Switzerland
TEST SYSTEM:
Freshly isolated bovine eyes were collected from the abattoir. After excess tissue was removed from the excised eyes, they were stored at room temperature in Hank's Balanced Salt solution containing penicillin/streptomycin and then transported for further preparations. The eyes were used immediately after delivery in the laboratory and within four hours after slaughtering.

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 (sclerea) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in complete minimum essential medium (cMEM) and were checked for defects.
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 posterior compartment had to be filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibrium, the corneas in the holder were incubated for about one hour at 32 +/- 2 deg. C in a water bath.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

other:

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% suspension in saline


VEHICLE (negative control and solvent)
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9%
- Lot/batch no. (if required): 773609
- Purity: no data
- Description: colorless clear liquid
- Stability of vehicle: stable under storage conditions
- Expiration date: Jan 2009
- Storage conditions: Refrigerator (range of 5 +/- 3°C)

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% dissolved in saline
- Description: colorless fine cristals
- Batch number: 054063/2
- Purity: >99.5%
- Storage conditions: at room temperature (range of 20 +/- 5 °C), light protected

Duration of treatment / exposure:

4 hours

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

3 corneas in each group (negative control, positive control and test item), 9 corneas total

Details on study design:

OPACITY READING:
At the end of the incubation period the medium was removed from both compartments and replaced by fresh cMEM, and the basal opacity was determined (t0min). For measurement, the posterior compartment was plugged while the anterior compartment remained unplugged.

TREATMENT OF CORNEAS
Fresh cMEM was filled into the posterior compartment, while the anterior compartment has received 0.75 mL aliquots of the test item or negative or positive control, respectively and were evenly distributed on the surface of the corneas.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the test substance was rinsed off from the application side by changed cMEM several times until precipitates of the test substance could be observed no longer, fresh cMEM was replaced in both compartments and the opacity was measured (t240min).
- Time after start of exposure: 240 minutes

APPLICATION OF FLUORESCEIN SODIUM DYE
To demonstrate possible treatment-induced transepithelial permeability of the cornea, the permeability test with fluorescein sodium dye was performed in a second step. Corneal permeability is quantified by measuring the amount of fluorescein sodium dye diffusing into the medium in the posterior chamber. Fresh cMEM was added to the posterior chamber and 1 mL of the fluorescein sodium dye solution, 0.5% dissolved in Dulbecco’s phosphate-buffered saline, was placed in the anterior compartment after removing the medium. The optical density of an aliquot of the mixed medium from the posterior chamber was measured by photometry at 490 nm. The dye solution is valid for use, if a dilution of the stock solution containing 10 µg/mL shows an optical density of 1.610 to 1.910.

OPACITY MEASUREMENT:
The opacitometer determined changes in the light transmission passing through the corneas and displayed a numerical opacity value. The opacitometer was calibrated with a standardized opaque polyester sheet as described in a manual, and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated corneas.
After recording the basal opacity of all corneas, the mean value of all corneas was calculated. No cornea deviated from this by more than +-3 units and no cornea was discarded. Sets of three corneas were used for treatment with the test substance, the negative and positive controls, respectively.
Medium was completely removed from the anterior compartment and replaced by the test substance, positive or negative control. The anterior compartment was plugged. The holder was turned to a horizontal position and slightly rotated to ensure uniform covering of the cornea with the test substance and was incubated in a horizontal positioning water bath at 32 deg. C +- 2 deg. C.

PERMEABILITY DETERMINATION:
Following the opacity readings after treatment, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the medium was removed from the anterior compartment and replaced by 1 mL of a fluorescein solution. Corneas were incubated again in a horizontal position for about 90 minutes in a water bath at 32 deg. +- 2 deg. C. Medium from the posterior compartment was removed with a 5 mL- syringe, well mixed and transferred to a cuvette of 10 mm path length, and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

SCORING SYSTEM:
- Opacity:
The change of opacity value of each treated cornea or positive or negative control corneas was calculated by subtracting the initial basal opacity from the post treatment opacity reading, for each individual cornea (t240min-t0min).
The average change in opacity of the negative control corneas was calculated and this value subtracted 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.
- Permeability:
The corrected OD490 value of each treated cornea or positive control corneas was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.
The mean corrected permeability values of each treatment group were calculated from the individual corrected permeability values of the treated corneas for each treatment condition.
- In Vitro Score Calculation:
The following formula was used to determine the In Vitro Score:
In-Vitro Score = opacity value + (15 X OD490 value)
The In-Vitro Score was calculated for each individual treatment and positive control cornea. The mean In Vitro Score value of each treated group was calculated from the individual In-Vitro Score Values:
Negative control:
In-Vitro Score = opacity value + (15 X OD490 value)
Positive control and test substance cornea:
In-Vitro Score = corrected opacity value + (15 X corrected OD490 value)
Depending on the score obtained, the test substance was classified into one of the following categories:
In-Vitro Score: (Proposed In-Vitro Irritation Scale)
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)

TOOL USED TO ASSESS SCORE:
- An opacitometer was used to determine the changes in light transmission passing through the corneas. 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 corneas.
- To demonstrate possible treatment-induced transepithelial permeability of the cornea, the permeability test with fluorescein sodium dye was performed in a second step using a spectrophotometer.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD = 0.8; range 1.1 to 2.5

Results 2

Irritation parameter:

cornea opacity score

Value:

1.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD=0.6; range 1 to 2

Results 3

Irritation parameter:

other: permeability score

Value:

0.014

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD=0.015; range 0.005 to 0.031

Other effects / acceptance of results:

Treatment of the corneas with the test substance resulted in a mean in-vitro score of 1.5 +/- 0.8 after 240 minutes incubation, ranging from 1.1 to 2.5. The net value of the opacity score ranged from 1 to 2, and the mean value was 1.3 +/- 0.6. The mean corrected permeability value of the corneas was 0.014 +/- 0.015, ranging from 0.005 to 0.031.
The in-vitro score of saline, used as the negative control, was 0.4 +/- 0 (0.3 to 0.4) with the mean opacity value of 0 +/- 0 (0) and the mean permeability value of 0.024 +/- 0.003 (0.021 to 0.026).
The in-vitro score of the positive control (imidazole, 20%, dissolved in saline) was 76.4 +/-1.9, confirming the validity of the study. The corrected mean value of the opacity was 56.0 +/- 3.6, ranging from 52 to 59. The corrected mean value of the permeability was 1.361 +/- 0.335, ranging from 1.167 to 1.748.

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 µ/mL showed an optical density (OD490) of 1.610 to 1.910. The value found by spectroscopy was 1.866.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the given test conditions, the test item T000293 is considered to be a non-eye irritant.