1-benzyl-4-piperidone

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-02-14 to 2000-02-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

non-GLP study, equivalent to OECD guideline OECD 437

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

The aim of this study was to assess the ocular irritancy potential of the test substance. The test utilized bovine corneas, isolated from eyes freshly collected in a local slaughterhouse. Two end-points, namely opacity and permeability, were measured. The objective values obtained from both parameters were combined, and the resulting in vitro score was compared to a previously established scale of ocular irritancy. This scale is composed of five broad categories: no irritation, mild, moderate, severe and very severe irritation.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-benzyl-4-piperidone

EC Number:

222-782-4

EC Name:

1-benzyl-4-piperidone

Cas Number:

3612-20-2

Molecular formula:

C12H15NO

IUPAC Name:

1-benzylpiperidin-4-one

Test material form:

liquid

Details on test material:

- Identification: JNJ-728195-AAA
- Physical state: liquid
- Appearance: clear yellowish liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Janssen Pharmaceutica N.V./ Prof-0001-214-1
- Expiration date of the lot/batch: no data
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in a closed labelled container
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: not applicable, test item was tested undiluted

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none, test item was tested undiluted

Test animals / tissue source

Species:

other: bovine eyes

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine corneas were isolated from eyes collected and kept in a plastic jar containing Hank's Balanced Salt Solution (HBSS). The eyes were used within three hours after killing the animals.
- Storage, temperature and transport conditions of ocular tissue: medium storage and transportation of the eyes to the laboratory was performed at room temperature
- Time interval prior to initiating testing: the eyes were used within three hours after killing the animals

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9% NaCl

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75 mL

Duration of treatment / exposure:

Corneas were incubated for 10 minutes at 32 °C

Duration of post- treatment incubation (in vitro):

After 10 minutes of treatment, the corneas were thoroughly rinsed to remove test item and incubated for 120 minutes, followed by opacity measurement. The permeability measurement of the corneas was performed following the opacity measurement after an incubation period of 90 minutes.

Number of animals or in vitro replicates:

Three corneas were selected for each treated group.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Before dissection, all eyes were carefully examined and those presenting defects, such as neovascularization, pigmentation, or scratches were discarded. During dissection great care was taken to avoid damage of corneal surfaces. Selected corneas were dissected with a 2-3 mm rim of sclera for easier handling and stored in a petri dish containing Hank’s Salt Solution until use. Corneas were mounted in holders, the endothelial side being applied on the O-ring of the posteriar part of the cornea holder. The anteriar part of the corneal holder was applied onto the posterior part with cornea and held in place with 3 screws. Compartments were filled (the posterior part first) with Minimal Essential Medium (MEM, Sigma) solution. The corneas were incubated for one hour in a water-bath at 32°C. After incubation, the medium was removed and both compartments were refilled with fresh medium.

TREATMENT METHOD:
The medium from the anterior compartment was removed and 0.75 mL of either the negative control, positive control or test item was introduced into the anterior compartment. After closing of the holes with caps, corneas were incubated in a horizontal position for 10 minutes at 32 deg C in a water bath.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After incubation, the test item and the controls were removed from the anterior compartment and the epithelium was washed twice at least 3 times with approximately 4 mL of MEM-solution until the medium was clear. The medium was then removed from the posterior compartment and both compartments were finally refilled with MEM-solution and again incubated at 32 ° C. After 2 hours of incubation, the MEM-solution of both compartments was replaced.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity was measured with an opacitometer and the difference in light transmission between the treated cornea and its own untreated control was then calculated. Croneas were rejected if they showed a background opacity grade greater than three.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a spectrophotometer (OD490)
The medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% sodium-fluorescein solution (sodium fluorescein was diluted in Dulbecco's phosphate buffered saline, Sigma). Corneas were incubated in a horizontal position for 90 minutes at 32°C in a water-bath. After incubation, medium from the posterior chamber was removed, and its optical density (OD) was determined spectrophotometrically at 490 nm.
Individual corneal opacity (at 2 hours and time zero) and permeability values were corrected with the mean opacity and permeability scores obtained for the negative control. Then, the mean (± SD) was calculated of the corrected opacity and permeability values.

SCORING SYSTEM:
- The objective values obtained for both parameters were combined into an equation, and the resulting in vitro eye irritancy scores were compared to a previously established scale of ocular irritancy:

In vitro irritancy = opacity value + 15 times OD490 value for permeability

<= 3.0 = non eye irritant
3.1-25.0 = mild eye irritant
25.1-55.0 = moderate eye irritant
55.1-80.0 = 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:

Test item after 10 minutes of exposure-1st test

Value:

10.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range of IVIS score of test item 10.2 - 11.3

Results 2

Irritation parameter:

cornea opacity score

Remarks:

mean

Run / experiment:

Test item after 10 minutes exposure-1st test

Value:

2.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range of corneal opacity score of test substance 1.7 to 3.7

Results 3

Irritation parameter:

other: permeability value mean

Run / experiment:

Test item after 10 minutes of exposure-1st test

Value:

0.556

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range of permability value of test item .431 - .641

Results 4

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

Test item after 10 minutes of exposure-2nd test

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range of IVIS score of test substance 0 - 1.0

Results 5

Irritation parameter:

cornea opacity score

Remarks:

mean

Run / experiment:

Test item after 10 minutes of exposure-2nd test

Value:

2.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range of corneal opacity score of test substance 1.7 - 3.7

Results 6

Irritation parameter:

other: permeability value mean

Run / experiment:

Test item after 10 minutes of exposure-2nd test

Value:

0.536

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range of permeability value of test substance .247 - .693

Other effects / acceptance of results:

mean in vitro irritancy score (range) 1st test:
negative control: 0.4 (-0.9 to 2.1)
positive control: 157.4 (152.3 to 160.7)

mean opacity scores (range) 1st test:
negative control: 0.3 (-1.0 to 2.0)
positive control: 113.4 (112.7 to 113.7)

mean permeability scores (range) 1st test:
negative control: 0.006 (0.005 to 0.007)
positive control: 2.934 (2.570 to 3.134)

mean in vitro irritancy score (range) 2nd test:
negative control: 0.3 (0 - 1.0)
positive control : 157.2 (138.1 to 180.2)

mean opacity scores (range) 2nd test:
negative control: 0.3 (0 - 1.0)
positive control : 107.4 (95.7 to 122.7)

mean permeability scores (range): 2nd test
negative control: 0.002 (0.001 to 0.003)
positive control: 3.318 (2.826 to 3.830)

- For all examined corneas (n=18, 9 per study), background opacity ranged between 0 and 3, indicating that no corneas had to be excluded from the experimental test design.

- In the first and the repeat study, each negative control cornea showed an opacity, permeability and an in vitro score which was situated within the laboratory range of historical data.
- In both studies, the corneas treated with the positive control N,N-dimethylformamide (100%) showed a marked increase in opacity and permeability. N,N-dimethylformamide (100%) was considered a very severe eye irritant in accordance with the historical data for the in vitro score of N,N-dimethylformamide (100%). Based on the findings in the negative and positive control group, it was concluded that the test conditions were optimal for the evaluation of the potential of the test substance to induce opacity and permeability.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The in vitro BCO-P eye irritation test classified the test item as a mild eye irritant when classification was made based on a combination of results obtained for opacity and permeability. This in vitro grade is related to a very small increase in corneal opacity and a small increase in permeability. Since the test item induced an IVIS of 10.7 in the first and second test, no prediction can be made on the GHS eye irritation or serious eye damage classification of the test item.