N-phenyl-N-piperidin-4-ylpropionamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-06-21 to 2005-08-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed equivalent/similar to OECD Test Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants) and 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) with one acceptable deviation: The purity of the test substance was not provided.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

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

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

N-phenyl-N-piperidin-4-ylpropionamide

EC Number:

216-543-3

EC Name:

N-phenyl-N-piperidin-4-ylpropionamide

Cas Number:

1609-66-1

Molecular formula:

C14H20N2O

IUPAC Name:

N-phenyl-N-piperidin-4-ylpropionamide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-1594255-AAA (T000425)
- Physical state: solid (powder)
- Appearance: white

Specific details on test material used for the study:

- Name of test material (as cited in study report): T425
- Substance type: no data
- Physical state: solid
- Analytical purity: no data
- Impurities (identity and concentrations): not applicable
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: 00454795
- Expiration date of the lot/batch: 2005-12-31
- Stability under test conditions: no data
- Storage condition of test material: at room temperature (range of 20 +/- 5 deg C), protected from light
- Other: solubility in water: 43 g/L, solubility in ethanol: >500 g/L

Test animals / tissue source

Species:

other: bovine eyes

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Freshly isolated bovine eyes were collected from the abattoir (Abattoir Basel, Schlachthofstrasse 55, CH-4055 Basel, Switzerland).

Test system

Vehicle:

physiological saline

Controls:

other: negative (solvent) and positive (Imidazole) eyes

Amount / concentration applied:

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

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9%
- Lot/batch no. (if required): 768707/1
- Purity: no data

Duration of treatment / exposure:

240 min

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

- Three corneas were treated with the test substance, the positive control substance, or the solvent.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Collection of bovine eyes
Freshly isolated bovine eyes were collected from the abattoir (Abattoir Basel, Schlachthofstrasse 55, CH-4055 Basel, Switzerland). 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 delivered the day before treatment and the isolated corneas were stored over night in the preservation medium in a refrigerator.

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.
Since the bovine 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 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.

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.

NUMBER OF REPLICATES 3

NEGATIVE CONTROL USED
Saline

POSITIVE CONTROL USED
imidazole, 20%, dissolved in saline

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL- aliquots, the test item was tested as a 20% suspension in saline. Incubation lasted 240 minutes.

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
The test substance was rinsed off from the application side by changing cMEM several times until precipitates of the test substance could no longer be observed. After that, fresh cMEM was replaced in both compartments and opacity was measured (t240min).

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometry (OD490)
- Others (e.g, pertinent visual observations, histopathology): no

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: equivalent to OECD Guideline 437

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Value:

212.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: very severe eye irritant

Remarks:

SD of 23.1

Results 2

Irritation parameter:

cornea opacity score

Value:

173.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD of 23.5

Results 3

Irritation parameter:

other: permability value

Value:

2.573

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD of 0.049

Other effects / acceptance of results:

- Treatment of the corneas with the test substance resulted in a mean in-vitro score of 212.3 ± 23.1 after 240 minutes incubation, ranging from 189.0 to 235.2. The net value of the opacity score ranged from 149.7 to 196.7, and the mean value was 173.7 ± 23.5. The mean corrected permeability value of the corneas was 2.573 ± 0.049, ranging from 2.527 to 2.624.
- The in-vitro score of saline, used as negative control, was -0.4± 0.6 (-0.8 to 0.3) with the mean opacity value of -0.7 ± 0.60 (-1 to 0) and the mean permeability value of 0.018 ± 0.003 (0.014 to 0.020).
- The in-vitro score of the positive control was 100.2 ± 18.6, proving the validity of the study. The corrected mean value of the opacity was 69.3 ± 12.1, ranging from 55.7 to 78.7. The corrected mean value of the permeability was 2.061 ± 0.518, ranging from 1.547 to 2.582.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under the experimental conditions, the test substance was considered to be a very severe eye irritant. Based on the CLP criteria, the substance is classified as category 1 (irreversible effects on the eye)