Ethyl 4-oxopiperidine-1-carboxylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2006-04-20 to 2006-04-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Well-documented 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 feb 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

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:

Ethyl 4-oxopiperidine-1-carboxylate

EC Number:

249-984-5

EC Name:

Ethyl 4-oxopiperidine-1-carboxylate

Cas Number:

29976-53-2

Molecular formula:

C8H13NO3

IUPAC Name:

ethyl 4-oxopiperidine-1-carboxylate

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study reports): JNJ-126971-AAA (T000509)
- Physical state: liquid
- Appearance: clear colourless to light yellow liquid

Test animals / tissue source

Species:

other: bovine eyes

Details on test animals or tissues and environmental conditions:

TEST SYSTEM:
Source: freshly isolated bovine cornea from 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 used immediately after delivery in the laboratory and within four hours after slaughtering.

PREPARATION OF THE CORNEAS:
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. each corneas was dissected from the eye using scalpel and rounded scissors. A rim about 2-3mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in complete minimum essential medium (cMEM) andwere checked finally with a view box for defects listed above. 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 must 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied: 0.75 mL
- Concentration (if solution): 0.9% saline
- Lot/batch no. (if required): no data
- Purity: no data
-description: colorless clear liquid
-charge no: 773609 (expiration date Jan 2009)
-storage conditions: refrigerator (range of 5+/-3°C)

POSITIVE CONTROL: 2-ethoxyethanol
-amount applied: 0.75 mL
-concentration: undiluted
-description: colorless liquid
-batch no: MA 09739MA
-storage conditions: at room temperature (range of 20+/-5°C), light protected

Duration of treatment / exposure:

10 minutes(+/- 30 seconds), in a water-bath at 32°C +/- 2°C

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

3 corneas per group
3 groups (test item, negative control, positive control)

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. For measurement, the posterior compartment was plugged while the anterior compartment remained unplugged.

TREATMENT OF CORNEAS:
Medium was completely removed from the anterior compartment and replaced by the test item, positive or negative control. The anterior compartment was unplugged. The holder was turned to a horizontal position and slightly rotated to ensure uniform covering of the cornea with the test item and will be incubated in a horizontal positioning a water-bath at 32°C +/-2°C.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): by changing cMEM three times in minimum.
- Time after start of exposure:10 min
-fresh cMEM was added and opacity was measured (t10). The corneas were then incubated for further two hours, followed by a third opacity reading (120).

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 medium was added to the posterior chamber and 1ml of the fluorescein sodium dye solution, 0.4% 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 a wave length of 490 nm. The dye solution is valid for sure, if a dilution of the stock solution containing 10 µg/ml of fluorescein sodium showed 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 °C +/- 2 °C.


PERMEABILITY DETERMINATION:
Following the opacity readings after treatment, the permeability endpoint was measured as an ndication 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 1ml of a fluorescein solution. Corneas were incubated again in a horizontal position for about 90 minutes in a water-bath at 32+/-2°C. Medium from the posterior compartment was removed with a 5ml-syring, well mixed and transferred to a cuvette of 10mm path length and the optical density at 490nm was determined with a spectrophotometer.

SCORING SYSTEM:
-opacity: the change of opcity value of each treated cornea or positive or negative control corneas was calculated by subtracting the initial based opacity from the post treatment opacity reading, for each individual cornea. The ten -minute values were not used for calculation, but indicated early effects. 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 treatmnet group was than calculated from the individual corrected opacity values of the treated corneas for each treatment condition.
-permeability: the corrected OD 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 was calculated from the individual correctedpermeability alues of treated corneas for each treatment condition.
-in vitro score calculation: formula used to determine the in vitro score of negative control: in vitro score = opacity value + (15xOD value). The in vitro score was calcualted 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 item cornea: in vitro score = corrected opacity value + (15xcorrected OD 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:

27.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 3.1

Results 2

Irritation parameter:

cornea opacity score

Value:

19.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 2.9

Results 3

Irritation parameter:

other: permeability score

Value:

0.511

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 0.124

Other effects / acceptance of results:

The in vitro score of the negative control using sodium chloride, 0.9% (saline) as reference was 1.3 +/- 0.9 (0.4 to 2.2) with the mean opacity value of 1.0+/-1.0 (0 to 2) and the mean permeability value of 0.018 +/-0.010 (0.012 to 0.029). The in vitro score of the positive control (2-ethoxyethanol, 20%, dissolved in saline) was 124.9 +/- 7.8 (117.0 to 132.6), proving the validity of the study. The corrected mean value of the opacity was 90.3 +/-6.8, ranging from 85 to 98. The corrected mean value of the permeability was 2.307 t+/-0.178, ranging from 2.132 to 2.487. Treatment of the corneas during ten minutes with the test item, followed by a recovery period of two hours, resulted in a mean in vitro score of 27.3 +/-3.1, ranging from 24.1 to 30.2. The net value of the opacity score ranged from 18 to 23, the mean value was 19.7+/-2.9. The mean corrected permeability value of the corneas was 0.511+/-0.124, ranging from 0.406 to 0.649.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

According to the in vitro irritation scale stated in the INVITTOX Protocol no 98 'The Bovine Corneal Opacity and Permeability Assay', dated Feb 1994, it is considered to be moderate eye irritant. Based on the criteria in the CLP regulation, the in vitro score assessed the test substance as inconclusive for classification.