Loperamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2006-03-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Non-GLP study performed according to 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 without deviations and similar to OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants) which was adopted after study completion.

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"

Deviations:

no

Remarks:

The testing facility indicated that the protocol was followed without deviations.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

The testing facility indicated that the protocol was followed without deviations.

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Loperamide

EC Number:

258-416-5

EC Name:

Loperamide

Cas Number:

53179-11-6

Molecular formula:

C29H33ClN2O2

IUPAC Name:

4-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]-N,N-dimethyl-2,2-diphenylbutanamide

Details on test material:

- Name of test material (as cited in study report): T001001; 4-(4-chlorophenyl)-4-hydroxy-N,N-dimethyl-alpha, alpha-diphenyl-1-piperidinebutanamide.- CAS Number: 53179-11-6- Physical state: solid - Analytical purity: 100%- Impurities (identity and concentrations): none- Purity test date: no data- Lot/batch No.: 00456462RT001001G1B591- Expiration date of the lot/batch: 2006-06-30- Stability under test conditions: no data- Storage condition of test material: room temperature (20±5°C), light protected- Other: solubility in water: <0.010 g/L.

Test animals / tissue source

Species:

other: bovine corneas

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

Test system: freshly isolated bovine corneaSource: Abattoir Basel, Schlachthofstrasse 55, CH-4055 Basel, SwitzerlandCollection of bovine eyes: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 preparation. The eyes were used immediately after delivery in the laboraotry 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 (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) and were checked finally with a view box for the defects listed above. 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 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±2°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.

Test system

Vehicle:

physiological saline

Remarks:

natrium chloratum 0.9 %

Controls:

other: positive (imidazole) and negative (physiol. saline) control eyes used

Amount / concentration applied:

TEST MATERIAL- Amount(s) applied (volume or weight with unit): 0.75 mL- Concentration (if solution): 20%Strong stirring with a magnetic stirrer resulted in a suspension. Until administration, the suspension was stirred with a magnetic stirrer. VEHICLE- Concentration (if solution): 0.9%

Duration of treatment / exposure:

240 min

Observation period (in vivo):

After the test item was rinsed off from the application side by changing cMEM several times until precipitates of the test item could be observed no longer, fresh cMEM was replaced in both compartments and opacity was measured (t240min). To demonstrate possible treatment-induced transepithelial permeability of the cornea, the permeability test with fluorescein sodium dye was performed in a second step.

Number of animals or in vitro replicates:

9 bovine eyes in total (3 test, 3 negative controls, 3 positive controls).

Details on study design:

REMOVAL OF TEST SUBSTANCE- Washing (if done): by rinsing with "complete minimum essential medium" (cMEM).- Time after start of exposure: 240 minutesOPACITY MEASUREMENT: 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 item, the negative and positive controls, respectively. Medium was completely removed from the anterior compartment and replaced by the test item, 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 item and will be incubated in a horizontal positioning a water-bath at 32±2°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±2°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. 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 in vitro score value of each treated group was calculated from the individual in vitro score valuesnegative control:in vitro score = opacity value + (15 x OD490 value)Positive control and test item cornea:in vitro score = corrected opacity value + (15 x corrected OD490 value)Depending on the score obtained, the test item was classified into one of the following categories:in vitro score 0 - 3: non eye irritantin vitro score 3.1 - 25: mild eye irritantin vitro score 25.1 - 55: moderate eye irritantin vitro score 55.1 - 80: severe eye irritantin vitro score > 80.1: very severe eye irritant

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean value after 240 minutes incubation

Value:

4

Positive controls validity:

valid

Remarks on result:

other: SD = 2.4 (opacity score: 4±2.6 ; permeability score: -0.002±0.011)

In vivo

Irritant / corrosive response data:

Treatment of the corneas with the test substance resulted in a mean in-vitro score of 4.0 ± 2.6 after 240 minutes incubation, ranging from 1.9 to 6.9. The net value of the opacity score ranged from 2 to 7; mean value 4.0 + /- 2.6. The mean corrected permeability value of the corneas was -0.002 + /- 0.011, ranging from -0.009 to 0.011.

Any other information on results incl. tables

The in-vitro score of saline, used as a negative control, was 0.3 ± 0 (0.3 to 0.4) with the mean opacity value of 0 + / - 0

(0) and the mean permeability value of 0.023 + / - 0.003 (0.020 to 0.025).

The in vitro score of the positive control (imidazole, 20%, dissolved in saline), was 76.8 + / - 1.8, proving 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 1.389 + / - 0.330, ranging from 1.193 to 1.770.

Before starting the permeability test, the sodium fluorescein dye solution was checked for quality. The dye solution is valid for use if the stock solution containing 10 µg/mL showed an optical density (OD490) of 1.610 to 1.910. The value found by spectroscopy was 1.866.

Applicant's summary and conclusion

Interpretation of results:

other: No prediction can be made.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Since the in vitro score was >3 and ≤55 (in vitro score cut-off values for identifying test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) and test chemicals as inducing serious eye damage (UN GHS Category 1), respectively), no prediction can be made regarding the classification of the test item.