cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolane-4-ylmethyl benzoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-10-27 to 2005-10-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Study was conducted according to INVITTOX protocol no. 98 "Bovine Corneal Opacity and Permeability Assay" and Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK, and similar to Draft OECD guideline 437 "The Bovine Corneal Opacity and Permeability (BCOP) Test Method for Identifying Ocular Corrosives and Severe Irritants". Dec 18, 2008 with the following acceptable deviation: no data was provided on the purity of the test substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: INVITTOX (UK) protocol no. 98 "The 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 (BCOP) Assay, 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:

cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolane-4-ylmethyl benzoate

EC Number:

262-765-9

EC Name:

cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolane-4-ylmethyl benzoate

Cas Number:

61397-56-6

Molecular formula:

C18H15BrCl2O4

IUPAC Name:

[(2R,4R)-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl]methyl benzoate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-16479671-AAA (T001095)
- 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: 00452023 ZT001095PUA121
- 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 (range of 20 +/- 5 deg C), light protected
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: test substance was tested at a concentration of 20% in saline. Strong stirring with a magnetic stirrer resulted in suspension.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

Test animals / tissue source

Species:

other: bovine corneas

Strain:

other:

Remarks:

not applicable

Details on test animals or tissues and environmental conditions:

Test system: freshly isolated bovine cornea
Source:. Abattoir Basel, Schlachthofstrasse 55, CH-4055 Basel, Switzerland.
Collection 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 preparations. The eyes were delivered the day before treatment and the isolated corneas were stored overnight in a preservation medium (Medium 199 supplemented with L-glutamine, Na-biocarbonate and Taurine) 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 fr om the eye using scalpel and rounded scissors. A rim of about 2-3 mm of tissue (sclera) was left fo r 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 defect s 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 (Oring) 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°C +/- 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:

other:

Remarks:

physiological saline natrium chloratum 0.9%

Controls:

other: negative (saline) and positive (imidazole, 20% in saline)

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): 20%
Until administration, the solution was stirred with a magnetic stirrer


VEHICLE
- Concentration (if solution): 0.9% sodium chloride in water
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Lot/batch no. (if required): Charge No. 773609
- Purity: no data

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

POSITIVE CONTROL - Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): >/=99.9% (Assay)

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

After 240 ± 1 minutes of treatment, the corneas were thoroughly rinsed to remove test item and incuba ted for 120 ± 10 minutes at 32 ± 1°C with fresh medium followed by opacity measurement. The permeability measurement of the corneas was performed following the opacity measurement after an incubation period of 90 minutes ± 5 minutes.

Number of animals or in vitro replicates:

Three corneas were selected at random for each treatment group

Details on study design:

TREATMENT METHOD:
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control or test item was introduced onto the epithelium of the cornea. Corneas were incubated for 240 minutes at 32 ± 2°C.

REMOVAL OF TEST SUBSTANCE
Preparation of the test item solution: The test item was tested at a concentration of 20% in saline. Strong stirring with a magnetic stirrer resulted in a solution. Until administration, the solution was stirred with a magnetic stirrer.

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

METHODS FOR MEASURED ENDPOINTS:
- OPACITY MEASUREMENT: After recording the basal opacity of all corneas, the mean value of all corneas was calcualted. No cornea deviated from this by more than +/-3 units and no cornea was discarded. Sets of three cor neas 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, positiv e 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°C +/- 2°C.

PERMEABILITY DETERMINATION:
-Following the opacity readings after treatment, the permeability endpoint was measured as an indicated of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the medium was removed from teh aniterior compartment and repalced by 1 mL of a fluorescein solution. Corneas were incubated again in a horizaontal 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 optical density at 490 nM (OD 490) was determined with a spectophotometer.

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 values:
Negative 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 irritant
in vitro score 3.1 - 25: mild eye irritant
in vitro score 25.1 - 55: moderate eye irritant
in vitro score 55.1 - 80: severe eye irritant
in vitro score > 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 240 minutes of exposure

Value:

0.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD +/- 0.6

Results 2

Irritation parameter:

cornea opacity score

Remarks:

mean

Run / experiment:

Test item as 240 minutes of expsure

Value:

0.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD +/- 0.6

Results 3

Irritation parameter:

other: permeability value mean

Run / experiment:

Test item after 240 minutes

Value:

0.011

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD +/- 0.007

Other effects / acceptance of results:

Other effects / acceptance of results
mean in vitro irritancy score (range):
negative control: 0.5 (-0.7 to 2.2)
positive control: 81.1 (70.5 to 95.1)

mean opacity scores (range):
negative control: 0.3 (-1 to 2)
positive control: 65.7 (59.7 to 71.7)

mean permeability scores (range):
negative control: 0.014 (0.011 to 0.018)
positive control: 1.029 (0.724to 1.561)

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 µg/mL showed an optical density (OD490) of 1.610 to 1.910. The value found by spectroscopy was 1.697. According to the results obtained in this experiment, the test was considered acceptable.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the given test conditions, the test substance is not considered to be an eye irritant.