3-(2-chloroethyl)(1H,3H)quinazoline-2,4-dione

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

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 without deviations.

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 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.

Principles of method if other than guideline:

not applicable

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-(2-chloroethyl)(1H,3H)quinazoline-2,4-dione

EC Number:

225-795-3

EC Name:

3-(2-chloroethyl)(1H,3H)quinazoline-2,4-dione

Cas Number:

5081-87-8

Molecular formula:

C10H9ClN2O2

IUPAC Name:

3-(2-CHLOROETHYL)QUINAZOLINE-2,4(1H,3H)-DIONE

Details on test material:

- Name of test material (as cited in study report): T001201
- Substance type: white to beige powder
- Physical state: powder
- Analytical purity: no data
- Purity test date: no data
- Lot/batch No.: 00464885RT001201G1A251
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: room temperature (range of 20 ± 5 °C), light protected
- Other:
Solubility:
Water: < 0.09 g/L
Methanol: 10 g/L
Dichloromethane: 10.2 g/L
Acetone: 26.7 g/L
Ethanol: 7.9 g/L
2-propanol: 1.3 g/L
N,N-Dimethylformamide: 290 g/L

Test animals / tissue source

Species:

other: freshly isolated bovine cornea

Strain:

other: not applicable

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.
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 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 (sclera) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in complete medium essential medium (cMEM) and were checked finally with a view box for defects listed before.
Since the bovine eyes were delivered in the afternoon, corneas were stored in a preservation medium overnight 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 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. During the whole experiment, cornea holders and medium were maintained in a water-bath at 32°C ± 2°C

Test system

Vehicle:

physiological saline

Controls:

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

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% imidazole, dissolved in saline

Duration of treatment / exposure:

240 min

Observation period (in vivo):

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

Number of animals or in vitro replicates:

Group 1 (negative control): 3 corneas
Group 2 (positive control): 3 corneas
Group 3 (test item): 3 corneas

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the test substance was rinsed off from the application side by changing cMEM solution several times until precipitates of the test substance could be observed no longer, fresh cMEM was replaced in both compartments and the opacity was measured (t240min).
- Time after start of exposure: 240 minutes

- Opacity measurement:
The experiment was performed to determine an irritation effect of the test item on the corneal opacity.
The opacitometer determined changes in the light transmission passing through the corneas, and displayed a numerical opacity value. This value was recorded in a table. 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 cornea.

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 was incubated in a horizontal positioning a water-bath at 32°C ± 2°C.

- Permeability determination
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 was added to the posterior chamber and 1 mL of the fluorescein sodium dye solution, 0.5% dissolved in Dulbecco's phosphate-buffered saline, was placed in the anterior compartment after removing the medium. Corneas were incubated again in a horizontal position for about 90 minutes in a water-bath at 32°C ± 2°C. The optical density of an aliquot of the mixed medium from the posterior chamber was measured by photometry at 490 nm. The dye solution was valid for use, if a dilution stock solution containing 10 µg/mL showed an optical density (OD490) of 1.610 to 1.910.

Evaluation of results:
Opacity:
The change of opacity value of each treated cornea or positive or negative control corneas was calculated by subtracting the initial basal opacity from the post treatment opacity reading, for each individual cornea (t240min-t0min).

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 treatment group was then calculated from the individual corrected opacity values of the treated corneas for each treatment condition.

Permeability:
The corrected OD490 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 were calculated from the individual corrected permeability values of the treated corneas for each treatment condition.

In Vitro Score Calculation:
In-Vitro Score = opacity value + (15 X OD490 value)

The In-Vitro Score was calculated 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 substance 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 (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)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Value:

1.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 1.5

Results 2

Irritation parameter:

cornea opacity score

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 1.2

Results 3

Irritation parameter:

other: permeability score

Value:

0.03

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/-0.025

Other effects / acceptance of results:

mean in vitro irritancy scores (range):
negative control: 0.5 ± 1.5 (-0.8 to 2.2)
positive control: 80.9 ± 12.4
test item: 1.4 ± 1.5 (-0.3 to 2.4)

mean opacity scores (range):
negative control: 0.3 ± 1.5 (-1 to 2)
positive control (mean corrected value): 65.7 ± 6.0 (59.7 to 71.1)
test item (mean corrected value): 1.0 ± 1.2

mean permeability scores (range):
negative control: 0.014 ± 0.002 (0.013 to 0.017)
positive control (mean corrected value): 1.018 ± 0.450 (0.724 to 1.536)
test item (mean corrected value): 0.030 ± 0.025 (0.002 to 0.051)

Any other information on results incl. tables

- Before starting the permeability test, the sodium fluorescein dye solution was checked for quality. The dye solution was valid for use if the stock solution containing 10 ug/mL showed an optical density (OD₄₉₀) of 1.610 to 1.910. The value found by spectroscopy was 1.643.

The test was considered valid as the positive control caused an in vitro score greater than 55.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was not considered to be an eye irritant under the given test conditions.