3-(2-hydroxyethyl)-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:

2006-03-30 to 2006-03-30

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 without deviations. Conducted in a manner consistent with OECD guideline 437.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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

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

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)

Deviations:

no

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

214-897-3

EC Name:

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

Cas Number:

1207-75-6

Molecular formula:

C10H10N2O3

IUPAC Name:

3-(2-hydroxyethyl)-1,2,3,4-tetrahydroquinazoline-2,4-dione

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00495665 RT001200G1A291
- Expiration date of the lot/batch: 2006-06-30
- 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: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: the test substance was tested at a concentration of 20% in saline. Strong stirring with a magnetic stirrer resulted in suspension. Until administration, the suspension was stirred with a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material): 20% suspension in saline

Test animals / tissue source

Species:

other: bovine corneas

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Test system: Freshly isolated bovine cornea
- Source: Abattoir Basel, Schlachthofstrasse 55, CH-4055 Basel, Switzerland.
- Collection of bovine eyes:
Freshly isolated bovine cornea 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 used immediately after delivery in the laboratory and within four hours after slaughtering.

Preparation of corneas: All eyes were carefully examined macroscopically for defects. Those pre senting 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 e xperiment were collected in complete minimum essential medium (cMEM) and were checked finally with a view box for the defect s listed above.
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 th e 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:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

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


VEHICLE/NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9%

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

Duration of treatment / exposure:

240 min

Duration of post- treatment incubation (in vitro):

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°C +/- 2°C.

Number of animals or in vitro replicates:

Three corneas per test group (test item, negative control and positive control)

Details on study design:

TREATMENT METHOD
- Fresh cMEM was filled into the posterior compartment, while the anterior compartment has received 0.75 mL-aliquots of the test item or negative or positive control. The incubation lasted 240 minutes. During the whole experiment, cornea holders and medium were maintained in a water-bath at 32°C +/- 2°C.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the test item 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

METHODS FOR MEASURED ENDPOINTS:
- OPACITY 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 di scarded. 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 t est item and will be incubated in a horizontal positioning a water-bath at 32°C +/- 2°C.
- The change of opacity value of each tretaed cornea or positive or negative control corneas was calculated by substracting the initial basal opacity from the post treatment opacity reading, for each individual cornea. The average change in opacity of the negative control corneas was calculated and this value was 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 opacity values of the treated corneas for eacht treatment condition.

PERMEABILITY DETERMINATION:
-Following the opacity readings after treatment, the permeability endpoint was measured as an indi cated 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 deter mined with a spectophotometer.
- 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 was calculated from the individual corrected permeability values of the treated corneas for each treatment condition.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
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:

-0.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range: -0.8 to 0.3; SD: +/- 0.6

Results 2

Irritation parameter:

cornea opacity score

Remarks:

mean

Run / experiment:

Test item after 240 minutes of exposure

Value:

-0.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range: -0.7 to 0.3; SD: +/- 0.6

Results 3

Irritation parameter:

other: permeability value mean

Run / experiment:

Test item as 240 minutes of exposure

Value:

-0.005

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range: -0.006 to -0.004; SD: +/- 0.001

Other effects / acceptance of results:

Other effects / acceptance of results
mean in vitro irritancy score (range):
negative control: -0.1 (-0.8 to 0.4)
positive control: 85.2 (82.5 to 90.1)
mean opacity scores (range):
negative control: -0.3 (-1 to 0)
positive control: 63.3 (58.3 to 68.3)
mean permeability scores (range):
negative control: 0.017 (0.012 to 0.026)
positive control: 1.458 (1.276 to 1.647)

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 1.613. 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.