Ibuprofen

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

7 September 2009

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

24 April 2002; for determination of eye irritation/corrosion

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ibuprofen

EC Number:

239-784-6

EC Name:

Ibuprofen

Cas Number:

15687-27-1

Molecular formula:

C13H18O2

IUPAC Name:

2-(4-isobutylphenyl)propanoic acid

Test material form:

solid

Specific details on test material used for the study:

- Name of test substance: Ibuprofen
- Test substance No.: 02/0156-6
- Batch ID: IB1S1290
- Purity: 99.7%
- pH: ca. 4.5 (undiluted test substance, minimally moistened with water); ca. 4 (20% aqueous preparation)

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim
- Characteristics of donor animals: age of animals: minimum 12 months, maximum 60 months
- Species: Bovine (eyes are obtained as a by-product of freshly slaughtered cattle)

Test system

Details on study design:

SELECTION AND PREPARATION OF CORNEAS & QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in specially designed corneal holders that consists of anterior and posterior chambers. Both chambers were filled to excess with prewarmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour. After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that show macroscopic tissue damage or an opacity value < 566 opacity units were discarded. Corneas with opacity values close to the median value of all corneas were selected as negative (vehicle) control. The remaining corneas were then distributed into treatment and positive control groups. Each corneal holder was uniquely identified with a number written on the chambers with a felt pen.

NUMBER OF REPLICATES
Each treatment group (test substance, vehicle control and positive control) consisted of 3 corneas.

SOLVENT CONTROL USED
Highly deionized water

POSITIVE CONTROL USED
20% (w/v) solution of Imidazole in highly de-ionized water

APPLICATION DOSE AND EXPOSURE TIME
750 μL of the 20% (w/v) test-substance preparation (non-surfactant), vehicle control or positive control was applied using a pipette. The corneas were incubated in a horizontal position at about 32 °C for approximately 4 hours (non-surfactant solids).

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
The vehicle control and positive control were removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red). Because the test substance could not be removed using a syringe, the epithelium was rinsed with the open chamber method.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: visual observations and opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria is not covered, repetition of the test was considered.
- A study is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the established upper limits.
- Since the IVIS per treatment group is determined from the mean of three single corneas, the variability between the corneas treated per test substance should be acceptably low. If the prediction is not clearly identified in all corneas, the test will be repeated.

DECISION CRITERIA
IVIS > 55: risk of serious damage to eyes
IVIS ≤ 55: no risk of serious damage to eyes

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

1st

Value:

28.7

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

2nd

Value:

32.7

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

1st

Value:

1.357

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

2nd

Value:

1.05

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 5

Irritation parameter:

in vitro irritation score

Run / experiment:

1st

Value:

49

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 6

Irritation parameter:

in vitro irritation score

Run / experiment:

2nd

Value:

48.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

Results of negative and positive controls see "any other information on results incl. tables"

Any other information on results incl. tables

First run

	Mean Opacity Value	Mean Permeability Value	In Vitro Irritancy Score
Test substance	28.7	1.357	49.0
Negative Control	2.2	0.020	2.5
Positive Control	81.6	3.738	137.6

Second run

	Mean Opacity Value	Mean Permeability Value	In Vitro Irritancy Score
Test substance	32.7	1.050	48.4
Negative Control	4.1	-0.005	4.0
Positive Control	63.2	3.466	115.2

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive to the eyes