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Eye irritation

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eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-04-26 - 2011-06-10
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
other: OECD 437
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
other: liquid

Test animals / tissue source

other: bovine eye
other: not applicable
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hank’s balanced salt solu-tion (supplemented with 0.01% streptomycin and 0.01% penicillin). Then the corneas were dissected and incubated with media at 32 ± 1°C in an incubation chamber for 1 hour.

Test system

unchanged (no vehicle)
Amount / concentration applied:
750 µL
Duration of treatment / exposure:
10 min.
Observation period (in vivo):
120 min
Number of animals or in vitro replicates:
3 bovine eyes
Details on study design:
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32°C ± 1°C.
On the day of the assay, the MEM without Phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32°C ± 1°C.
The same was performed with the MEM with Phenol red but without the sodium bicar-bonate.
After the arrival of the corneas they were examined and only corneas which were free from defects were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM without Phenol red was filled. The holders were then incubated for one hour in the incubation chamber at 32°C.
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used.
The baseline opacity was measured by placing the holder with the cornea in a spectral photometer and recording the absorption at 570 nm. Opacity is calculated from the meas-ured absorption following the equation stated in chapter “Evaluation”, page 14.
For each treatment group (negative control, positive control and test item), three replicates were used. 750 µl negative control resp. test item resp. positive control solution were ap-plied to each replicate.
According to the characteristics of the test item, the following treatment procedure was performed:
7.3.1 Closed Chamber Method
The “closed chamber-method” is used for non-surface-active liquids. Non-surface-active liquids are used neat.
The respective substance (negative control, positive control or test item) was applied by pipetting 750 µL of the appropriate solution through the refill hole in the holder on the cor-nea. The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with test item.
750 µL of the test item were tested neat.
Exposition time on the corneas was 10 min at 32°C ± 1°C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for an additional two hours at 32°C ± 1°C (post-incubation).
After the post-incubation, the cMEM without phenol red was renewed in both chambers. Then, final opacity value of each cornea was recorded (again by measurement at 570 nm). The cMEM without Phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution (concentration 4 mg/ml) was added to the front chamber.
The chambers were then closed again and incubated for 90 min at 32 ± 1 C. After incuba-tion, the content of the posterior chamber was thoroughly mixed. Then, the permeability was measured with the spectral photometer as optical density of the liquid at 490 nm.

Results and discussion

In vivo

Irritation parameter:
other: IVIS (in vitro irritation score)
Time point:
other: 10 min
not specified

Applicant's summary and conclusion

Interpretation of results:
highly irritating
Migrated information Criteria used for interpretation of results: OECD GHS
It can be concluded that 3 -Methylpyrazole possesses very severe eye irritation potential.
Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of 3-Methylpyrazolby quantitative measurements of changes in opacity and permeability in a bovine cornea.

The test item 3-Methylpyrazolwas brought onto the cornea of a bovine eye which previously had been incubated with cMEM without Phenol red at 32±1°C for one hour and whose opacity had been determined. The test item was incubated on the cornea for 10 minutes at 32±1°C. After removal of the test item and two hours post-incubation, opacity and permeability values were measured.

Physiological sodium chloride solution was used as negative control, sodium hydroxide (10% solution in demineralised water) was used as positive control.

The positive control induced a very severe irritation on the cornea, mean IVIS was 215.7930.

The negative control showed no irritation, mean IVIS was -0.2160.

The test item was tested pure. A mean IVIS of 85.7370 was calculated, corresponding to a classification as very severely eye irritant.

No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

It can be concluded that 3 -Methylpyrazole possesses very severe eye irritation potential.