Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 6th, 2007 to September 19th, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to OECD and in accordance with GLP. The study material is well characterized

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
White powder stored at room temperature and protected from exposure to light.

Test animals / tissue source

Species:
other: Bovine
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Bovine eyes were obtained froma local abattoir as a by-product from freshly slaughtered animals (J.W. TREUTH & SONS, Inc. Baltimore, MD). The eyes
were excised and then placed in Hanks' Balanced Salt Solution, containing Penicillin/Streptomycin (HBSS), and transported to the laboratory on ice
packs. Immediately upon receipt of the eyes in the laboratory, preparation of the corneas was initiated.

Test system

Vehicle:
physiological saline
Controls:
yes
Amount / concentration applied:
As instructed by the Sponsor, the test article was administered to the test system as a 20% (w/v) dilution in 0.9% USP saline. The test article dilution
was prepared by weighing 1211 mg of the test article into a prelabeled conical tube. Saline was added until a 20% (w/v) dilution was achieved and the
conical was vortexed for approximately 1 min. prior to application.
Duration of treatment / exposure:
Treatment of corneas

Immediately prior to treatment, the medium was removed from the anterior compartment of the
holder using a pipette tip attached to a vacuum pump. Seven hundred and fifty µl (750 µl) of test substance, negative control (0.9%
sodium chloride solution) or positive control material (Imidazole 20% w/w solution) was
introduced in to the anterior part of the holder. The test substance at 20% w/w formed a white
paste and was added to the anterior compartment using a 2 ml syringe with the end cut off. The
test substance was applied via the anterior window and gently spread over the cornea with a
spatula. Following application the anterior compartment was plugged.

For the negative and positive control the holder was slightly rotated to ensure uniform distribution of the test
substance over the surface of the cornea. Each holder was then incubated in a horizontal position
at 32°C ± 2°C for 4 hours ± 5 minutes in a waterbath.

Following incubation, the test substance was removed and the epithelial surface of the cornea
washed, at least three times or until the wash medium was clear, with cMEM added via the holes
on the top of the holder. After each wash, the wash medium was removed using a pipette tip
attached to a vacuum pump. For the test substance, the anterior window was removed and the
cornea gently washed five times. The anterior window was then replaced. Following
completion of the wash step, the medium was removed from both compartments which were refilled
with fresh cMEM. The posterior compartment was re-plugged and the opacity of each
cornea measured and recorded. The opacity values obtained at this stage were used in
calculating the final In Vitro Irritancy Score.
Observation period (in vivo):
Opacity measurements performed immediately after exposure period ends.
Number of animals or in vitro replicates:
5 corneas
Details on study design:
After the tratment of the corneas, described above, the opacity of each cornea measured and recorded. The opacity values obtained at this stage were used in calculating the final In Vitro Irritancy Score.

The next stage is to perform the permeability assay:

Following the final opacity measurement, the medium was removed from the anterior compartment of the holder. One ml (1ml) of the sodium fluorescein solution was added to the anterior compartment using a micropipette, the compartment was plugged and the corneas incubated in a horizontal position at 32°C ± 2°C for 90 ± 5 minutes in a waterbath.

Followingthis incubation, the medium in the posterior compartment was mixed by drawing approximately 2.5 ml gently up and down a 5 ml syringe, with a needle attached, three times. An aliquot of the mixed medium from the posterior compartment was removed and transferred to a 1cm path length cuvette.

A spectrophotometer was adjusted to read at 490nm (OD490) and a sample of cMEM read (OD =0.065). The spectrophotometer was blanked using this solution prior to reading the permeability samples. Any solution giving an OD490 value above 1.8 was diluted 1 in 5 with cMEM.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: Cornea opacity
Basis:
mean
Time point:
other: 4 hrs.
Score:
8.5
Irritation parameter:
other: Permeability
Basis:
mean
Time point:
other: 4 hrs.
Score:
0.002
Irritation parameter:
other: In Vitro Score
Basis:
mean
Time point:
other: 4 hrs.
Score:
8.5

Any other information on results incl. tables

The pH of the test sample formulation, measured using universal pH sticks, was approximately 4.5

Opacity:

  • The change in the opacity of each cornea was calculated by subtracting the initial basal opacity from the post-treatment opacity measurement.
  • The mean change in opacity for the negative control corneas was calculated and was subtracted from the change in opacity of each treated cornea to obtain the corrected opacity value.
  • The mean corrected opacity change value of each treatment group (of three corneas) was calculated from the individual corrected opacity values of the treated corneas.

Permeability:

  • The corrected permeability value (OD490) of each treated cornea was calculated by subtracting the mean negative control cornea value from the permeability value of each cornea.
  • The mean corrected permeability value of each treatment group was calculated from the individual corrected permeability values of the treated corneas.

The In Vitro Irritancy Score was calculated using the following formula:

  • In Vitro Irritancy Score = Corrected Opacity Value + (15 x Corrected OD490 Value)
  • The In Vitro Irritancy Score was calculated for each individual treatment and positive control cornea.
  • The mean In Vitro IrritancyScore value for each treatment group was calculated from the In Vitro IrritancyScores of each individual cornea in the treatment group.

A substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

Applicant's summary and conclusion

Interpretation of results:
slightly irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test article recorded having an in vitro score of 8.5, classifying it as a mild irritant per criteria of test method.