Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Feb - 13 Mar 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted according to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted 2009
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
adopted 2010
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solution
Details on test material:
- Name of test material (as cited in study report): GSID 3056-1
- Physical state: solid, brownish
- Analytical purity: 60.8 area-% (HPLC; analytical report 11L00523)
- Lot/batch No.: 1470 VB04
- Storage condition of test material: room temperature
- Stability under storage conditions over the study period: guaranteed by the sponsor (sponsor holds this responsibility)

Test animals / tissue source

Species:
other: bovine eyes, isolated bovine cornea
Strain:
not specified
Details on test animals or tissues and environmental conditions:
- Bovine eyes: Schlachthof Bensheim, Bensheim, Germany
- Cornea Holder: Supplier - LAB Research, Veszprem, Hungary
- Opacitometer: Kit BASF-OP2.0, BASF Ludwigshafen, self-construction

Test system

Vehicle:
water
Controls:
other: negative control: concurrent vehicle; positive control: 20% (w/v) imidazole solution
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20 %
Duration of treatment / exposure:
4 hours
Observation period (in vivo):
none
Number of animals or in vitro replicates:
3 cornea per group
Details on study design:
REMOVAL OF TEST SUBSTANCE
- after 4 hours

SCORING SYSTEM:
- Measurement of corneal opacity: Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer:
- Determination of permeability: For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (5 mg/mL for solid test substances) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

DATA EVALUATION
The mean corneal opacity and permeability values of each treatment group were used to calculate an In Vitro Irritancy Score (IVIS).
The opacity change per cornea was calculated by subtracting the initial from the final opacity.
• opacity change per cornea = final opacity - initial opacity
Subsequently, the corrected opacity change was calculated by subtracting the mean opacity change of the negative control.
• corrected opacity change = opacity change - mean opacity change of NC
Finally, the mean opacity value for each test substance could be determined as the mean of all corrected opacity changes per treatment group.
• mean opacity value = mean of all corrected opacity changes per group

CALCULATION ON THE IN VITRO IRRITANCY SCORE (IVIS)
The IVIS could be calculated per treated cornea and finally the mean IVIS per treatment group ± standard deviation was determined:
• IVIS per cornea = corrected opacity change + 15 * corrected permeability OD change
• IVIS per treatment group = mean opacity value + 15 * mean permeability OD value

ACCEPTANCE CRITERIA
- IVIS > 55 <=>  risk of serious damage to the eyes
- IVIS <= 55 <=> no risk of serious damage to the eyes
In case one of the above given acceptance criteria is not covered, repetition of the test is 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 no clear prediction is possible, e.g. different predictions are obtained for single corneas, the test will be repeated.

Results and discussion

In vivo

Results
Irritation parameter:
other: IVIS (in vitro irritancy score)
Basis:
mean
Remarks:
Cornea 1-3
Time point:
other: 4 hours
Score:
-1.9
Irritant / corrosive response data:
The test item did not induce effects indicative of serious eye damage in the in vitro test under the conditions chosen. Negative and positive control values were in range of historical control data.
Other effects:
none

Any other information on results incl. tables

Table2: BCOP test results

 Test Substance  Mean Opacity Value  Mean Permeability Value  In Vitro Irritancy Score
 test item  -2.0  0.007  -1.9
 NC  5.5  -0.001  5.5
 PC  80.2  2.804  122.2

Applicant's summary and conclusion

Conclusions:
The test method according to the regulatory accepted protocol at the time of reporting does not allow for the evaluation of eye irritation. The result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed.