Registration Dossier

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:
other: GLP compliant guideline study, available as unpublished report, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
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)
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 1152/2010
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Aminopropyl Vinyl Ether
- Test-substance No.: 12/0567-1
- Lot/batch No.: C520/070/2012/2
- Physical state: liquid
- Purity: 99.6 corrected area%
- Homogeneity: The test substance was homogeneous by visual inspection
- Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility
- pH ca. 11 (undiluted test substance)

Test animals / tissue source

Species:
other: Isolated bovine cornea
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- Source: Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim

The test system (target tissue) is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 ± 1

Test system

Vehicle:
water
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
Duration of treatment / exposure:
4 hours
Observation period (in vivo):
Measurement of final corneal opacity: None
Determination of permeability: 90 ± 5 min
Number of animals or in vitro replicates:
3 corneas per treatment group
Details on study design:
EXPERIMENTAL PROCEDURE
Preparation of the bovine corneas and measurement of initial corneal opacity
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 pre-warmed 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 pre-warmed 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 < 512 opacity units were discarded. Corneas with opacity values close to the median value of all corneas were selected as negative control (NC). The remaining corneas were then distributed into treatment and positive control groups.
Each corneal holder was uniquely identified with a number on the chambers.

Application of the test substance and washing
Each treatment group (test substance, NC and PC) consisted of 3 corneas.
Before application the medium in the anterior chamber was removed using a syringe.
750 μL of the undiluted liquid test substance was applied into the anterior chamber using a pipette.
Control tissues were concurrently applied into the anterior chamber with 750 μL of de-ionized water (negative control, NC) or with 750 μL of 1% (w/v) solution of sodium hydroxide in de-ionized water (positive control, PC) using a pipette.
The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes (liquids and surfactants). The test substance, NC and PC were then 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).

Post-exposure incubation for liquid test substances and surfactants
The corneas were incubated for further 2 hours at about 32 °C. After the incubation period the medium was removed and both chambers were then refilled with fresh Eagle’s MEM.

Measurement of final 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 (4 mg/mL for liquid test substances and surfactants) 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 measured data of the opacitometer and spectrophotometer were copied electronically into EXCEL data spreadsheets which carried out the
necessary calculations.

Calculation of the corneal opacity value
First, the opacity was calculated using the opacitometer specific algorithm, with a and b device specific and with Io being the illuminance (lux)
through the empty corneal holder with windows and liquid and I being the illuminance (lux) through the holder with the cornea.

• opacity value = a * Io/I + b

Then 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 of permeability value
First, the OD490 value was calculated by subtracting the mean blank OD490 (blank = Eagle´s MEM w/o phenol red) from the OD490 of each cornea.

• OD490 value = OD490 - mean blank OD490

If the OD490 value of the treated cornea was above 1.5, the OD490 of a 1:5 dilution was used to calculate the OD490 value:

• OD490 value = 5 * (OD490 of a 1:5 dilution - mean blank OD490)

Subsequently, the corrected OD490 value was calculated by subtracting the mean OD490 value of the negative control.

• corrected OD490 value = OD490 value - mean OD490 value of NC

Finally, the mean OD490 value for each test substance could be determined as the mean of all corrected OD490 values per treatment group.

• mean OD490 value = mean of all corrected OD490 values per group

Calculation of 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
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 no clear prediction is possible, e.g. different predictions are obtained for single corneas, the test will be repeated.

EVALUATION OF RESULTS
The following rules of assessment apply:
IVIS Prediction
> 55 risk of serious damage to the eyes
≤ 55 no risk of serious damage to the eyes

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: Opacity score
Basis:
mean
Score:
156
Irritation parameter:
other: Permeability score
Basis:
mean
Score:
4.771
Irritation parameter:
other: In Vitro Irritancy score (IVIS)
Basis:
mean
Score:
227.6
Irritant / corrosive response data:
Based on the observed results and applying the evaluation criteria it was concluded, that causes serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.

Any other information on results incl. tables

in vitro test

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye)
Remarks:
Migrated information Criteria used for interpretation of results: EU