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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reliable GLP guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Ludwigshafen, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
Please refer to confidential details on test material.

Test animals / tissue source

Species:
other: bovine eyes
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
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: 12 - 60 months).

Test system

Vehicle:
other: De-ionized water
Controls:
other: not applicable
Amount / concentration applied:
- Amount applied: 750 µL
- Concentration: 20% (w/v) suspension in de-ionized water
Duration of treatment / exposure:
4 hours
Observation period (in vivo):
4 hours
Number of animals or in vitro replicates:
3 bovine eyes/test material
3 bovine eyes/positive control
3 bovine eyes/negative control
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 consist 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 < 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 (PC) groups. Each corneal holder was uniquely identified with a number on the chambers.

Application of the test substance and washing
Before application the medium in the anterior chamber was removed using a syringe. A volume of 750 μL of the 20% (w/v) test-substance preparation (non-surfactant) was applied directly to the epithelial surface of the cornea using a pipette (open chamber method). Control tissues were concurrently applied into the anterior chamber with 750 μL of de-ionized water (NC) or with 750 μL of 20% (w/v) solution of Imidazole in de-ionized water PC) using a pipette. The corneas were incubated in a horizontal position at about 32 °C for approximately 4 hours (non-surfactant solids). The 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). Because the test substance could not be removed using a syringe, the epithelium was rinsed with the open chamber method.

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 (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).

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 was 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 was 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 was 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 > 55, prediction: risk of serious damage to the eyes
IVIS ≤ 55, prediction: no risk of serious damage to the eyes

HISTORICAL CONTROL DATA
Historical control values of negative and positive controls, gathered over an appropriate time period are used to derive suitable acceptance criteria for the test system.

Results and discussion

In vivo

Results
Irritation parameter:
other: IVIS
Basis:
other: mean from 3 corneas
Time point:
other: 4 hours
Score:
5.4
Reversibility:
other: not applicable
Remarks on result:
other: The test item did not cause serious eye damage

Any other information on results incl. tables

The IVIS value determined for the test item did not indicated a test item-related risk of serious damage to eyes. The PC item demonstrated appriate sensitivity of the test system.

Table1: Test results

 

Opacity score (mean+/- SD)

Permeability score (mean+/- SD)

IVIS (mean +/- SD)

Test substance

5.5 +/- 1.6

-0.004 +/- 0.002

5.4 +/- 1.6

NC

1.5 +/- 3.2

0.201 +/- 0.358

4.5 +/- 3.9

PC

72.2 +/- 6.4

3.847 +/- 0.959

129.9 +/- 16.4

IVIS: In Vitro Irritance score

PC: positive control

NC: negative control

SD: Standard deviation

BCOP: bovine corneal opacity and permeability

The mean permeability score of the NC is out of the historical range, because the value of one cornea is exceptionally high. Therefore NC-corrections of the permeability values and consistently opacity values were not performed for the PC and test substance. Due to the unambiguous results of the test substance even without NC-subtraction and because all other acceptance criteria were met in the test, the evaluation of the study is not expected to be influenced by this deviation.

Applicant's summary and conclusion