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Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 16 September 2015 to 18 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 437. The study was conducted on the registered substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
The positive control In Vitro Irritancy Score fell slightly short of the expected historical values. This was thought not to have affected the purpose or integrity of the study.
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
yes
Remarks:
The positive control In Vitro Irritancy Score fell slightly short of the expected historical values. This was thought not to have affected the purpose or integrity of the study.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Cordierite
EC Number:
603-401-4
Cas Number:
1302-88-1
Molecular formula:
Mg2[Al4O3(SiO3)5]
IUPAC Name:
Cordierite
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: Cordierite, sintered, synthetic
CAS Number: 1302-88-1
Physical state: Pale brown powder
Substance type: UVCB
Storage conditions: Room temperature in the dark

Test animals / tissue source

Species:
other: bovine cornea
Strain:
other: bovine cornea
Details on test animals or tissues and environmental conditions:
Not applicable

Test system

Vehicle:
physiological saline
Controls:
other:
Amount / concentration applied:
TEST MATERIAL
- Concentration (if solution): 20% w/v solution in 0.9% w/v sodium chloride solution.

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9% w/v sodium chloride solution
- Lot/batch no. (if required): 3011424
- Purity: 0.9%
Duration of treatment / exposure:
Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
Not applicable
Details on study design:
The negative control item, 0.9% w/v sodium chloride solution, was used as supplied.
The positive control item, Imidazole, was used as a 20% w/v solution in 0.9% w/v sodium chloride solution.

Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer (Appendix 1). The average opacity for all corneas was calculated.
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea.
Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
2.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

In vivo

Irritant / corrosive response data:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

Any other information on results incl. tables

The In Vitro irritancy scores are summarized as follows:

Treatment In Vitro Irritancy Score
Test Item 2.5
Negative Control 2.8
Positive Control 65.1

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item produced an IVIS ≤ 3 when tested under a Bovine Corneal Opacity and Permeability (BCOP) Assay implying that it is not categorized as irritant to the eye. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.
Executive summary:

The potential of the test item to be irritant to the eye was determined in accordance with the OECD Guideline for Testing of Chemicals 437. The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS)

The BCOP produced an IVIS ≤ 3 implying that it is not categorized as an irritant to the eye. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.