Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to internationally recognised guideline; under GLP conditions.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Ocular Irritation Assay for Irritant/Non-Irritant classification using the EpiOcular Human Tissue Model.
Deviations:
no
Principles of method if other than guideline:
This method has been designed to predict and classify the occular irritation potential of a chemical by assessment on its effect on EpiOcular, a non-ocular human epithelial cell model. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after test material exposure. The EpiOcular Eye Irritation Test (EIT) protocol has been pre-validated by COLIPA and is currently under validation by ECVAM (validation still ongoing).
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 11-HN
- Physical state: solid
- Expiration date of the lot/batch: 19/11/2013
- Storage condition of test material: room temperature

Test animals / tissue source

Species:
other: in vitro: EpiOcular human tissue model
Strain:
other: n/a
Details on test animals or tissues and environmental conditions:
The test was performed with the EpiOcular corneal model (MatTek). The model consists of normal, human-derived epidermal keratinocytes that have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. The epidermal cells, which are cultured on specially prepared cell culture inserts using serum free medium differentiate to form a multi-layered structure which closely parallels the corneal epithelium.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: additional tissue was available for negative and positive control groups.
Amount / concentration applied:
Test Material:
50 mg of test material was applied directly to the surface of the EpiOcular tissue.

Positive control:
50 ul methyl acetate.

Negative control:
50 ul of distilled water.
Duration of treatment / exposure:
90 minutes
Details on study design:
Pre-test: to check the MTT reducing ability of the test material, 50mg of the test material was mixed with 500 ul of MTT medium and incubated for 2 hours at room temperature.

Main test: The test was performed on each dose group in duplicate, together with negative and positive controls. After dosing, the tissues were incubated for 90 +/- 5 minutes at 37 +/- 1 degrees C, 5% CO2 / 95% air. After the exposure period, tissues were rinsed with DPBS to remove test material. Tissues were then transferred to an post-soak plate (with prewarmed assay medium) and incubated for 12 minutes at room temperature. These tissues were further transferred to a post treatment plate (containing prewarmed assay medium) and incubated for 18 +/- 0.25 h at 37 +/- 1 degrees C, 5% CO2 / 95% air. After this incubation period, determination of the cytotoxic effect was performed. Tissues were transferred into an MTT assay plate (containing prewarmed MTT solution) and incubated for 180 +/- 10 minutes at 37 +/- 1 degrees C, 5% CO2 / 95% air. After incubation, tissues were transferred to 'extraction plates' containing 2ml of isopropanol and kept overnight (in sealed bags to reduce evaporation of isopropanol) in the dark at 2-8 degrees C. Tissue were then discarded and the OD of the extracts were measured using a spectrophotometer.

Results and discussion

Any other information on results incl. tables

Pre-test: The test material did not cause the MTT to change colour to blue/purple indicating that the test material does not have MTT-reducing capability.

Results of the main test are shown in the table below:

Name Negative Control Positive Control Test Material
Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2
Mean OD 550nm (blank corrected) 0.997 1.173 0.015 0.013 0.738 0.754
Total mean OD 550nm of 2 replicate tissues (blank corrected) 1.085 0.014 0.746
SD OD 550nm 0.1 0 0.01
Relative tissue viability (%) 91.9 108.1 1.4 1.2 68 69.5
SD tissue viability (%) 11.5 0.1 1.1
Mean tissue viability (%) 100 1.3 68.8

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
Under the conditions of this study, the test material showed no irritant effects producing a mean relative tissue viability of 68.8%. Therefore the test material is considered to be a non-irritant. The positive and negative control groups achieved the expected result and confirm the validity of this study.
Executive summary:

This study protocol is based on pre-validated ocular Irritation Assay for Irritant/Non-Irritant classification using the EpiOcular Human Tissue Model guideline. EpiOcular tissue were exposed to 50 mg of test material (applied directly to the tissue without vehicle) for 90 minutes. After exposure tissues were rinsed and further incubated for an additional 12 minutes at room temperature then 180 minutes at 37 degrees C. After this incubation period, determination of the cytotoxic effect was performed by transferring tissues to plates containing MTT medium and incubated for 2 hours. Extraction of tissue contents were performed by placing tissues in isopropanol overnight. Tissues were discarded and the OD of the extracts were measured using a spectrophotometer to determine cytotoxicity. Under the conditions of this study, the test material showed no irritant effects producing a mean relative tissue viability of 68.8%. The test material is considered to be a non-irritant. The positive and negative control groups achieved the expected result and confirm the validity of this study.