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Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
other: SkinEthic Reconstituted Human Corneal model
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
The study was performed between 08 July 2008 and 10 July 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
SkinEthic Reconstituted Human Corneal Ephitelium model
Deviations:
not specified
Principles of method if other than guideline:
The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994))

Test material

Constituent 1
Chemical structure
Reference substance name:
aluminium(3+) nickel(2+) λ²-cobalt(2+) lithium(1+) tetraoxidandiide
EC Number:
700-042-6
Cas Number:
177997-13-6
Molecular formula:
LiNiCoAlO2 where stoichiometry of Ni+Co+Al equals 1 and ranges are Li = 0.9 – 1.3, Co = 0.01 – 0.2, Ni = 0.75 – 0.99 and Al = 0.01 – 0.2
IUPAC Name:
aluminium(3+) nickel(2+) λ²-cobalt(2+) lithium(1+) tetraoxidandiide

Test animals / tissue source

Species:
other: The SkinEthic RHC model consists of transformed human keratinocytes
Strain:
other: The SkinEthic RHC model consists of transformed human keratinocytes of the cell line HCE
Details on test animals or tissues and environmental conditions:
TEST ANIMALS: Not applicable.

Tissue: The purpose of this study was to determine the eye irritation potential of the test materials using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France)
The SkinEthic RHC model consists of transformed human keratinocytes of the cell line HCE that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye.



ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature
- Humidity (%): Not stated.
- Air changes (per hr): Not stated.
- Photoperiod (hrs dark / hrs light): Not stated.


IN-LIFE DATES: Not applicable.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg of test material.
- Concentration (if solution): Not applicable.


Duration of treatment / exposure:
Triplicate tissues were treated with 30 mg of the test material for 10 minutes.
Observation period (in vivo):
Not applicable.
Number of animals or in vitro replicates:
Not applicable.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the relevant exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco’s Phosphate Buffered Saline (DPBS). Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated ‘holding plate’ containing 300 µl of maintenance medium (at room temperature) until all the tissues were rinsed.
- Time after start of exposure: 10 minutes.


SCORING SYSTEM:
The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues.
After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured (OD540). Data are presented in the form of % viability (MTT conversion relative to negative controls).



TOOL USED TO ASSESS SCORE:
The test material was classified according to the following criteria:
i) If the % relative mean tissue viability was ≥ 60% the test material was considered to not likely to be a severe ocular irritant.
ii) If the % relative mean tissue viability was < 60% the test material was considered to be irritant.

Results and discussion

In vitro

Results
Irritation parameter:
other: % viability
Remarks:
mean value
Run / experiment:
10 minutes exposure period
Value:
7
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other:
Other effects / acceptance of results:
According to the protocol followed the test material was considered to be Irritant (I). It was considered unnecessary to proceed with tissue histopathology.

Any other information on results incl. tables

Assessment of Eye Irritation Potential

        

The mean OD540 values and mean viabilities for each treatment group are given in Table 1.

The relative mean viability of the test material treated tissues after a 10 minute exposure was 7.0%.

It was considered unnecessary to proceed with tissue histopathology.

Qualitative Evaluation of Tissue Viability (MTT Uptake Visual Assessment)

The qualitative evaluation of tissue viability is presented in Table 2

The test material treated tissues appeared white and were considered to be indicative of dead tissue. The negative control treated tissues appeared blue and were considered to be indicative of viable tissue. The positive control material treated tissues appeared blue/white and were considered to be indicative of semi‑viable tissue.

Table1               Assessment of Eye Irritation Potential – Viability of RHC Tissues

Material

Mean OD540

% Viability

Negative Control

0.981

100*

Positive Control

0.454

46.3

Test Material

0.069

7.0

* = The mean viability of the negative tissues is set at 100%

Table2               Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material

Score

Tissue 1

Tissue 2

Negative Control

-

-

Positive Control

+

+

Test Material

++

++

MTT Visual Scoring Scheme of SkinEthic Tissues

-       =   Blue tissue (viable)

+      =   Blue/White tissue (semi viable)

++    =   Tissue completely white (dead)

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to the protocol followed the test material was considered to be Irritant (I).
Executive summary:

Introduction. 

The purpose of this study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

Methods. 

The experimental design of the study consists of a test for Direct Reduction of MTT by the test materials, followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test material for 10 minutes. Triplicate tissues treated with 30 µl of Solution A served as the negative control and triplicate tissues treated with 30 µl of 1% w/v Sodium Dodecyl Sulphate served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured (OD540). Data are presented in the form of % viability (MTT conversion relative to negative controls).

The test material was classified according to the following criteria:

i)                   If the % relative mean tissue viability was ≥ 60% the test material was considered to not likely to be a severe ocular irritant.

ii)                  If the % relative mean tissue viability was < 60% the test material was considered to be irritant.

Results. 

The relative mean viability of the test material treated tissues after a 10 minute exposure was 7.0%.

It was considered unnecessary to proceed with tissue histopathology.

Quality criteria. 

The quality criteria required for acceptance of results in the test were satisfied.

Discussion. 

Under the conditions of the test the test material was considered to be an irritant (I). However, a subsequent in vivo skin irritation test identified the test material as being corrosive to the skin. Therefore the corrosive results of the in vivo skin irritation test should also be taken into consideration when concluding the eye irritation potential of the test material.

Conclusion. 

According to the protocol followed the test material was considered to be Irritant (I).