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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:
key study
Study period:
03 April 2012 to 05 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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:
other: SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories, Nice France) (10 minute exposure)
Principles of method if other than guideline:
The SkinEthic HCE model consists of transformed human corneal epithelial cells of the cell line HCE that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. The test item is applied directly to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium.

The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic HCE model and are sufficiently cytotoxic to cause cell death in the underlying cell layers.

Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in the test tiem treated tissues (quantitative measurement of tissue viability) relative to the negative control.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Cis-hex-3-en-ol, LFA
- Physical state: clear, colourless liquid
- Analytical purity: 98.95 %
- Lot/batch No.: 1Z50650
- Expiration date of the lot/batch: 06 June 2015
- Storage condition of test material: room temperature in the dark under nitrogen

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
SkinEthic HCE Model (0.5 cm2)
Supplier: SkinEthic Laboratories, Nice, France
Date received: 03 April 2012

On arrival, the SkinEthic HCE tissues (Day 6 cultures), were stored at room temperature prior to transferring into 24-well plates designated ‘arrival plates’ containing 300 µL of maintenance medium. It was important to ensure that there were no air bubbles present under the tissue inserts. The tissues were incubated overnight at 37 °C, 5 % CO2 in air.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
Test item: 30 µL of the test item
Negative control: 30 µL of Solution A
Positive control: 30 µL of 2 % w/v SDS
Duration of treatment / exposure:
Main test: 10 min
Number of animals or in vitro replicates:
Triplicate tissues were treated with the test item.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the relevant exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++. Rinsing was achieved by fillieng and emptying each tissue insert using a constant stream of DPBS to gently remove any residual test item. 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 min

SCORING SYSTEM:
The mean OD 540 values of the duplicate tissues were calculated. Each of these OD540 values had already been corrected for blanks by the microplate reader.

The relative mean tissue viability (percentage of the negative control) was calculated as follows:

Relative mean tissue viability (%)= (mean OD540 of test item/ mean OD540 of negative control) x 100

The mean tissue viability for the test item was compared to the negative control and classified according to the following:

Relative mean tissue viability Prediction
(percentage of negative control)
Tissue viability <60 Irritant (I)
Tissue viability ≥60 Non-irritant (NI)

Results and discussion

In vitro

Results
Irritation parameter:
other: relative mean viability (%)
Run / experiment:
Mean of two tissues
Value:
2.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Any other information on results incl. tables

Assessment of direct test item reduction of MTT

The MTT solution containing the test item remained yellow which indicated that they test item did not directly reduce MTT.

Assessment of eye irritation potential

Assessment of eye irritation potential- viability of HCE tissues

Item

OD540of individual tissue

Mean OD540

Relative mean viability (%)

Negative control

0.872

0.850

100*

0.828

Positive control

0.081

0.082

9.6

0.082

Test item

0.025

0.025

2.9

0.025

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

The relative mean viability of the test item treated tissues after a 10-min exposure period was 2.9 %.

 

It was considered unnecessary to proceed with tissue histopathology.

Assay acceptance criterion

The quality criterion required for the acceptance of results in the test was satisfied.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Remarks:
(CLP)
Conclusions:
The purpose of this study was to determine the eye irritation potential of the test item using the Skin Ethic reconstructed Human Corneal Epithelium model after a treatment period of 10 minutes. The relative mean viability of the test item treated tissues after a 10-minute exposure period was 2.9 %. According to the study plan followed the test item was considered to be a irritant.
Executive summary:

Introduction: The purpose of the study was to determine the eye irritation potential of the test item using the SkinEthic reconstructed Human Corneal Spithelim model 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 (3 -[4,5 -dimethylthizol-2 -yl]-2,5 -tetrazolium bromide) by the test item followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 µl of the test item 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 2% w/v Sodium Dodecyl Sulphate (SDS) 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 absorbancy of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. Teh optical density was measured at 540 nm (OD540). Data are presented in the form of percentage viability (MTT conversion relative to negative controls).

The test item was classified according to the following criteria:

i) If the percentage relative mean tissue viability was ≥60% the test item was considered to be non-irritant (NI).

ii) If the percentage relative mean tissue viability was <60% the test item was considered to be an irritant (I).

Results: The relative mean viability of the test item treated tissues after a 10 -minute exposure period as 2.9%.

It was considered unnecessary to proceed with tissue histopathology.

Quality criterion: The quality criterion required for acceptance of results in the test was satisfied.

Conclusion: The test item was considered to be an irritant (I).