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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:
The study was performed between 06 July 2010 and 08 July 2010.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: In vitro eye irritation study used as an indication of whether a substance is likely to be an irritant to the eye. Pre-validated study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: SkinEthic Reconstituted Human Corneal model
Deviations:
not applicable
Principles of method if other than guideline:
The purpose of this study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France) 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.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification: CAS No 77-58-7
Description: clear colourless liquid
Batch number: LA6K05N001
Date received: 26 March 2010
Expiry date: 26 March 2011
Storage conditions: approximately 4°C in the dark under nitrogen

The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.

Test animals / tissue source

Species:
other: The SkinEthic RHC model consists of transformed human keratinocytes of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye.
Strain:
other: n/a
Details on test animals or tissues and environmental conditions:
SKINETHIC HCE MODEL
Supplier: SkinEthic Laboratories, Nice, France
Date Received: 06 July 2010

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: Triplicate tissues were treated with 30 µl of solution A to serve as negative controls and triplicate tissues were treated with 30 µl of 1% w/v SDS to serve as positive controls.
Amount / concentration applied:
30 µl of the test material
Duration of treatment / exposure:
10 minutes
Number of animals or in vitro replicates:
n/a
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).
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
The mean OD540 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 viabilities (percentage of the negative control) were calculated as follows:

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

The mean tissue viabilities for the test material was compared to the respective untreated negative control and classified according to the following table:
Relative Mean tissue viability (percentage of negative control) Prediction
Tissue viability <60 Irritant (I)
Tissue viability ≥60 Non-Irritant (NI)

TOOL USED TO ASSESS SCORE: The optical density was measured (quantitative measurement of tissue viability) at 540nm (OD540) using the Anthos 2001 microplate reader.


Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: % Viability
Basis:
mean
Time point:
other: 10 minutes
Score:
91.3
Reversibility:
not specified
Irritation parameter:
other: Mean OD540
Basis:
mean
Time point:
other: 10 minutes
Score:
0.889
Reversibility:
not specified
Irritant / corrosive response data:
The relative mean viability of the test material treated tissues after a 10 minute exposure was 91.3%.

Any other information on results incl. tables

Assessment of Direct Test Material Reduction of MTT

The test material was not able to directly reduce MTT.

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 91.3%.

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 and negative control material treated tissues appeared blue which was considered to be indicative of viable tissue. The positive control material treated tissues appeared blue/white which was considered to be indicative of semi-viable tissue.

Assay Acceptance Criterion

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

Table 1              Assessment of Eye Irritation Potential – Viability of RHC Tissues

Material

Mean Tissue Viability

Mean OD540

Viability (%)

Negative Control

0.995

0.974

100*

0.953

Positive Control

0.342

0.396

40.7

0.449

Test Material

0.906

0.889

91.3

0.871

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


Table 2              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:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to the protocol followed the test material was considered to be a Non-Irritant (NI).
Executive summary:

The purpose of this study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France) 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.

The experimental design of the study consists of a test for direct reduction of MTT (3 [4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) by the test material followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 µl 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. The 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 material was classified according to the following criteria:

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

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

The relative mean viability of the test material treated tissues after a 10 minute exposure was 91.3%.

It was considered unnecessary to proceed with tissue histopathology.

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

According to the protocol followed the test material was considered to be a Non Irritant (NI).