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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 23 March 2010 and 25 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

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

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The protocol followed was considered to be a reliable alternative to the in vivo rabbit Draize eye irritation test in a pre-validation study. This study, using human derived keratinocytes which form a corneal epithelial tissue reconstruct, has been recommended by ECVAM for inclusion in a formal international validation study designed to offer a stand alone replacement to the in vivo test. Validation is expected to commence in 2010.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
other: Reconstructed Human Corneal Model
Strain:
other: Reconstructed Human Corneal Model
Details on test animals or tissues and environmental conditions:
Not applicable

Test system

Vehicle:
other: No vehicle used
Controls:
no
Amount / concentration applied:
TEST MATERIAL

-The test Material was applied neat.

-Amounts(s) applied (volume or weight with unit):
Triplicate tissues were treated with 30 µl of the test material.

-Concentration (if solution):
The test material was used as supplied.

VEHICLE
No vehicle used.
Duration of treatment / exposure:
10 Minutes.
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
Not applicable
Details on study design:
TEST SITE
-Area of exposure:
Triplicate tissues were treated with 30 µl of the test material.

-% coverage:
The test material was applied topically to the corresponding tissues ensuring uniform covering.

-Type of wrap used:
None used.

REMOVAL OF TEST SUBSTANCE
-Washing (if done):
At the end of the relevant exposure period, each tissue insert was 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.

-Time after start of exposure:
10 Minutes post exposure.

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

mean OD540 of test material / mean OD540 of negative control x 100 = relative mean tissue viability (percentage of negative control)

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

Tissue viability <60 = Irritant (I)
Tissue viability =60 = Non-Irritant (NI)

Results and discussion

In vitro

Results
Irritation parameter:
other: percentage viability of cells
Value:
45.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

In vivo

Irritant / corrosive response data:
The relative mean viability of the test material treated tissues was 45.8% after a 10-minute exposure.

Any other information on results incl. tables

RESULTS

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 45.8%.

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 OD540 of Individual Tissues

Mean OD540

of Duplicate Tissues

±SD

Viability (%)

Negative Control

0.878

0.888

± 0.014

100*

0.898

Positive Control

0.029

0.029

± 0.000

3.3

0.029

Test Material

0.420

0.407

± 0.018

45.8

0.394


*=      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:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
According to the protocol followed the test material was considered to be an Irritant (I).
Executive summary:

Introduction. 

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

Methods. 

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).

Results. 

The relative mean viability of the test material treated tissues after a 10 minute exposure was 45.8%.

It was considered unnecessary to proceed with tissue histopathology.

Quality criteria. 

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

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