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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 September 2008 and 04 September 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 not effect the quality of the relevant results.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: SkinEthic Reconstituted Human Corneal model
Deviations:
no
Principles of method if other than guideline:
The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic RHC model and are sufficiently cytotoxic to cause cell death in the underlying cell layers.
GLP compliance:
yes (incl. QA statement)
Remarks:
The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
other: SkinEthic Reconstituted Human Corneal 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 material using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes.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


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37°C
- 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): 30mg of test material
- Concentration (if solution): Not applicable


VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable
- Concentration (if solution): Not applicable
- Lot/batch no. (if required): Not applicable
- Purity: Not applicable
Duration of treatment / exposure:
For the main test, triplicate SkinEthic 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:. Following rinsing, the tissues (two per group) were transferred to a pre labelled 24-well plate designated ‘MTT Loading plate’ containing 300 µl of a 0.5 mg/ml MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 37°C, 5% CO2 in air.
At the end of the incubation period, the tissues were visually examined and the degree of MTT staining evaluated (qualitative evaluation of tissue viability).The optical density was measured (quantitative measurement of tissue viability) at 540nm (OD540) using the Anthos 2001 microplate reader.


TOOL USED TO ASSESS SCORE:
Relative Mean tissue viability
(% negative control) Prediction
Tissue viability <60 Irritant (I)
Tissue viability ≥60 Non-Irritant (NI)

Results and discussion

In vivo

Results
Irritation parameter:
other: % viability
Basis:
mean
Time point:
other: 10 minutes
Score:
30.1
Max. score:
30.1
Reversibility:
not specified
Remarks on result:
other: The relative mean viability of the test material treated tissues after a 10 minute exposure was 30.1%.
Irritant / corrosive response data:
According to the protocol followed the test material was considered to be Irritant (I).
Other effects:
The test material treated tissues appeared white and therefore were considered to be indicative of dead tissue. The negative control treated tissues appeared blue and were therefore considered to be indicative of viable tissue. The positive control material treated tissues appeared blue/white and therefore were considered to be indicative of semi viable tissue.

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 OD540values 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 30.1%.

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

CONCLUSION

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

Assessment of Eye Irritation Potential – Viability of RHC Tissues

Material

Mean Tissue Viability

Mean OD540

% Viability

Negative ControlÅ

0.955

0.917

100*

0.878

Positive ControlÅ

0.212

0.249

27.2

0.286

Test Material

0.273

0.276

30.1

0.278

Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material

Score

Tissue 1

Tissue 2

Negative ControlÅ

-

-

Positive ControlÅ

+

+

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, SkinEthic Laboratories,,) 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 material 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 be non‑irritant.

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

Results. The relative mean viability of the test material treated tissues after a 10 minute exposure was 30.1%.

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 Irritant (I).