Registration Dossier

Administrative data

Endpoint:
eye irritation
Remarks:
other: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 January 2012 - 12 January 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The purpose of this study was to determine the eye irritation potential of the test item using the SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes.

The SkinEthic HCE model consists of transformed human corneal epithelial cells 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. 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 item treated tissues (quantitative measurement of tissue viability) relative to the negative control.

One tissue for each treatment group was retained for possible tissue histopathology.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Description: off white powder
Storage conditions: room temperature in the dark

Test animals / tissue source

Species:
other: Reconstructed Human Corneal Epithelium Model
Strain:
other: Not applicable - test was conducted in vitro
Details on test animals or tissues and environmental conditions:
Not applicable. Test was conducted in vitro.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
30 mg
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
No animals.
The test was carried out in vitro.
Details on study design:
The test item was used as supplied.

NEGATIVE AND POSITIVE CONTROLS
The negative control item, Solution A, was used as supplied
Composition of Solution A for 1 litre:
Na2HPO4 0.142 g/L
Glucose 1.802 g/L
HEPES 7.149 g/L
KCl 0.224 g/L
NaCl 7.597 g/L

The positive control item, Sodium Dodecyl Sulphate 2% w/v, was prepared in sterile distilled water.

SKINETHIC HCE MODEL
Supplier: SkinEthic Laboratories, Nice, France

METHODS
PRE-TEST
-Assessment of Direct Test Item Reduction of MTT:
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT to formazan, thus mimicking dehydrogenase activity of the cellular mitochondria of viable cells. This property of the test item is only a problem, if at the time of the MTT test (after the chemical has been rinsed off) there are still sufficient amounts of the test item on or in the tissues. To identify this possible interference, the test item was checked for its ability to reduce MTT directly.
30 mg of test item was added to 1 mL of a 0.5 mg/mL MTT solution and incubated at 37°C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution turned blue, the test item was presumed to have reduced the MTT.
The test item was found to have the ability to directly reduce MTT and therefore the following procedure, employing freeze-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues, was required.
In addition to the normal test procedure, the MTT reducing test item was applied to one freeze-killed tissue. One freeze-killed tissue remained untreated. The untreated freeze-killed tissue demonstrates a small amount of MTT reduction due to residual reducing enzymes.
Freeze-killed tissues were prepared by placing untreated SkinEthic HCE tissues in a freezer (-14 to -30°C) overnight. Once killed, the tissues were stored in the freezer.

-Receipt of Tissues:
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.

-Preparation of Tissues:
Using sterile techniques, 1 mL of maintenance medium at room temperature, was dispensed into the appropriate number of wells of 6-well plates designated 'treatment plates'. Each well was labelled with details of the treatment and the appropriate exposure time. Separate treatment plates were used for the test item, negative and positive controls to avoid the possibility of cross contamination occurring. Before treatment, the 7-Day old tissues were transferred from the 'arrival plates' into the wells of the 'treatment plates' containing the maintenance medium.

MAIN TEST
Triplicate tissues were treated with 30 mg of the test item for 10 minutes. The tissues were dosed at regular timed intervals to allow for the period taken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Triplicate tissues were treated with 30 µL of solution A to serve as negative controls and triplicate tissues were treated with 30 µL of 2% w/v SDS to serve as positive controls. The plates were incubated at 37°C, 5% CO2 in air during the exposure time.
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) without Ca ++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert using a constant soft 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. 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 inserts were rinsed twice with phosphate buffered saline and blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labelled 24-well plate designated `MTT extraction plate' containing 0.75 mL of Isopropanol in each of a sufficient number of wells. An extra 0.75 mL of lsopropanol was added onto each tissue and the plate sealed to prevent lsopropanol evaporation. The plate was wrapped in aluminium foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue.
At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 µL tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded. For each tissue triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of lsopropanol alone was added to three wells designated as 'blanks'. The optical density was measured (quantitative measurement of tissue viability) at 540nm (OD540) using the Anthos 2001 microplate reader.

-Tissue Histology:
One tissue for each treatment group was retained for possible tissue histopathology.
The tissues were carefully cut out of the polycarbonate inserts with a sharp scalpel. The tissues were carefully cut in half. Both halves were placed into a pre-labelled 1.5 mL Eppendorf tube containing 1 mL of 10% Formalin and stored at room temperature.

INTERPRETATION OF RESULTS
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 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

When a test item was shown to directly reduce MTT, and therefore freeze-killed tissues were employed, the results of the MTT assay are corrected as follows:
True viability = mean OD tvt - (OD tkt - OD ukt)
where
OD = optical density at 540 nm
tvt = treated viable tissues
tkt = treated killed tissues
ukt = untreated killed tissues

If the direct reduction by the test item was greater than 30% of the negative control value, additional steps must be taken into account, or the test item was considered to be incompatible with this test system.
If the direct reduction of MTT by the test item was less than 30% of the negative control value, the net OD of the test item treated killed control will be subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
The mean tissue viability for the test item was compared to the negative control and classified as follows:
Relative Mean Tissue Viability Prediction
(percentage of negative control)
Tissue viability <60 Irritant (I)
Tissue viability ≥60 Non-Irritant (NI)

ASSAY ACCEPTANCE CRITERION
The results of the assay are considered acceptable if the following assay acceptance criterion was achieved:
Assay Acceptance Criterion: Positive Control
The assay meets the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is <60% relative to the negative control treated tissues.

Results and discussion

In vivo

Results
Irritation parameter:
other: mean % viability
Basis:
mean
Time point:
other: 10 minutes
Score:
62.7
Reversibility:
other: not applicable
Remarks on result:
other: In vitro result using the Skinethic Reconstructed Human Corneal Epithelial Model
Irritant / corrosive response data:
-Assessment of Direct Test Item Reduction of MTT:
The MTT solution containing the test item turned blue which indicated that the test item directly reduced MTT and therefore the MTT viability assay was performed in parallel on viable and freeze-killed tissues.

-Assessment of Eye Irritation Potential:
The individual and mean OD540 values and mean viabilities for each treatment group are given in Table 1.
The relative mean viability of the test item treated tissues after a 10-Minute exposure period was 62.7%.
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.

Any other information on results incl. tables

Table 1: Assessment of Eye Irritation Potential - Viability of HCE Tissues

Item

Mean Tissue Viability

Mean OD540

Mean OD540 of tissues corrected for MTT direct reduction (-2.80)

% Viability

Negative Control

1.546

1.328

Not applicable

100*

1.110

Positive Control

0.209

0.212

Not applicable

16.0

0.214

Test Item

1.097

1.112

0.832

62.7

1.126

Corrected viability of treated killed tissues = 0.361 (tkt) - 0.081 (ukt) = 0.280

 

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

OD540 = optical density at 540 nm

tkt = treated killed tissues

ukt = untreated killed tissues

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: If the percentage relative mean tissue viability was ≥60% the test item was considered to be a non-irritant (NI)
Conclusions:
According to the study plan followed, the test item was considered to be a Non-Irritant (NI)
Executive summary:

According to the study plan followed, the test item was considered to be a Non-Irritant (NI). The method used was in vitro, determining the eye irritation potential of the test item using the SkinEthic reconstructed Human Corneal Epithelium 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.