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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:
25 August 2009 to 27 August 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP. Study follows an appropriate protocol for in vitro eye irritation potential without any deviations from the study design.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The aim of the 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. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Manganese oxide
EC Number:
215-695-8
EC Name:
Manganese oxide
Cas Number:
1344-43-0
Molecular formula:
MnO
IUPAC Name:
manganese oxide
Details on test material:
- Name of test material : Manganese (II) oxide
- Molecular formula : MnO
- Substance type: Green powder
- Physical state: Solid
- Analytical purity: Certificate of analysis included within report
- Impurities (identity and concentrations): MnO2 0.46 %, Fe 56 ppm, Ins ac 15 ppm, H2O105 0.01%
- Composition of test material, percentage of components: Mn 77.8 %
- Purity test date: 29/09/08
- Lot/batch No.: 104833-06
- Storage condition of test material: Room temperature in the dark
- Other: Particles size < 45 µm 0.40%

Test animals / tissue source

Species:
other: Reconstituted Corneal Epithelium model
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Not applicable

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied : Tissues were treated with 30 mg of the test material.

VEHICLE
Test material was used as supplied

CONTROLS:
- Amount(s) applied: 30 µL of Solution A was applied as a negative control and 30 µL of of SDS 1.0% (w/v) as a positive control. Solution A was comprised of Na2HPO4 0.142 g/L, Glucose 1.802 g/L, HEPES 7.149 g/L, KCl 0.224 g/L and NaCl 7.597 g/L.
Sodium Dodecyl Sulphate (SDS) was prepared as a 1% w/v solution in sterile distilled water.
Duration of treatment / exposure:
Cultures were exposed for 10 minutes to the test material.
Observation period (in vivo):
Skin cultures were examined after three hours.
Number of animals or in vitro replicates:
All test substances were tested in triplicate (including controls)
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Cultures were rinsed 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 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 tissues were rinsed..
- Time after start of exposure: 10 minutes

STAINING PROCEDURE:
Staining: After rinsing, the tissues (two per group) were transferred into a pre-labelled 24-well plate containing 300 µL of a 0.5 mg/mL MMT solution prepared in maintenance medium. The MMT loading plate was placed into an incubator for approximately three hours at 37 °C, 5 % CO2 in air.


SCORING SYSTEM:

- Tissue viability (OD): After incubation with MMT, the tissues were visually examined and the degree of MMT staining was evaluated (qualitative evaluation of tissue viability). The inserts were blotted on absorbent paper to remove residual MMT and transferred to a pre-labelled 24-well plate containing 0.75 mL of Isopropanol in each of a sufficient number of wells. An extra 0.75 mL of Isopropanol was added onto each tissue and the plate sealed to prevent Isopropanol evaporation. The plate was wrapped in aluminium foil and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue.

- Histology: If deemed necessary, the histopathology of the remaining insert was examined. 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 Isopropanol alone was added to three wells designated as blanks. The optical density was measured (quantitative measurement of tissue viability) at 540 nm (OD540) using Anthos 2001 microplate reader. One tissue for each treatment groups was retained for assessment of tissue histopathology. Tissues were cut out of the polycarbonate inserts with a sharp scalpel. The tissues were 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.
To determine histopathological changes the tissues are observed for any changes in thickness or organisation of the cells. The negative control tissues should have a constant thickness devoid of terminally differentiated cell, and feature a regular and compact shape. Cells must maintain attachment via multiple desmosomes. Positive control tissues should have a disintegration of most of the upper cell layers of the epithelial tissue. The remaining basal cells should be loosely attached to the polycarbonate substratum.

-Interpretation of data: Quantitative MMT Assessment (percentage of viable tissue)
The relative mean tissue viabilities are compared to the mean of the untreated negative control tissues (n = 2). The relative mean viabilities are calculated using the following: (mean OD540 of test material/mean OD540 of negative control) x 100

TOOL USED TO ASSESS SCORE: Anthos 2001 microplate reader.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: Mean tissue viability.
Run / experiment:
#1
Value:
0.989
Vehicle controls validity:
other: Not applicable.
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
After 10 minutes exposure.
Irritation parameter:
other: Mean tissue viability.
Run / experiment:
#2
Value:
1.004
Vehicle controls validity:
other: Not applicable.
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
After 10 minutes exposure.
Other effects / acceptance of results:
The relative mean viability of the test material treated tissues after a 10 minute exposure was 98.7%.

-Interpretation of data: Quantitative MMT Assessment (percentage of viable tissue)
The relative mean tissue viabilities are compared to the mean of the untreated negative control tissues (n = 2). The relative mean viabilities are calculated using the following: (mean OD540 of test material / mean OD 540 of negative control) x 100.

The test material was found to not directly reduce MMT. It was deemed unnecessary to examine tissue histopathology.

Any other information on results incl. tables

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

 

Material

Mean Tissue Viability

Mean OD540

Viability (%)

Negative control

1.042

1.010

100*

0.977

Positive control

0.282

0.218

21.6

0.154

Test material

1.004

0.997

98.7

0.989

* 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

-

-

- = Blue tissue (viable)

+ = Blue/White tissue (semi viable)

++ = Tissue completely white (dead)

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Criteria used for interpretation of results: other: Criteria set out in the protocol of this study.
Conclusions:
According to the protocol followed, the test material was found to be a Non-Irritant (NI) to the reconstituted human corneal epithelial model, SkinEthic.

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