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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
There are no official national or international guidelines for the EpiOcularTM test yet; however, the study was performed according to the methods described in the following publications:
- MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct: Procedure details, Version 3.1a of February 10, 2010.
- Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing In Vitro Eye Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/Inter Laboratory Evaluation of EpiOcular Assay for Chemicals. Poster # 378, Society of Toxicology, March 2009.
In addition the study follows the testing strategy for determination of eye irritation/corrosion as given in the following OECD guideline:
- OECD Guideline for Testing of Chemicals No. 405, April 24, 2002 (“Acute Eye Irritation/Corrosion”)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Zinkphthalocyaninsulfonat
- Test-substance No.: 11/0369-1
- Lot/batch No.: 176647

Test animals / tissue source

Species:
other: EpiOcular™ OCL-200 kit
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- Source: MatTek Corp., Ashland MA, USA

Three dimensional human cornea model
The EpiOcular™ model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37
- Photoperiod: dark

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: historical control values
Amount / concentration applied:
TEST MATERIAL:
- Amount(s) applied (volume or weight with unit): bulk volume of 50 μL
Duration of treatment / exposure:
90 minutes
Observation period (in vivo):
18 hours
Details on study design:
EXPERIMENTAL PROCEDURE
Direct MTT reduction
To assess the ability of the test material to directly reduce MTT a pretest was performed as described below.
The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (highly de-ionized water) was tested concurrently.
If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.
The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.
In case that direct MTT reduction occurred, two freeze-killed control tissues were treated with, each, the test article and the negative control, in the same way as described in section “Basic procedure”, additionally.

Basic procedure
Two tissues were treated with the test substance, the PC and NC, respectively.
There are two separate protocols for liquids and solids, differing in exposure time and postincubation period. Due to the physical condition of the test substance the protocol for solids was applied.

Pre-incubation of the tissues
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at standard culture conditions for 16 – 24 hours (pre-incubation).

Pretreatment of the tissues
After the pre-incubation the tissues were pre-treated with 20μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.

Application of the test substance
Using a sharp spoon, a bulk volume of 50 μL of the test material was applied covering the whole tissue surface.
Control tissues were concurrently applied with 50 μL of highly de-ionized water (NC) or with 50 μL of methyl acetate (PC).
After application, the tissues were placed into the incubator until the total exposure time of 90 minutes (solids) was completed.

Removal of the test substance and postincubation period
To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion) in order to remove residual test substance.
After 12 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium.
Subsequently, the tissues were incubated at standard culture conditions for 18 hours (solids) (postincubation period).

MTT incubation
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation.
The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

Data evaluation

Principle
The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is an irritant.

Calculation of individual and mean optical densities
The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way is calculated.

Tissue viability
The quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 NC value in percent for each exposure time.

ACCEPTANCE CRITERIA

Assay acceptance criterion for the NC
The absolute OD570 of the NC-tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.

Acceptance criteria for the PC
Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.

Assay acceptance criterion for tissue variability
Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the difference of the viability ≤ 20%.

EVALUATION OF RESULTS
The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile water. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.
At present no prediction is performed if the mean relative tissue viability with a test material is > 50 ≤ 60% as the cut off value is currently being evaluated to lie in this range.

Mean tissue viability
(% of negative control) Prediction
≤ 50 irritant
> 50 ≤ 60 no prediction
> 60 non-irritant

Results and discussion

In vivo

Results
Irritation parameter:
other: viability [% of NC]
Basis:
mean
Score:
74
Remarks on result:
other: 1st test run: mean OD570: 1.067
Irritant / corrosive response data:
Due to the ability of the test substance to reduce MTT directly, a KC was applied in parallel.
However, the result of the KC did not indicate an increased MTT reduction (difference to KC of NC is not greater than 0.1). Thus the KC was not used for viability calculation.
Although a slight discoloration of the tissues was observed after the washing procedure, no extraction of the test substance into the isopropanol was obtained and thus there was no interference with the colorimetric test. This was demonstrated by the CC, the single tissues representing OD570 values of 0.009 and 0.005 (data not shown). Due to these very low OD570 values the CC was not used for viability calculation.

Based on the observed results and applying the evaluation criteria it was concluded, that Zinkphthalocyaninsulfonat does not show an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU