Registration Dossier

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:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
EpiOcular(TM) test

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
no guideline followed
Version / remarks:
There are no official national or international guidelines for the EpiOcularTM test yet; however,
the test 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.
Principles of method if other than guideline:
The test is based on the experience that irritant chemicals produce cytotoxicity in human
reconstructed cornea after a short term topical exposure. The test is designed to predict an
eye irritation potential of a chemical by using the three dimensional human cornea model
EpiOcularTM. After application of the test material to the surface of the EpiOcularTM tissue the
induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is
expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial
dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanolextraction
of the formazan from the tissues, the optical density of the extract is determined
spectrophotometrically. Optical density of the extracts of test-substance treated tissues is
compared to values from negative control tissues and expressed as relative tissue viability.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
Identification: NM01
Bach No.: 492-14-01
Purity: 99.08 area-% (190 nm) 98.17 area-% (200 nm)
Molecular Weight: 200.24 g/mol
Physical state, appearance: White, solid
Expiry Date: 21 January 2016
Storage Conditions: At room temperture, moisture protected

Test animals / tissue source

Species:
other: human reconstructed cornea in vitro model

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
50 μL
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2
Details on study design:
To assess the ability of the test material to directly reduce MTT a pretest was performed. 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 (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 where direct MTT reduction occurred, two freeze-killed control tissues each were treated with the test article and the negative control, in the same way as described in thefollowing section.

Basic procedure
Several test substances were tested in parallel within the present test (test no. 50) using the same control tissues (NC and PC). Two tissues were treated with each, the test substance, the PC and the NC. 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 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.
Pretreatment of the tissues
After the pre-incubation, the tissues were pre-treated with 20 μL 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 sterile 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 6 hours 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 25 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 (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
The OD570 values determined for the various tissues are measures of their viability. The ratio 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 was an irritant.
Calculation of individual and mean optical densities
The corrected measured OD570 value for each individual tissue was 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 was calculated.
Tissue viability
The quantification of tissue viability is presented as the ratio of the mean OD570 divided by the respective OD570 NC value in percent.

Decision criteria of EpiOcular.
Mean tissue viability (% of negative control)
≤ 60: irritant
> 60: non-irritant

Results and discussion

Results of ex vivo / in vitro study
Irritation parameter:
other: tissue viability in % of negative control based on fluorescein leakage
Value:
110
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Executive summary:

The eye irritating potential of the test substance was determined in the in vitro EpiOcular Eye Irritation Test.

The potential of NM01 to cause ocular irritation was assessed by a single topical application of 50 μL bulk volume (about 9 mg) of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™).

Two EpiOcular™ tissue samples were incubated with the test substance for 6 hours followed by a 18-hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

The EpiOcular™ eye irritation test showed the following results:

The test substance is not able to reduce MTT directly.

The mean viability of the test-substance treated tissues was 110%.

Based on the observed results for the EpiOcular Test alone and applying the evaluation criteria it was concluded, that NM01 does not show an eye irritation potential under the test conditions chosen.