Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant non-guideline study, available as unpublished report, fully adequate for assessment

Data source

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

Materials and methods

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, October 2, 2012 ("Acute Eye Irritation/Corrosion")
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Sovermol Pol 1100
- Test-substance No.: 14/0220-1
- Physical state: liquid
- Lot/batch No.: L82

Test animals / tissue source

Species:
other: EpiOcular™ OCL-200 kit
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
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 EpiOcular tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts and are commercially available as kits, containing 24 tissues on shipping agarose.
- Source: MatTek Corp., Ashland MA, USA
- Tissue for MTT-reduction control: OCL-200 tissue that is killed by freezing at -20°C
- Assay medium: Dulbecco's modified eagle's medium (DMEM); for the assay and for diluting MTT
- Wash buffer: Dulbecco's phosphate buffered saline (PBS)
- Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
- Extracting agent: Isopropanol p.a.
- Negative control: De-ionized water, sterile
- Positive control: Methyl acetate
- MTT reduction control: De-ionized water, sterile or test substance
- Color control: Test substance

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
Duration of treatment / exposure:
30 minutes
Number of animals or in vitro replicates:
2
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 liquids 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 pipette, 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 30 minutes 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 (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.

Application of measurements using killed control tissues
In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the testsubstance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is
able to produce some formazan net OD570 KC is calculated by subtracting the mean OD570 KC of the NC from the mean OD570 KC of the test substance. In case the mean net OD570 KC is greater than 0.1 it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.

Application of measurements using color control tissues
The OD570 values measured in the color control tissues (CC) will be used to correct the mean OD570 of the test-substance treated tissues (mean OD570 CC corrected). The mean net OD570 CC will be subtracted from the respective mean OD570 to result in the mean OD570 CC corrected, only when interference of the test substance in the colorimetric test is noticed. The mean OD570 CC corrected represents the formazan production without the absorbance of the colored test substance.

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%.

Acceptance criteria for the KC
The OD570 of the killed control tissues treated as negative control should be ≤ 30% of the NC.

Acceptance criteria for the CC
The OD570 of the color control tissues should be comparable, thus representing a reproducible amount of test substance residues.

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
≤ 60 irritant
> 60 non-irritant

Results and discussion

In vivo

Results
Irritation parameter:
other: viability [% of NC]
Basis:
mean
Score:
102
Remarks on result:
other: mean OD570: 2.007
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.

The mean OD570 of the test-substance treated and MTT-incubated tissues was 2007 corresponding to a mean tissue viability of ca. 102%.

Any other information on results incl. tables

Test Substance

 

Tissue 1

Tissue 2

Mean KC

Mean CC

Mean*

Inter-tissue variability [%]

NC

Mean OD570

1.955

1.989

0.038

-

1.972

 

Viability [% of NC]

99.1

100.9

-

-

100

1.7

Test item

Mean OD570

2.105

1.910

0.056

-

2.007

 

Viability [% of NC]

106.7

96.9

-

-

102

9.9

PC

Mean OD570

0.422

0.516

-

-

0.469

 

Viability [% of NC]

21.4

26.1

-

-

24

4.7

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information
Conclusions:
Based on the observed results and applying the evaluation criteria it was concluded, that the test item does not show an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.
Executive summary:

The potential of Sovermol Pol 1100 to cause ocular irritation was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissue samples per test run were incubated with the test substance for 30 minutes followed by a 2-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 able to reduce MTT directly. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing. The mean viability of the test-substance treated tissues was 102%.

Based on the observed results for the EpiOcular Test alone and applying the evaluation criteria it was concluded, that Sovermol Pol 1100 does not show an eye irritation potential under the test conditions chosen.