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Diss Factsheets

Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 07 October 2014 and 09 October 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be a reliability 1 as it has been conducted according to the Assessment of Ocular Irritation Potential using the SkinEthic Reconstructed Human Corneal Epithelial Model and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2014

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
This method was designed to be compatible with the following:

Prevalidation of a new in vitro reconstructed human cornea model to assess the eye irritating potential of chemicals (Van Goethem et al, 2006).

The SkinEthicTM HCE model, although not yet formally validated, has undergone successful pre-validation. The method followed forms part of a currently ongoing Colipa/ECVAM Eye Irritation Validation Study designed to offer a replacement to the in vivo rabbit eye test.

The experimental design of the study consists of a test for direct reduction of MTT (3 [4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) by the test item followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with the test item for 10 minutes. At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues were taken for MTT-loading. After MTT-loading the tissues were removed and immersed in isopropanol for extraction of formazan crystals out of the MTT loaded tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT conversion relative to negative controls).
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methyl-1-benzofuran-5-ol
EC Number:
807-272-7
Cas Number:
7182-21-0
Molecular formula:
C9H8O2
IUPAC Name:
3-methyl-1-benzofuran-5-ol
Test material form:
other: Solid
Details on test material:
Identification: IFF TM 11-0230
Physical state/Appearance: Beige solid
Storage Conditions: Room temperature in the dark

Test animals / tissue source

Species:
other: SkinEthic Reconstructed Human Corneal Epithelial Model
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
Test System
Human Corneal Epithelium (HCE)
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 07 October 2014
HCE Tissues (0.5cm2) batch number: 14-HCE-040
Maintenance Medium lot number: 14-MIMA-042

Test system

Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
Triplicate tissues were treated with 30 mg of the test item
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
3 hours
Number of animals or in vitro replicates:
Not applicable
Details on study design:
Test System
Human Corneal Epithelium (HCE)
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 07 October 2014
HCE Tissues (0.5cm2) batch number: 14-HCE-040
Maintenance Medium lot number: 14-MIMA-042

Test Item Formulation and Experimental Preparation
The test item was used as supplied.

Preparation of Negative and Positive Control Items
The negative control item, Solution A, was used as supplied. The positive control item, Sodium Dodecyl Sulphate 2% w/v, was prepared in sterile water.

Study Design
Pre-Test – Assessment of Direct Test Item reduction of MTT
The MTT assay, a colorimetric 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 ºC) overnight. Once killed, the tissues were stored in the freezer.

Receipt and Preparation 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.

Main Test
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 labeled 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. 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-labeled 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 were transferred to a pre-labeled 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-labeled 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 isopropanol was added onto each tissue and the plate sealed to prevent isopropanol evaporation. The plate was wrapped in aluminum 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-labeled 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 562nm (OD562) using the Anthos 2001 microplate reader.

Results and discussion

In vivo

Results
Irritation parameter:
other: Relative mean viability (%)
Basis:
mean
Time point:
other: 10-minute exposure
Score:
43.2
Remarks on result:
other: Irritant
Irritant / corrosive response data:
The relative mean viability of the test item treated tissues after a 10-Minute exposure period was 43.2%.

Any other information on results incl. tables

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.

 

Assay Acceptance Criterion

The relative mean tissue viability for the positive control treated tissues was 17.8% relative to the negative control treated tissues.

The quality criterion required for the acceptance of results in the test was satisfied.

Assessment of Eye Irritation Potential – Viability of HCE Tissues

Item

OD562of
Individual Tissue

Mean OD562

Mean OD562of tissues corrected for MTT direct reduction

(-0.056)

Relative Mean Viability (%)

Negative ControlÅ

0.795

0.815

na

100*

0.815

0.834

Positive ControlÅ

0.144

0.145

na

17.8

0.148

0.143

Test Item

0.392

0.408

0.352

43.2

0.412

0.419

Corrected viabilityof treated killed tissues

=

0.082 (tkt)-0.026 (ukt) = 0.056


 

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was classified as irritant.
Executive summary:

The eye irritation potential of the test substance, TM 11-0230 was assessed using the SkinEthic reconstructed Human Corneal Epithelium model. The relative mean viability of the test item treated tissues after a 10-Minute exposure period was 43.2%. The test item was therefore considered to be an irritant.