Reaction mass of (6E)-Dec-6-enal and (7E)-Dec-7-enal and (8E)-Dec-8-enal and (8Z)-Dec-8-enal

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 30 September 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 Epithelium model and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The purpose of this study was to determine the eye irritation potential of the test item using the SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories, Lyon, 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.
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).

The mean tissue viability for the test item was compared to the negative control and classified.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (6E)-Dec-6-enal and (7E)-Dec-7-enal and (8E)-Dec-8-enal and (8Z)-Dec-8-enal

Molecular formula:

C10H18O

IUPAC Name:

Reaction mass of (6E)-Dec-6-enal and (7E)-Dec-7-enal and (8E)-Dec-8-enal and (8Z)-Dec-8-enal

Test material form:

other: Liquid

Details on test material:

Identification: IFF TM 11-212 (FRET 08-0334)
Physical state/Appearance: Clear colourless liquid
Storage Conditions: Approximately 4 °C in the dark

Test animals / tissue source

Species:

other: SkinEthic reconstructed Human Corneal Epithelium model

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

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:

yes

Amount / concentration applied:

30 µL

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

No data

Number of animals or in vitro replicates:

Not applicable

Details on 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 µL 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.


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 µL 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 minutes

Score:

83.7

Remarks on result:

other: Non-irritant

Irritant / corrosive response data:

Assessment of Direct Test Item Reduction of MTT
The MTT solution containing the test item remained yellow which indicated that the test item did not directly reduce MTT.


Assessment of Eye Irritation Potential
The relative mean viability of the test item treated tissues after a 10-Minute exposure period was 83.7%.

Any other information on results incl. tables

Assay Acceptance Criterion

The relative mean tissue viability for the positive control treated tissueswas 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	Relative Mean Viability (%)
Negative ControlÅ	0.795	0.815	100*
	0.815
	0.834
Positive ControlÅ	0.144	0.145	17.8
	0.148
	0.143
Test Item	0.683	0.682	83.7
	0.671
	0.692

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to the study plan followed the test item was considered to be Non-Irritant.
EU DSD (67/548/EEC): Not classified for irritation.
EU CLP (1272/2008/EC)/UN GHS: Not classified for irritation.

Executive summary:

The eye irritation potential of the test substance, TM 11-212 (FRET 08-0334), 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 83.7 %. The test item was therefore considered to be non-irritant.