monoesters of C16 and C18 (branched and linear) fatty acids with decan-1-ol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 - 12 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

monoesters of C16 and C18 (branched and linear) fatty acids with decan-1-ol

EC Number:

953-178-5

Molecular formula:

not applicable, substance is UVCB

IUPAC Name:

monoesters of C16 and C18 (branched and linear) fatty acids with decan-1-ol

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The SkinEthic HCE model consists of transformed human corneal epithelial cells of the
cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue
(mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the
human eye. The test item is applied directly to the culture surface, at the air interface, so
that undiluted and/or end use dilutions can be tested directly. The model consists of an
airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum-free
and chemically defined medium.

SKINETHIC HCE MODEL
Supplier: SkinEthic Laboratories, Nice, France
Date Received: 10 May 2011

Receipt 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.
Preparation of Tissues
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 labelled 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

tissues were treated with 30 pl of the test item
tissues were treated with 30 pl of solution A to serve as negative controls
tissues were treated with 30 pl of 2% w/v SDS to serve as positive controls

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

3 hours

Number of animals or in vitro replicates:

Triplicate

Details on study design:

METHODS
Pre-Test
Assessment of Direct Test Item Reduction of MTT
The MTT assay, a colourimetric method of determining cell viability, is based on
reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
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 room
temperature in the dark 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 not to have directly reduced the MTT. However it was noted
that in a preceding in vitro skin irritation test, Harlan Laboratories Ltd project number
41100729, the test item had demonstrated the ability to directly reduce MTT. Therefore,
for precautionary reasons the following procedure, employing freeze-killed tissues that
possess no metabolic activity but absorb and bind the test item like viable tissues, was
performed.
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°C) overnight. Once killed, the tissues were stored in the freezer.

Main Test
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-labelled 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 (two per group) were transferred to a
pre-labelled 24-well plate designated WTI 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 tissues were visually examined and the degree of
MTT staining evaluated (qualitative evaluation of tissue viability). The inserts were rinsed
twice with phosphate buffered saline and blotted on absorbent paper to remove residual
MTT solution and transferred to a pre-labelled 24-well plate designated NTT 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 aluminium 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-labelled 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 540nm (OD540) using the Anthos 2001 microplate
reader.
Tissue Histology
One tissue for each treatment group was retained for possible tissue histopathology.
The tissues were carefully cut out of the polycarbonate inserts with a sharp scalpel. The
tissues were carefully cut in half. Both halves were placed into a pre-labelled 1.5 ml
Eppendorf tube containing 1 ml of 10% Formalin and stored at room temperature.

INTERPRETATION OF RESULTS
The mean OD540 values of the duplicate tissues were calculated. Each of these OD540
values had already been corrected for blanks by the microplate reader.
The relative mean tissue viability (percentage of the negative control) was calculated as
follows:
Relative mean viability (%)= (mean OD540 of test item/ mean OD540 of negative control) x 100

ASSAY ACCEPTANCE CRITERION
The results of the assay are considered acceptable if the following assay acceptance
criterion was achieved:
Assay Acceptance Criterion: Positive Control
The assay meets the acceptance criterion for an acceptable test if the relative mean
tissue viability for the positive control treated tissues is <60% relative to the negative
control treated tissues.

Results and discussion

In vitro

Results

Irritation parameter:

other: Relative Mean Viability (%)

Value:

104.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Assessment of Direct Test Item Reduction of MTT
An assessment found the test item was able to directly reduce MTT in a preceding
in vitro skin irritation test, Harlan Laboratories Ltd project number 41100729. Therefore,
an additional procedure using water-killed tissues was performed during the
determination of skin irritation potential. However, the results obtained showed no
degree of interference due to direct reduction of MTT occurred. It was therefore
considered unnecessary to use the results of the freeze-killed tissues for quantitative
correction of results or for reporting purposes.

Assessment of Eye Irritation Potential
The relative mean viability of the test item treated tissues after a 10-Minute exposure
period was 104.4%.
It was considered unnecessary to proceed with tissue histopathology.

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

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008

Executive summary:

Introduction.

The purpose of this study was to determine the eye irritation potential of

the test item using the SkinEthic Reconstructed Human Corneal model (HCE, SkinEthic

Laboratories, Nice, 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.

Methods.

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 30 µl of the test item for

10 minutes. Triplicate tissues treated with 30 µl of Solution A served as the negative

control and triplicate tissues treated with 30 µl of 2% w/v Sodium Dodecyl Sulphate

served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues

(two per group) were taken for MTT loading. The remaining tissues were retained for

possible histopathology. Following MTT loading the reduced MTT was extracted from

the 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 540 nm

(OD540). Data are presented in the form of percentage viability (MTT conversion relative

to negative controls).

The test item was classified according to the following criteria:

i) If the percentage relative mean tissue viability was ≥60% the test item was

considered to be non-irritant (NI).

ii) If the percentage relative mean tissue viability was <60% the test item was

considered to be an irritant (I).

Results.

The relative mean viability of the test item treated tissues after a 10-Minute

exposure period was 104.4%.

It was considered unnecessary to proceed with tissue histopathology.

Quality criteria.

The quality criteria required for acceptance of results in the test were

satisfied.

Conclusion.

According to the study plan followed the test item was considered to be a

Non-Irritant (NI).