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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Mar - 04 Apr 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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

The EPISKINTM model is a three-dimensional reconstructed human epidermis model
consisting of adult human-derived epidermal keratinocytes seeded on a dermal
substitute consisting of a collagen type I matrix coated with type IV collagen. A highly
differentiated and stratified epidermis model is obtained after a 13-Day culture period
comprising of the main basal, supra basal, spinous and granular layers and a functional
stratum corneum.
EPISKINTM MODEL KIT
Date received : 29 March 2011

Vehicle:

unchanged (no vehicle)

Details on test system:

PROCEDURE
Pre-Test
Assessment of Direct Test Item Reduction of MTT
MTT dye metabolism, cell viability assay
The MTT assay, a colourimetric method of determining cell viability, is based on
reduction of the yellow tetrazolium salt (344,5-dimethylthiazol-2-y1]-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, thus mimicking dehydrogenase activity of the cellular
mitochondria. This property of the test item is only a problem, if at the time of the MTT
test (after rinsing) there are still sufficient amounts of the test item present on or in the
tissues. In this case, the true metabolic MTT reduction and the false direct MTT
reduction can be differentiated and quantified.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly
reduce MTT and at the same time is present on or in the tissues when the MTT viability
test is performed. To identify this possible interference, each test item is checked for the
ability to directly reduce MTT according to the following procedure:
10 µl of the test item was added to 2 ml of a 0.3 mg/ml MTT solution freshly prepared in
assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for
3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue, the test item is presumed to have
reduced the MTT.

The test item was shown to directly reduce MTT in the direct MTT reduction test. There
was a possibility that if the test item could not be totally rinsed off the tissues, any
residual test item present on or in the tissue may directly reduce MTT and could have
given rise to a false negative result. Therefore the determination of skin irritation
potential was performed in parallel on viable and water-killed tissues.
This step was a functional check which employs water-killed tissues that possess no
metabolic activity but absorb and bind the test substance like viable tissues.
Water-killed tissues were prepared by placing untreated EPISKINTM tissues in a 12-well
plate containing 2.2 ml of sterile distilled water in each well. The tissues were Incubated
at 37°C, 5% CO2 in air for 48 hours ± 1 hour. At the end of the incubation the water was
discarded. Once killed the tissues were stored in a freezer (-14 to -30°C) for up to
6 months. Before use each tissue was thawed by placing in 2.2 ml of maintenance
medium for approximately 1 hour at room temperature.
In addition to the normal test procedure, each MTT reducing test substance was applied
to a water-killed tissue. In addition, one water-killed tissue remained untreated. The
untreated water-killed control showed a small amount of MTT reduction due to residual
reducing enzymes within the killed tissue.

Pre-Incubation (Day 0: tissue arrival)
2 ml of maintenance medium, warmed to approximately 37°C, was pipetted into the first
column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred
into the maintenance medium filled wells (3 units per plate). A different 12-well plate was
used for the test item and each control item. The tissues were incubated at 37°C,
5% CO2 in air overnight.
Main Test
5.2.1 Application of Test Item and Rinsing (Day 1)
2 ml of maintenance medium, warmed to approximately 37°C, was pipetted into the
second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes.
The test item was applied topically to the corresponding tissues ensuring uniform
covering. 10 µl of the test item was applied to the epidermis surface. Triplicate tissues
treated with 10 µl of PBS served as the negative controls and triplicate tissues treated
with 10 µl of SDS 5% w/v served as the positive controls. To ensure satisfactory contact
with the positive control item the SDS solution was spread over the entire surface of the
epidermis using a pipette tip (taking particular care to cover the centre). After 7 minutes
contact time the SDS solution was re-spread with a pipette tip to maintain the distribution
of the SDS for the remainder of the contact period. The plate(s) were kept in the
biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps
and rinsed using a wash bottle containing PBS with Ca' and Mg'. Rinsing was
achieved by filling and emptying each tissue insert for approximately 40 seconds using a
constant soft stream of PBS to gently remove any residual test item. The rinsed tissues
were transferred to the second column of 3 wells containing 2 ml of maintenance
medium in each well. The rinsed tissues were incubated at 37°C, 5% CO2 in air for
42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42-Hour post-exposure incubation period each 12-well plate was placed
onto a plate shaker for 15 minutes to homogenise the released mediators in the
maintenance medium. 1.6 ml of the maintenance medium from beneath each tissue was
transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30°C for possible
inflammatory mediator determination.
2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into
the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the
MTT filled wells, being careful to remove any excess maintenance medium from the
bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated
for 3 hours at 37°C, 5% CO2 in air. At the end of the 3-Hour incubation period each
tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was
made using the EPISKINTM biopsy punch. The epidermis was carefully separated from
the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed
into labelled 1.5 ml micro tubes containing 500 µl of acidified isopropanol, ensuring that
both the epidermis and collagen matrix were fully immersed. Each tube was plugged to
prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were
refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of
formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex
mixer to produce a homogenous coloured solution.
For each tissue, duplicate 200 µl samples were transferred to the appropriate wells of a
pre-labelled 96-well plate. 200 µl of acidified isopropanol alone was added to the two
wells designated as 'blanks'. The optical density was measured (quantitative viability
analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 µl of the test item was applied to the epidermis surface.
Triplicate tissues treated with 10 µl of PBS served as the negative controls.
triplicate tissues treated with 10 µl of SDS 5% w/v served as the positive controls

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

incubated at 37°C, 5% CO2 in air for 42 hours.

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

experiment 1

Value:

94.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

experiment 2

Value:

94.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

experiment 3

Value:

91.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Direct MTT Reduction
An assessment found the test item was able to directly reduce MTT. 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 water-killed tissues for quantitative correction of
results or for reporting purposes.

Test Item, Positive Control Item and Negative Control Item
The relative mean viability of the test item treated tissues was 93.6% after a 15-Minute
exposure period.

Quality Criteria
The relative mean tissue viability for the positive control treated tissues was ≤40%
relative to the negative control treated tissues and the standard deviation value of the
percentage viability was ≤18%. The positive control acceptance criterion was therefore
satisfied.
The mean OD540 for the negative control treated tissues was ≥0.6 and the standard
deviation value of the percentage viability was ≤18%. The negative control acceptance
criterion was therefore satisfied.
The standard deviation calculated from individual percentage tissue viabilities of the
three identically treated tissues was ≤18%. The test item acceptance criterion was
therefore satisfied.

Any other information on results incl. tables

Mean OD₅₄₀ Values and Percentage Viabilities for the Negative

Control Item, Positive Control Item and Test Item

Item	OD540of tissues	Mean OD540 of triplicate tissues	± SD of 0D540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of
						Relative
						mean
						viability (%)
Negative Control Item	0.777	0.75	0.024	103.6	100*	3.2
	0.732			97.6
	0.74			98.7
Positive Control Item	0.05	0.049	0.006	6.7	6.6	0.1
	0.049			6.5
	0.049			6.5
Test Item	0.711	0.702	0.012	94.8	93.6	1.5
	0.706			94.1
	0.689			91.9

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

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 test was to evaluate the skin irritation potential of the

test item using the EPISKIN^TM reconstructed human epidermis model after a treatment

period of 15 minutes followed by a post-exposure incubation period of 42 hours. The

principle of the assay was based on the measurement of cytotoxicity in reconstructed

human epidermal cultures following topical exposure to the test item by means of the

colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of

the yellow MTT tetrazolium salt (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyktetrazolium

bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item

treated tissues relative to the negative controls. The concentration of the inflammatory

mediator IL-lα in the culture medium retained following the 42-Hour post-exposure

incubation period is also determined for test items which are found to be borderline nonirritant

based upon the MTT reduction endpoint. This complimentary end-point will be

used to either confirm a non-irritant result or will be used to override the non-irritant

result.

Method

Triplicate tissues were treated with the test item for an exposure period of

15 minutes. At the end of the exposure period each tissue was rinsed before incubating

for 42 hours. At the end of the post-exposure incubation period each tissue was taken

for MTT-loading. The maintenance medium from beneath each tissue was transferred to

pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator

determination. After MTT loading a total biopsy of each epidermis was made and placed

into micro tubes containing acidified isopropanol for extraction of formazan crystals out of

the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and

duplicate 200 pl samples were transferred to the appropriate wells of a pre-labelled

96-well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item

treated tissues relative to negative control tissues).

Results

The relative mean viability of the test item treated tissues was 93.6% after the

15-Minute exposure period.

Quality criteria

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

satisfied.

Conclusion

The test item was considered to be Non-Irritant (NI).