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Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
not specified
this test was not carried out initially to meet the REACH registration requirement, hence the fact that it is not carried out under GLP.

Test material

Constituent 1
Reference substance name:
Docosan-1-ol, icosan-1-ol, octadecan-1-ol and their reaction products with betaine and ethanesulphonic acid
Docosan-1-ol, icosan-1-ol, octadecan-1-ol and their reaction products with betaine and ethanesulphonic acid
Test material form:
solid: bulk

In vitro test system

Test system:
human skin model
Source species:
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
unchanged (no vehicle)
Details on test system:
Inserts (filter + epidermis) were gently detached from the agar and if necessary the bottom of the insert was wiped on an absorbent paper in order to avoid leaving agar pieces onto the polycarbonate membrane.
Inserts were then placed into wells (6 wells culture plate) previously filled with 1 ml of growth medium at room temperature (the absence of air bubbles was checked). Cultures were incubated overnight at 37°C, 5% C02.

16 µl ± 0.5 µl of the references item were deposited with a positive displacement micropipette on the surface of the epidermises and a 7.5 mm diameter nylon mesh (except for PBS) is gently applied on the surface of the epidermis using meezers.

16 mg ± 2 mg of test item (grinded with mortar) were applied to the surface of the tissue previously moistened with 10 µl deionised sterile water in order to ensure good contact with the test item.
The epidermises were incubated in 0.3 ml of maintenance medium (24 wells plate) at room temperature for 42 minutes ± 1 minute.

The epidermises were rinsed with 25 ml of PBS by epidermis (25 x 1 ml using a distributor). The residual PBS was eliminated by tapping the epidermis on absorbent paper and the epidermis was gently wiped with a cotton swab.
The epidermises were incubated in 2 ml of growth medium in 6 wells plate at 37°C, 5% C02 for 42 hours ± 1 hour (the absence of air bubbles was checked).

At the end ofthe incubation period, the plates were stirred approximately 2 minutes at 300 rotations per minute in order to homogenize the released mediators or enzymes in the culture medium.
All epidermises were incubated in 0.3 ml of maintenance medium at 1 mg/ml MTT in 24 wells plate.
After 3 hours 5 minutes incubation at 37oC, 5% C02, outside ofinserts was rinsed With 2 ml of PBS.
Extraction was performed by placing the epidermises into wells filled with 0.8 ml of isopropanol then covered with 0.7 ml isopropanolfor 2 hours 5 minutes under gentle agitation at room temperature.
The inserts were pierced and removed from wells then the extraction solution was homogeneized by pipeting. The absorbances were measured in triplicate on 200 µl MTT extract in 96 wells plate. Absorbances are measured at 540 nm against isopropanol as blank
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
16 mg ± 2 mg of the test item pure, as well as the reference items, were tested on three epidermises.
Preliminary tests to the study were conducted in order to include or not additional controls:
• Assessment ofthe MTT reduction potential by the test item:
16 mg ± 2 mg of the test item (grinded with mortar) were added to 0.3 ml of 1 mg/ml MTT maintenance medium, mixed and incubated 3 hours 5 minutes at 37oc, protected from the light. A blue/purple colouration indicates a non-specific reduction induced by the test item (untreated MTT medium was used as control). The colour was assessed visually at the end of the exposure time.
No colour was observed. No additional controlfor the direct MTT reduction by the test item is required.
• Assessment of the test item colouring potential:
16 mg ± 2 mg of the test item (grinded with mortar) were added to 0.3 ml of water and 16 mg ± 2 mg of the test item were added to 2 ml of isopropanol and mixed around 15 minutes at room temperature.
The colour was assessed by absorbance reading at 540 nm against water or isopropanol as blanks. The absorbances are not significant (< 0.08). No additional control for coloured test item is required.
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3 replicates for test item
3 replicates for negative control
3 replicates for positive control

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
Run / experiment:
ca. 94
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Executive summary:

Under the retained experimental conditions (OECD 439 guideline) and according to the CLP regulation, the test item COSMEGREEN ES 1822+ code D-20/01686 tested pure must not be classified. No symbol, risk phrase, no signal word or hazard statement is required.