9-Octadecen-1-ol, (Z)-, phosphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-02-06 to 2013-02-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: CLP and Guideline compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

, 22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

, 06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

UVCB: not applicable

IUPAC Name:

UVCB: not applicable

Constituent 2

Reference substance name:

UVCB: not applicable

IUPAC Name:

UVCB: not applicable

Constituent 3

Reference substance name:

9-Octadecen-1-ol, (Z)-, phosphate

EC Number:

253-455-4

EC Name:

9-Octadecen-1-ol, (Z)-, phosphate

Cas Number:

37310-83-1

Molecular formula:

C18H37O4P

IUPAC Name:

[(9Z)-octadec-9-en-1-yloxy]phosphonic acid

Test material form:

liquid: viscous

Test animals

Species:

other: not applicable (reconstituted human epidermis)

Strain:

other: not applicable (reconstituted human epidermis)

Details on test animals or test system and environmental conditions:

EpiSkinTM Small Model (EpiSkinTMSM), EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are 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 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Source: SKINETHIC Laboratories; 4, rue Alexandre Fleming, 69007 – LYON, France.
Batch No.: 13-EKIN-004
Expiry date: 11 February 2013

Test system

Type of coverage:

other: not applicable (reconstituted human epidermis)

Preparation of test site:

other: not applicable (reconstituted human epidermis)

Vehicle:

water

Controls:

not required

Amount / concentration applied:

The test item is a viscous liquid therefore exact weighing of treatment volume was not performed. A sufficient amount of the test item was used that evenly covered the whole epidermal surface. The test substance was gently spread onto the skin with a flat curved spatula.

Duration of treatment / exposure:

15 min

Observation period:

Not applicable (reconstituted human epidermis)

Number of animals:

Not applicable (reconstituted human epidermis). However, in this assay 3 replicates per test item and 3 replicates negative controls + 3 replicates positive controls were used. Furthermore 3 replicates of additional control for coloured test item (i.e. for non-specific OD evaluation) were used.

Details on study design:

Pre-incubation (day [-1]):
The “maintenance medium” was pre-warmed to 37 °C. The appropriate number of assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed above the media in a separately prepared well. Contact of the epidermis units with the media was assured. The well was then incubated overnight at 37 °C in an incubator with 5 % CO2.

Application (day 0):
The test item is a viscous liquid therefore exact weighing of treatment volume was not performed. A sufficient amount of the test item was used that evenly covered the whole epidermal surface. The test substance was gently spread onto the skin with a flat curved spatula.

Positive and negative control
Test Item
A volume of 20 μL positive control (SDS 5 % aq.) or negative control (1 x PBS) was applied to the skin surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Exposure (day 0):
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature.

Rinsing (day 0):
After the incubation time, the EPISKIN-SM units were removed and rinsed thoroughly with PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis).

Post-incubation (day 0-2):
After rinsing the units were placed into the plate wells filled with fresh pre-warmed “Maintenance Medium” (2 mL/well) below them and then incubated for 42 hours at 37 °C in an incubator with 5 % CO2.

MTT test after 42 hours incubation (day 2):
After the 42 hours incubation the EPISKIN-SM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours at 37 °C in an incubator with 5 % CO2 protected from light.

Formazan extraction (day 2):
At the end of incubation with MTT a formazan extraction step was undertaken:
A defined disk of epidermis from each replicate was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 μL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).

The capped tubes were thoroughly mixed by using a Vortex Mixer to achieve a good contact of all of the material to the acidified isopropanol. Then the mixture was incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurements (day 2):
Following the formazan extraction, 2 × 200 μL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD’s of each well were recorded using a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at a wavelength of 570 nm while using an acidified isopropanol solution blank (6 × 200 μL).

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Optical density (OD) values

Value:

1.115

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 47 hours. (migrated information)

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The results obtained from this in vitro skin irritation test using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. According to the current OECD Guideline No. 439, Reaction product of oleyl alcohol with polyphosphoric acid is considered as non-irritant to skin and is therefore not classified.

Executive summary:

In this in vitro skin irritation test using the EPISKIN model, the test item Reaction product of oleyl alcohol with polyphosphoric acid did not show significantly reduced cell viability in comparison to the negative control. All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. According to the current OECD Guideline No. 439, Reaction product of oleyl alcohol with polyphosphoric acid is considered as non-irritant to skin and is therefore not classified.