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Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-03-06 to 2012-04-11
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
according to guideline
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. QA statement)
BASF SE 67056 Ludwigshafen, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized
EC Number:
Cas Number:
Molecular formula:
Unspecified (UVCB)
Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized
Test material form:
Specific details on test material used for the study:
- Name of test material: Sovermol 1055
- Substance type: organic
- Physical state: liquid, clear,
- Lot/batch No.: CE80580015
- Stability under test conditions: stable under test conditions
- Storage condition of test material: at room temperature
- The CAS RN cited in the test report is 91051-90-0 (chemical name: Fatty acids, tallow, Me esters, epoxidized). However, after the finalization of the study, it became evident that the test material is better described by CAS RN 158318-67-3 (Fatty acids, C16-18 and C18-unsatd., Me esters, epoxidized). The test material is the same as defined in section 1.

In vitro test system

Test system:
human skin model
Source species:
Cell type:
other: EpiSkin SM model
Details on test system:

Pre-incubation (day [-1]):
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.

Application (day 0):
Three tissues were treated with the test substance, the PC and NC, respectively. Thirty microliter (30 μL) of the undiluted test substance was applied using a pipette. Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards.

Exposure (day 0):
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.

Rinsing (day 0):
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab.

Post-incubation (day 0-2):
Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.

MTT test after 42 hours incubation (day 2):
After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation.

Formazan extraction (day 2):
At the end of incubation with MTT a formazan extraction step was undertaken:
The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Amount(s) applied: a sufficient amount to cover evenly all the epidermal surface (30 μL per EpiSkin SM unit)
Duration of treatment / exposure:
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. Undiluted test item was applied.
Duration of post-treatment incubation (if applicable):
MTT test after 42 hours post-incubation
Number of replicates:
3 EpiSkin SM for test item incubation
3 EpiSkin SM for positive control
3 EpiSkin SM for negative control

Test animals

Details on test animals or test system and environmental conditions:
- Human skin
- Source: MatTek In Vitro Life Science Laboratories, Bratislava

The EpiSkin SM model has been validated for irritation testing in an international trial and is considered to be suitable for this study.

- EpiSkin SM kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed via the MTT cell viability test using the cytotoxic test compound sodium dodecyl sulphate (SDS).

- Units: 24 Epi-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells ∅ 1 cm
- Wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca 2+, Mg 2+ (MatTek In Vitro Life Science Laboratories, Bratislava and Biochrom, Germany)
- Medium: Dulbecco's modified eagle's medium (DMEM); for the assay and for diluting MTT (MatTek In Vitro Life Science Laboratories, Bratislava /Sigma, Germany)

Test system

unchanged (no vehicle)

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 55-65 min exposure time after 42 hours
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
- Acceptance criteria met for positive control: 42 hours after 55-65 min exposure time the positive control viability was 3% compared to the untreated control.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met