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Diss Factsheets

Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
July 17, 2017 to February 27, 2018
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)
Version / remarks:
28 July 2015
according to guideline
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
6 July 2012
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Okra, ext.
EC Number:
EC Name:
Okra, ext.
Cas Number:
Molecular formula:
not applicable
Hibiscus abelmoschus, ext.
Specific details on test material used for the study:
-4407/Delip Hibiscus seed
- Lot/batch No.of test material: 0016468911
- Purity test date: 100 % UVCB
- Homogeity: The test substance was homogeneous by visual inspection.
- Appearance: Solid / beige

- Storage condition of test material: Room temperature

In vitro test system

Test system:
human skin model
EpiDerm(TM) model
Source species:
Cell type:
non-transformed keratinocytes
Cell source:
other: cultured
unchanged (no vehicle)
Details on test system:
- Laminar flow bench: HERAsafe KS 18 (Thermo ELECTRON CORPORATION)
- CO2 incubator: Heraeus BBD 6220
- Incubation conditions: 37°C ± 1°C, 5% ± 1%CO2, 90% ± 5%humidity
- Spectrophotometer: SunriseTM Absorbance Reader For the determination of the optical density of colored extracts.Measurement using a filter wavelength 570 nm without reference filter
- EpiDerm™ 200 kit: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia; containing: 24 EPI-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® ø 1 cm
- Assay medium: EPI-100-NMM assay medium
- MTT diluent: Dulbecco's modified eagle's medium (DMEM) based medium used for diluting MTT (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany)
- Wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca 2+, Mg 2+ (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Biochrom, Germany)
- Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany), 1.0 mg / mL MTT diluent
- Extracting agent: Isopropanol p.a.

The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ø) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
- Tissue model: EPI-200
- Tissue Lot Number: 23305
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

- The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.
- To assess the ability of the test material to directly reduce MTT a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed as described below.
- The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently.
- If the color of the MTT solution or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.
- In case that direct MTT reduction occurred, three freeze-killed control tissue (KC) were treated with, the test article and the negative control

Several test substances were tested in parallel within the present test using the
same control tissues (NC and PC).
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.
Three tissues were treated with the test substance, the PC and NC, respectively.
25 μL sterile PBS was applied first. Thereafter, a bulk volume of ca. 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid.
Control tissues were concurrently treated with 30 μL of sterile PBS (NC) or with 30 μL of 5% SDS (PC). A nylon mesh was placed carefully onto the tissue surface afterwards.
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
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.
Subsequently, the tissues were placed into 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. After the post-incubation 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. 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 4 microtiter wells filled with isopropanol for each microtiter plate.

In case one of the below given acceptance criteria was not met, repetition of the test was considered.
- Barrier function and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDermTM batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD guidelines. Lower acceptance limit: ET50 = 4.0 hours; Upper acceptance limit: ET50 = 8.7 hours
- Acceptance criteria for the negative control (NC): The absolute OD570 of the negative control tissues in the MTTtest is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 0.8. The mean OD570 of the NC should not exceed 2.8.
- Acceptance criteria for the positive control (PC): 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≥ 20% is acceptable.
- Acceptance criteria for the variability of the tissues: For every treatment three tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD of %-viability is ≤ 18%.

- Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.
- A single test composed of at least three tissue replicates should be sufficient for a test chemical when the result is unequivocal. However, in cases of borderline results, such as nonconcordant replicate measurements and/or mean percent tissue viability equal to ±5% of the cut-off value, a second test should be considered, as well as a third one in case of discordant results between the first two tests.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
25 μL sterile PBS was applied first (vehicle). Thereafter, a bulk volume of ca. 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid.

Control tissues were concurrently treated with 30 μL of sterile PBS (NC) or with 30 μL of 5% SDS (PC). A nylon mesh was placed carefully onto the tissue surface afterwards.
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hour post-incubation period
Number of replicates:
3 replicates

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Irritation test: 1 hour exposure
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
- Acceptance criteria for negative control are met.
- Acceptance criteria for positive control are met.
- Acceptance criteria for variability between replicate measurements are met.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
The substance is not irritating.