Registration Dossier

Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Aug 2016 to 28 Jun 2017
1 (reliable without restriction)

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
according to
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
6 July 2012
GLP compliance:
yes (incl. certificate)
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Test material form:
Specific details on test material used for the study:
- Name as cited in study report: Orasol Red 335
-Test-substance No.: 16/0125-1
- Batch identification: 002-141706
- Content: 96 g/100 g (100 g/100 g minus water content)
- Physical state / colour: Solid / red
- Storage conditions: Room temperature

In vitro test system

Test system:
human skin model
Three dimensional human epidermis model: EpiDerm model
Source species:
Cell type:
non-transformed keratinocytes
Cell source:
other: cultured
Justification for test system used:
The present test is based on the experience that corrosive and irritant chemicals produce cytotoxicity in human reconstructed epidermis after a short term topical exposure.
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 coloured extracts. Measurement using a filter wavelength 570 nm without reference filter
- EpiDermTM 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
- Tissue for MTT reduction control: EPI-200 tissue that is killed by freezing at -20°C
- 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 Ca2+, Mg2+ (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 (EpiDermTM 200), containing 24 tissues on shipping agarose.
- Tissue model: EPI-200
- Tissue Lot Number: 23348
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

- The direct reduction of MTT by a test substance interferes with the colour 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 colour 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.
- Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in parallel.

- The colour of a test substance may interfere with the colour density produced by metabolic capacity of the tissue and would falsify the test results when residues of the test substance remain on the tissues after washing and are extracted by the isopropanol.
- Due to the colour of the test substance a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed as follows: the test substance was applied to a KC tissue, incubated for 1 hour and removed by washing in the same way as in the main experiment. Thereafter extraction in isopropanol was performed and the OD570 of the extract was determined spectrophotometrically.
- Based on the result of the pretest it was judged that application of colour control tissues is not necessary.

- Several test substances were tested in parallel within the present test using the same control tissues (negative control, NC and positive control, 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. In addition three killed control tissues were used for the test substance and NC, respectively, in order to detect direct MTT reduction.
- A nylon mesh was placed carefully onto the tissue surface of the NC, NC KC and PC after application.
- 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. The tissues used as colour control were placed into assay medium without MTT.
- 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.

- Principle: The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is irritant.
- Calculation of individual and mean optical densities: The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of three tissues treated in the same way is calculated.
- Application of measurements using killed control tissues: In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the test-substance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.
- Tissue viability: The quantification of tissue viability is presented as the quotient of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent for each exposure time.

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 MTT test 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%.
- Acceptance criteria for the killed controls (KC): The OD570 of the tissues for the KC of the NC should be ≤ 0.35. The OD570 value for direct MTT-reduction of a test substance should be ≤ 30% of the OD570 of the NC.
- Acceptance criteria for the colour controls (CC): The OD570 value for the colour control of a test substance should be ≤ 30% of the OD570 of the NC.

- 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 non-concordant 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.

See table in 'Any other information on materials and methods incl. tables'
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
25 µL sterile PBS was applied first. Thereafter, a bulk volume of ca. 25 µL (about 8 mg) 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, NC KC) or with 30 µL of 5% SDS (PC) or test substance (KC). A nylon mesh was placed carefully onto the tissue surface of the NC, NC KC and PC afterwards.
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hour post-incubation period
Number of replicates:
Three tissues were treated with the test substance, the PC and NC, respectively. In addition three killed control tissues were used for the test substance and NC.

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
other: Viability after correction based on killed controls
Other effects / acceptance of results:
- Direct-MTT reduction: The results of the KC tissues indicate an increased MTT reduction (mean viability 0.6 % of NC). Thus for the test substance the final mean viability is given after KC correction.
- Colour interference with MTT: Based on the result of the pretest it was judged that application of colour control tissues is not necessary.

- 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