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Toxicological information

Skin irritation / corrosion

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Administrative data

skin corrosion: in vitro / ex vivo
in vitro
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
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
This deviation had no impact on the outcome of the study.
GLP compliance:

Test material

Details on test material:
- Name of test material: Fluidized Bed Combustion (FBC) Bottom Ash
- Substance type: technical product
- Physical state: solid
- Appearance: Light grey solid powder with some small black particle
- Chemical structure: Complex product of oxides
- Main components: SiO2 - 27,78%, Fe2O3 - 5,98%, CaO (total) - 35,65%, Na2O - 0,25%, P2O5 - 0,38%, CO2 - 0,5%, CaO (free) - 19,91%, Al2O3 - 11,16%, TiO2 - 2,65%, MgO - 0,44%, K2O - 0,42%, SO3 (sulphate) - 2,84%
- Impurities: Metals: As, Be, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Tl, V, Zn - sum < 0,1%
- Lot/batch No.: FBC/230309/T2
- Expiration date of the lot/batch: 03/2024
- Stability under test conditions: stable
- Storage: The substance will be stored in PE container at room temperature.

Test system

Amount / concentration applied:
The test substance (25 mg) was grinded in mortar with pestle. Then it was placed atop the tissue previously moistened with 25 μl of sterile water to improve contact of the tissue surface with the test chemical. The moistened material was spread on the tissue surface.

Water for injection - Ardeapharma a.s., Lot No. 0101030309
Duration of treatment / exposure:
3 min, 60 min
Details on study design:
The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Ashland, USA) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis.
The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing 24 tissues on shipping agarose together with the necessary amount of culture media.

Some test substances may interfere with the MTT endpoint if it is able to directly reduce MTT. Therefore, before exposure, functional checks for this possibility was performed as follows: 25 mg of the test substance is added to 1 mL MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistured) for 60 min. At the end of the exposure time, the presence and intensity of the staining (if any) is observed. If the solution changes color from red to blue, other steps to correction have to be done.

On the day of receipt, EpiDerm tissues are conditioned by incubation to release transport stress related compounds and debris. After pre-incubation, tissues are topically exposed to the test chemicals for 60 minutes. Three tissues are used per test chemical and for the positive control (PC) and negative control (NC). Tissues are then thoroughly rinsed, blotted to remove the test substances, and transferred to fresh medium.
After a 24 hr incubation period, the medium is replaced by fresh one. Tissues are incubated for another 18 hours. Afterwards, the MTT assay is performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/ml). After a 3 hr MTT incubation, the blue formazan salt formed by cellular mitochondria is extracted with 2.0 ml/tissue of isopropanol and the optical density of the extracted formazan is determined using a spectrophotometer at 570 nm.

OD570 is measured on a spectrophotometer Libra S22. Isopropanol serves as a blank.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min

Any other information on results incl. tables


Direct MTT reduction

Before the test itselfdirect MTT reduction was assayed.

After 60 min incubation of the test substance with MTT medium, colour was compared with control solution of MTT medium only. Due to presence of dark suspended test substance colour was not clearly appreciable. After filtration no changes in colouring were observed. 

The test substance did not reduce MTT directly.

MTT test

OD570 measuring was performed after overnight extraction.


The average viability of affected tissues was  94.6 % of negative control average value after 3 min treatment and 103.2 % after 60 min. These values are higher than critical values (50 and 15% respectively). The substance could be regarded as non-corrosive.

Applicant's summary and conclusion

Interpretation of results:
other: non-corrosive
Criteria used for interpretation of results: EU
Under the above-described experimental design, the test substance Fluidized Bed Combustion (FBC) Bottom Ash was non corrosive in In vitro Skin Corrosion Test on EpiDermTM tissues.
Executive summary:

Test substance Fluidized Bed Combustion (FBC) Bottom Ash was assayed for the in vitro skin corrosion in human epidermal model EpiDermTM. The test was performed according to Method B.40. Skin corrosion (in vitro),Council Regulation (EC) No. 440/2008. Published in O.J. L 142, 2008.

The test substance (25 mg) was grinded in mortar with pestle andplaced atop the tissue previously moistened with 25 μl of sterile water. Length of exposition was 3 and 60 minutes. Nine tissues were used for the experiment in each time, three per test substance (TS), three for positive control (PC) and three for negative control (NC).

After rinsing, tissues were incubated with MTT for three hours and extracted overnight subsequently at room temperature without shaking.OD570 of isopropanol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

In the experiment arrangement given above, the test substance Fluidized Bed Combustion (FBC) Bottom Ash was not corrosive in EpiDermTMmodel.