Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 April - 27 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Yellow to brown, aromatic liquid
- Storage: At +10°C to +25°C under inert gas, kept in a tightly closed container and stored in a dry place, protected from heat and direct sunlight.

In vitro test system

Test system:
human skin model
Source species:
other:
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDermTM (EPI-200, Lot no. 23338) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic
Source strain:
other: Reconstructed human epidermis model (see details below)
Details on animal used as source of test system:
Not applicable
Justification for test system used:
The reconstructed human epidermis model system is suitable to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water.
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiDerm is a three-dimensional reconstructed human epidermis model, comprised of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

The principle of the reconstructed human epidermis model test is based on the premise that irritant substances are able to penetrate the stratum corneum by diffusion and are cytotoxic to the cells in the underlying layers. Cell viability was measured by dehydrogenase conversion of the vital dye MTT (3-[4,5- Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue), into a blue formazan salt that was quantitatively measured after extraction from tissues. Irritant substances were identified by their ability to decrease cell viability below defined threshold levels (i.e.≤ 50% for UN GHS category 2 irritants).

The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by the manufacturer for viral, bacterial, fungal and mycoplasma contamination. MatTek corporation determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDerm lot. The ET50 must fall within a range established based on a historical database of results.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL of undiluted test item were applied to the skin model with a surface area of 0.63 cm2 to uniformly cover the skin surface. Three replicate tissues were employed. At the end of the exposure period, the test item was carefully washed from the skin surface with D-PBS.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL D-PBS was added to each of the three negative control skin units.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL SDS was added to each of the three positive control skin units.
- Concentration (if solution): 5% aqueous solution
Duration of treatment / exposure:
Exposure for 60 minutes: 35 minutes at 37°C, 5% CO2 and 95% humidity followed by 25 minutes at room temperature.
Duration of post-treatment incubation (if applicable):
The post-treatment incubation period of the rinsed tissues in fresh assay medium was 42 hours.
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
Score is a percentage (%) of negative control.
Run / experiment:
Mean
Value:
90.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The mean OD of the NC of 3 tissues was 1.773 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5.
Positive controls validity:
valid
Remarks:
The viability of cells treated with the PC was 6.0% of the NC and fulfilled the acceptance criterion of ≤ 20%.
Remarks on result:
no indication of irritation
Remarks:
If the mean relative viability <=50% the test substance is considered to be irritant to skin.
Other effects / acceptance of results:
The standard deviation of all triplicates determined was below the limit of acceptance of 18%. Hence, all acceptance criteria required were fulfilled.

Any other information on results incl. tables

No discolorations were noted in the test for colour change under aqueous conditions. Additionally, no change of colour was noted in the test for MTT interference potential. Therefore the test item did not interact with the MTT measurement and no additional test had to be performed.

 

The summary of the OD results is given below:

   Optical density (n=3 tissues)  SD  % OD540 compared to the control
 Negative control (D-PBS)  1.773  0.0907  -
 "Karstedt concentrate"  1.607  0.091  90.6
 Positive control (5% SDS)  0.106  0.012  6.0

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro EpiDerm assay conducted in accordance with OECD Test Guideline 439 and to GLP, "Karstedt concentrate" was non-cytotoxic and, hence, non-irritant to skin.
Executive summary:

“Karstedt concentrate” was tested for skin irritation potential in an in vitro EpiDerm assay, using reconstructed human epidermis, conducted in accordance with OECD Test Guideline 439 and to GLP. The mean cell viability following exposure to the test substance was calculated to be greater than 50% (90.6% of the negative controls), and it was therefore considered to be non-cytotoxic and predicted to be non-irritant to skin. The positive and negative controls were considered valid.

Under the conditions of this assay, “Karstedt concentrate” would not be classified as "irritant" according to EU CLP criteria (EC 1272/2008).