Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 24, 2011 - August 26, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
other: Skinethic Skin irritation test -42bis Standard operating procedure (SOP) 2009
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
To reduce animal testing, this alternative in vitro method was used. The human skin RHE-model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model
- Tissue batch number(s): 11 022A 0802
- Date of initiation of testing: August 24, 2011

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: minimum volume of 25 mL PBS using a multi pipette


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: spectrophotometer
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES:
3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
One experiment in triplicate

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after exposure is less than 50% or equal to 50 %.
- The test substance is considered to be non-irritant to skin if the viability after exposure is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied:
16 mg

NEGATIVE CONTROL
- Amount applied:
16 µL

POSITIVE CONTROL
- Amount applied: 16 µL
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 1/experiment 1
Value:
1.681
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 2/experiment 1
Value:
1.79
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 3/experiment 1
Value:
1.798
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system:
no
- Direct-MTT reduction:
no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
Yes
- Acceptance criteria met for positive control:
Yes
- Acceptance criteria met for variability between replicate measurements:
Yes

Any other information on results incl. tables

Results

 Dose group

 treatment interval  optical density tissue1  optical density tissue 2  optical density tissue 3  MEAN optical density  mean relative viability [%] Standard deviation 
 negative control  42 min  1.768  1.598  1.878  1.748  100  8.06
 positive control  42 min  0.057  0.049  0.057  0.054  3.11  8.32
 test item  42 min  1.681  1.790  1.798  1.756  100.47  3.74

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance is to be considered as non-irritating to the skin under the conditions of this experiment.
Executive summary:

In an in vitro skin irritation model, substance was tested. The applied cellline (RHE (Reconstructed Human Epidermis) is produced by SkinEthic by culturing human adult keratinocytes on a polycarbonate filter in such a quality that a functional stratum corneum is available and thermal differentation takes place. The test was conducted under GLP and according to OECD guideline 439, the EU Method B.46 and the Standard Operating Procedure of SkinEthic's (Skin irritation test -42bis).

Each test is performed in triplicates. 16µl of controls were directly applied to the tissue. Prior 16mg of test substance were applied, 10 µl of water were sprayed to the tissue. After treatment of (treatment time 42minutes at roomtemperature) substances were removed and tissues were incubated for 424 hours.

For testing validity of the Skin model, a positive control (5% sodiumdodecylsulfate in deionized water) and a negative control (PBS buffer) were used.

After topical exposure of test substance, positive control, or negative control cell viability is measured in a MTT assay. In this assay cell viability is measured by conversion of MTT to a formazan salt by a dehydrogenase enzyme.

As the test item has a very high cell viability of (100%) passing the threshold value of 50%, this item is to be considered as non-irritant. The test is valid since the criteria for the positive control (cell viability < 40%; 3,1% in this test) and the negative control (optical density between 1.2 and 2.5; 1.75 in this itest) are met.