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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 April 2017 to 01 June 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
no
Remarks:
See'Justification for information'

Test material

Constituent 1
Reference substance name:
Active enzyme protein of sedolisin (EC no. 848318-58-1, CAS no. 814-114-0, EC name Sedolisin, Enzyme class no 3.4.21.100 )
Molecular formula:
Not available
IUPAC Name:
Active enzyme protein of sedolisin (EC no. 848318-58-1, CAS no. 814-114-0, EC name Sedolisin, Enzyme class no 3.4.21.100 )
Constituent 2
Reference substance name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available
IUPAC Name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 3
Reference substance name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 4
Reference substance name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Constituent 5
Reference substance name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Test material form:
liquid
Details on test material:
- Lot/batch No.: PPF38268
- Expiration date of the lot/batch: 22-april-2025
- Storage condition of test material: Frozen

In vitro test system

Test system:
human skin model
Remarks:
SKINETHICTM RHE tissue model.
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Vehicle:
unchanged (no vehicle)
Details on test system:
According to INVITTOX Protocol SKINETHIC™ Skin Corrosivity Test
SKIN PREPARATION
- Procedure used: SKINETHICTM RHE tissue model. The cells were removed from packaging plate and transferred to 6-well plates containing 1 mL growth medium.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37ºC and 5% CO2
- Temperature of post-treatment incubation (if applicable): Same
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: The nylon mesh was removed from tissue surface and rinsed once with 25 mL PBS. The bottom of the insert and the atypical side of the tissue was dried.
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Yes, but not specified.
- Wavelength: 570 nm
- Filter: Not specified
- Filter bandwidth: Not specified
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
Negative Control: Negative Control ≥ 1.2 OD and SD is ≤ 18%.
Positive Control: Expressed as % of Neg. Cont. ≤ 40 % and SD is ≤ 18%.
Test Substance: Difference in viability between two replicas is ≤ 18%
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 μL
- Concentration (if solution): used as is

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): used as is
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): used as is
Duration of treatment / exposure:
42 min.
Duration of post-treatment incubation (if applicable):
42 ± 1 hours.
Number of replicates:
Triplicate tissues each for test substance, negative control Phosphate Buffered Saline and positive control 5% Sodium Dodecyl Sulphate.

Test system

Type of coverage:
open
Preparation of test site:
other: Not necessary since they are skin membranes
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 μL
- Concentration (if solution): used as is

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): used as is
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): used as is
Duration of treatment / exposure:
42 minutes
Details on study design:
TEST SITE
- Area of exposure: not specified
- Type of wrap if used: Nylon mesh

REMOVAL OF TEST SUBSTANCE
- Washing (if done): With PBS
- Time after start of exposure: Exposure time 42 min, post-exposure time 42 ± 1 h.

SCORING SYSTEM:
- Method of calculation: OD measurement with a spectrophotometer

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: Not specified
- Colour interference with MTT: Not specified

The experiment was considered valid if:
Negative Control: Negative Control ≥ 1.2 OD and SD is ≤ 18%.
Positive Control: Expressed as % of Neg. Cont. ≤ 40 % and SD is ≤ 18%.
Test Substance: Difference in viability between two replicas is ≤ 18%

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The acceptance criteria was fulfilled and the assay was deemed valid and the control test substances performed as expected. Sedolisin, batch PPF38268 did not cause any skin irritation and is classified as being a non-irritant (No category) under the conditions applied in this assay.
Executive summary:

The purpose of the study was to evaluate the skin irritation potential of sedolisin, batch PPF38268. This was done by employing SKINETHICTM RHE tissue model.

Skin irritation refers to the production of reversible damage to the skin following application of a test substance.

The irritation potential of the test substance is assessed by two methods:

• Visual evaluation of the color of the cell in the wells.

• The MTT spectrophotometric measurement at 570 nm on Enspire.

Both methods are based on the take-up of MTT in living cells and the hence the conversion of MTT to a blue dye that can be measured spectrophotometrically.

The test substance was given as is in amounts of 16 μL for 42 min, followed by an inclubation period of 42 ± 1 h.

The acceptance criteria was fulfilled and the assay was deemed valid and the control test substances performed as expected. Sedolisin, batch PPF38268 did not cause any skin irritation and is classified as being a non-irritant (No category) under the conditions applied in this assay.