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Toxicological information

Skin irritation / corrosion

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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:
09-11-2016 to 01-12-2016
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Adopted 28 July 2015.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate issued by Health Care Inspectorate of the Ministry of Health, The Netherlands.

Test material

Constituent 1
Reference substance name:
Nuclease, deoxyribo-
EC Number:
232-667-0
EC Name:
Nuclease, deoxyribo-
Cas Number:
9003-98-9
Molecular formula:
n.a.
IUPAC Name:
Nuclease, deoxyribo-
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
Test material form:
liquid
Details on test material:
- Lot/batch No.: PPW42035
- Expiration date of the lot/batch: No specific expiry date. At least 10 years or as long as enzyme activity is preserved.
- Storage condition of test material: Refrigerated in the dark.

In vitro test system

Test system:
human skin model
Remarks:
EpiDerm™ reconstructed skin membranes
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN PREPARATION
- Procedure used: EpiDerm™ reconstructed skin membranes. The keratinocytes were plated on chemically modified, collagen-coated, 9 mm ID cell culture inserts (surface area 0.64 cm2).
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: each skin model was removed from the well, rinsed with PBS to remove the study substance (and mesh), blotted dry and transferred to a 6-well plate containing medium.
- 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
The in vitro skin irritation potential of the test substance was determined from the relative mean tissue viabilities compared to the negative control tissues, using the following prediction model:
Mean tissue viability (% of negative control): ≤ 50 %, classification and labelling required, irritant or corrosive.
Mean tissue viability > 50 %, Non-irritant (No Category).
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL
- Concentration (if solution): used as is (7.2% TOS)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (7.2% TOS)
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (7.2% TOS)
Duration of treatment / exposure:
60 min.
Duration of post-treatment incubation (if applicable):
42 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 reconstructed 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): 30 μL
- Concentration (if solution): used as is (7.2% TOS)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (7.2% TOS)
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (7.2% TOS)
Duration of treatment / exposure:
60 minutes
Details on study design:
TEST SITE
- Area of exposure: 0.64 cm2
- % coverage: 100%
- Type of wrap if used: Nylon mesh

REMOVAL OF TEST SUBSTANCE
- Washing (if done): With PBS
- Time after start of exposure: Exposure time 60 min, post-exposure time about 42 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: No reduction was observed.
- Colour interference with MTT: None observed.

The experiment was considered valid if:
- the OD of the viable negative control was ≥ 0.8 and ≤ 2.8.
- skin models treated with the positive control showed mean tissue viability ≤ 20% compared to the negative control.
The test group was considered valid if the standard deviation (SD) calculated from individual tissue viability percentages of the three replicates was ≤ 18%.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The mean viability of the EpiDerm™ reconstructed skin membranes was 88 ± 1 % compared to the negative control group.
Based on the results obtained in the present study, deoxyribonuclease, batch PPW42035 was considered as non-irritant (UN GHS No Category).
Executive summary:

This study was performed to determine the in vitro skin irritation potential of deoxyribonuclease, batch PPW42035 using EpiDerm™ reconstructed skin membranes. The skin membranes were topically exposed to the test substances for 60 min. Viability of the epidermal cells was assessed using the MTT test at ca. 42 h post-exposure. Negative and positive controls were run in parallel. The general principle for the detection of viability via the MTT test is the conversion of the yellow tetrazolium salt (MTT) to the blue/purple coloured product formazan by mitochondrial enzymes. The formation of formazan was measured using a spectrophotometer. The acceptance criteria of the negative and positive control were met and therefore, the study was considered valid.

The mean viability of the skin membranes was 88 ± 1 % compared to the negative control group.

Based on the results obtained in the present study, deoxyribonuclease, batch PPW42035 was considered as non-irritant (UN GHS No Category).