Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 September 2016 - 3 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
23 July 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
other: liquid

In vitro test system

Test system:
human skin model
Source species:
other: human
Cell type:
other: epidermal keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France
Source strain:
other: not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM
- Tissue batch number(s): 16-EKIN-039
- Production date/Shipping date: no data
- Delivery date: 27 September 2016
- Date of initiation of testing: 29 September 2016

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3 hours at 37 °C
- Spectrophotometer: not specified
- Wavelength: 562 nm (without reference fildeter)
- Filter: no data
- Linear OD range of spectrophotometer: no data

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not required as no MTT colour interference expected (assessment done).

DECISION CRITERIA
- A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Applied volume: 10 μL
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Relative mean tissue viability compared to the negative control tissues (=100%)
Value:
109.2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks:
5.9% viability
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: No colour changes observed
- Colour interference with MTT: No colour changes observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: No data

ACCEPTANCE OF RESULTS:
-The relative mean tissue viability for the positive control treated tissues was 5.9% relative to the negative control treated tissues and the standard deviation value of the viability was 1.7%. The positive control acceptance criteria were therefore satisfied.
-The mean OD562 for the negative control treated tissues was 1.013 and the standard deviation value of the viability was 2.8%. The negative control acceptance criteria were therefore satisfied.
-The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 12.7%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Mean OD562Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item OD562 of
tissues
Mean OD562
of triplicate
tissues
± SD of
OD562
Relative
individual
tissue
viability (%)
Relative
mean
viability (%)
± SD of
Relative
mean
viability (%)
Negative
Control Item
0.981 / 1.031 / 1.028 1.013 0.028 96.8 / 101.8 / 101.5 100* 2.8
Positive
Control Item
0.078 / 0.045 / 0.058 0.060 0.017 7.7 / 4.4 / 5.7 5.9 1.7
Test Item 1.242 / 0.986 / 1.090 1.106 0.129 122.6 / 97.3 / 107.6 109.2 12.7

OD=Optical Density

SD=Standard deviation

*=The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:
other: not a skin irritant
Remarks:
in accordance with EU CLP (1272/2008 and its amendments)
Conclusions:
Under the conditions of this test, the relative mean tissue viability for the test item determined to be 109.2%. This value is well above the 50% viability threshold for irritancy. Based on the results obtained, it can be concluded that Muguesia is not an irritant to skin.
Executive summary:

The possible skin irritation potential of Muguesia was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 10 μL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 5.9% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. Under the conditions of this test, the relative mean tissue viability for the test item determined to be 109.2%. This value is well above the 50% viability threshold for irritancy. Based on the results obtained, it can be concluded that Muguesia is not an irritant to skin.