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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-11-15 to 2011-11-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 439 (In vitro skin irritation: reconstructed human epidermis test method)
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
Cell source:
other: The Episkin model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix loaded with type IV collagen.
Vehicle:
unchanged (no vehicle)
Details on test system:
MTT DYE METABOLISM, CELL VIABILITY ASSAY
- The MTT assay is a colourmetric method of determining cell viability based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
- One limitation of the assay is possible interference of the test material with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if, at the time of the MTT test (after rinsing), there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

TEST FOR DIRECT MTT REDUCTION
- To identify possible interference, the test material was checked for ability to directly reduce MTT.
- 10 μL of test material was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium.
- The solution was incubated in the dark at 37 ºC, 5 % carbon dioxide in air, for 3 hours.
- Untreated MTT solution was used as a control.
- If the MTT solution containing the test item turns blue, the test material is presumed to have reduced MTT.

EPISKIN MODEL KIT
- Supplier: SkinEthic Laboratories, Nice,.
- Date received: 2011-11-15.

PREPARATION OF NEGATIVE AND POSITIVE CONTROL ITEMS, MTT AND ACIDIFIED ISOPROPANOL
- The negative control item was used as supplied.
- The positive control item was prepared as a 5 % w/v aqueous dilution.
- A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
- A 0.04 N concentration of hydrochloric acid in isopropanol was prepared when required.
 
PRE-INCUBATION (DAY ZERO: TISSUE ARRIVAL)
- 2 mL of maintenance medium, warmed to approximately 37 ºC, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate.
- Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate).
- A different 12-well plate was used for the test material and control item.
- The tissues were incubated at 37 ºC, 5 % carbon dioxide in air, overnight.
 
MAIN TEST (APPLICATION OF TEST ITEM AND RINSING (DAY 1)
- 2 mL of maintenance medium, warmed to approximately 37 ºC, was pippetted into the second column of 3 wells of 12-well plate.
- Triplicate tissues were treated with the test item for an exposure period of 15 minutes.
- 10 μL of the test item was applied to the epidermis surface.
- Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5 % w/v served as the positive controls.
- To ensure satisfactory contact with the positive control item, the SDS solution was spread over the entire surface of the epidermis using a pipette tip, taking particular care to cover the centre.
- After 7 minutes contact time, the SDS solution was re-spread with a pipette to maintain the distribution of the SDS for the remainder of the contact period.
- The plate(s) were kept in a biological safety cabinet at room temperature for 15 minutes.
- At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++.
- Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test material.
- Rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well.
- The rinsed tissues were then incubated at 37 ºC, 5 % carbon dioxide in air, for 42 hours.
 
MTT LOADING AND FORMAZAN EXTRACTION (DAY 3)
- Following the 42 hour post-exposure incubation period, each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium.
- 1.6 mL of maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at minus 14 to minus 30 ºC for possible inflammatory mediator determination.
- 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plate(s).
- Tissues were transferred to the MTT filled wells and care was taken to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper.
- Tissues were incubated for 3 hours at 37 ºC, 5 % carbon dioxide in air.
- At the end of the three hour incubation period, each tissue was placed onto absorbent paper to dry.
- A total biopsy of the epidermis was made using the Episkin biopsy punch.
- The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed.
- Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer.
- The tubes were refrigerated at 1 to 10 ºC until day six of the investigation, allowing the extraction of formazan crystals from MTT-loaded tissues.
 
ABSORBANCE / OPTICAL DENSITY MEASUREMENTS (DAY 6)
- At the end of the formazan extraction period, each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
- For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate.
- 200 μL of acidified isopropanol alone was added to the two wells designated as blanks.
- Optical density was measured (quantitative viability analysis) at 540 nm (without reference filter) using an Anthos 2001 microplate reader.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 μL of the test item was applied to the epidermis surface.
Duration of treatment / exposure:
15 minutes.
Duration of post-treatment incubation (if applicable):
42 hours.
Number of replicates:
Triplicate

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of triplicate runs
Value:
68.8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: the test item was non-irritant
Other effects / acceptance of results:
DIRECT MTT REDUCTION
- The MTT solution containing the test item did not turn blue, which indicated that the test material did not directly reduce MTT.
 
TEST MATERIAL, POSITIVE CONTROL AND NEGATIVE CONTROL
- Individual and mean OD540 values, standard deviations and tissue viabilities for the test material, negative control and positive control are shown in Table 1 (attached).
- Mean viabilities and standard deviation of the test material and positive control relative to the negative control are also given in Table 1 (attached).

The relative mean viability of the test item treated issues was 68.8% after a 15-Minute exposure period.
 
QUALITY CRITERIA
- Relative mean tissue viability for the positive control treated tissues was 4.5 % relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.4 %. The positive control acceptance criterion was therefore satisfied.
- Mean OD540 for the negative control treated tissues was 0.927 and the standard deviation value of the percentage viability was 8.7 %. The negative control acceptance criterion was therefore satisfied.
- Standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 13.7 %. The test item acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
other: not-irritating
Conclusions:
The test material was determined to be non-irritant.