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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:
2012-03-06 to 2012-03-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-conducted and documented study performed under GLP according to current guideline.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
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
Test material form:
solid: flakes
Details on test material:
- Substance type: organic peroxide
- Physical state: flaked solid
- Analytical purity: >99.0%
- Expiration date of the lot/batch: 2012-10-01
- Storage condition of test material: -20 °C in dark

Test animals

Species:
human
Details on test animals and environmental conditions:
The EPISKIN(TM) 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 coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Test system

Type of coverage:
other:
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened wtih 5 µl of sterile distilled water to improve contact between the solid test item and the epidermis.
Duration of treatment / exposure:
Triplicate tissues were treated wtih the test item for an exposure period of 15 minutes. Culbecco's Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ was used as the negative control. Sodium Dodecyl Sulphate (SDS) 5% w/v was used as the positive control.
Observation period:
Rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
Number of animals:
not applicable
Details on study design:
Pre-incubation: 2 ml maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium felled wells (3 units per plate). A different 12-well plate wasused for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.
Application of test item and rinsing (Day 1): 2 ml maintenance medium, warmed to approximately 37 °C, was pipettted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. the test item was applied topically to the corresponsding tissues ensuring uniform covering. Approximately 10 mg of the test item ws applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test item and theepidermis. Triplicate tissues treated with 10 µl of DPBS served as the negative controls and triplicate tisues treated with 10 µl of SDS 5% w/v served as the positive controls. The plates were kept in the biological safety cabinet at romo temperature for 15 minutes.

At the end of the exposure period, each tissue was removed from teh well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved uby filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 ml of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

Following the 42-hour post-exposure incubation period, each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the medium. 1.6 ml of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 °C 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). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN(TM) biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 ml micro tubes containing 500 µl acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed throughly on a vortex mixer. The tubes we3re refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of the formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extration period each tube was mixed throughly 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". The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
99.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

In vivo

Irritant / corrosive response data:
The relative mean viability of the test item treated tissues was 99.5% after a 15-Minute exposure period.
Other effects:
The relative mean tissue viability for the positive control treated tissues was 7.8% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 0.7%. The positive control acceptance criterion was tehrefore satisfied.

The mean OD540 for the negative control treated tissues was 0.847 and the standard deviation value of the percentage viability was 5.8%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of three identically treated tissues was 3.3%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Tes test item is considered a Non-Irritant (NI).
Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN(TM) reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-expousre incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures folowing topical exposure to thetest imte by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 -[4,5 -dimethylthiazol-2 -yl]-2,5 -diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tisssues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 -hour post-exposure incubatino period is also determined for test items whicha re found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Triplicate tissues were treatd wtih the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for posssible inflammatory mediator determinatino. AFter MTTT loading a total biopsy of each epidermis wasmade and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were tranferred to the appropriate wells of a pre-labelled 96 -well plate. The optical density was measured at 540 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 99.5% after the 15 -minute exposure period. The quality criteria requried fora cceptance of results in the test were satisfied.

The test item was considered to be Non-Irritant (NI).