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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:
15-21 November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD439 and under GLP conditions.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Method B.46 of Commission Regulation 440/2008/EC (In vitro skin irritation)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guidelines for the Testing of Chemicals No. 439 "In Vitro Skin Irritation" (adopted 22 July 2010)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Cashmeran
- Molecular formula: C14H22O
- Molecular weight: 206.33
- Physical state: Liquid
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
human
Strain:
other: In vitro skin model (EPISKIN standard model)
Details on test animals and environmental conditions:
THE EPISKIN MODEL
- Supplier: SkinEthic Laboratories, Nice, France
- After receiving the EPISKIN, the tissue was pre-incubated overnight in maintenance medium at 37°C, 5% CO2 in air.

ENVIRONMENTAL CONDITIONS: all incubations, with the exception of the test substance incubation of 15 minutes at room temperature:
- Temperature (°C): 37
- 5% CO2 in air
- Photoperiod: dark

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: The test item was applied topically to the corresponding tissues ensuring uniform covering.
Vehicle:
unchanged (no vehicle)
Controls:
other: POSITIVE CONTROL: Sodium Dodecyl Sulphate (SDS), prepared as a 5% w/v aqueous dilution. NEGATIVE CONTROL: Dulbecco's Phosphate Buffered Saline (DPBS) with Ca++ and Mg++, used as supplied
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): Undiluted substance

POSITIVE CONTROL: Sodium Dodecyl Sulphate (SDS), prepared as a 5% w/v aqueous dilution
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. After 7 minutes contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period.

NEGATIVE CONTROL: Dulbecco's Phosphate Buffered Saline (DPBS) with Ca++ and Mg++, used as supplied
Duration of treatment / exposure:
Exposure: 15 minutes
Incubation: 42 hours
Number of animals:
A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control respectively.
Details on study design:
TEST SITE: EXPOSURE OF THE TISSUES
After receiving the EPISKIN, the tissue was pre-incubated overnight in maintenance medium at 37°C, 5% CO2 in air.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): 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++.
- Time after start of exposure: 15 minutes.

SCORING SYSTEM: The principle of the assay was based on the measurement of cytotoxicity (irritancy) in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability was 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 tissue relative to the negative controls.

TOOL USED TO ASSESS SCORE: The amount of extracted formazan was determined spectrophotometrically at 540 nm in duplicate (without a reference filter) using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other:
Value:
10.8
Remarks on result:
other:
Remarks:
Basis: mean. Max. score: 100.0. Reversibility: other: Not applicable. Remarks: SD: ± 2.9%. (migrated information)
Irritation / corrosion parameter:
other:
Value:
0.101
Remarks on result:
other:
Remarks:
Basis: mean (of triplicate tissue). Max. score: 0.934. Reversibility: other: Not applicable. Remarks: SD: ± 0.027. (migrated information)

In vivo

Irritant / corrosive response data:
The test item was considered to be irritant.
Other effects:
No other effects observed

Any other information on results incl. tables

The positive control had a mean cell viability of 5.5% after 15 minutes exposure. The negative control was set at 100%.

The standard deviation of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The relative mean tissue viability after 15 minutes treatment with Cashmeran compared to the negative control tissue was 10.8%. Since the mean relative tissue viability for Cashmeran was below 50% after 15 minutes treatment, Cashmeran is considered to be irritant. It is concluded that this test is valid and that Cashmeran is irritant in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

This report describes the ability of Cashmeran to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-TM)). The possible skin irritation potential of Cashmeran was tested through topical application for 15 minutes. The study procedures described in this report were based on the OECD Guidelines for the Testing of Chemicals No. 439 "In Vitro Skin Irritation" (adopted 22 July 2010) and Method B.46 of Commission Regulation (EC) No. 440/2008/EC.

Skin tissue was treated with Cashmeran by topical application of 10 µL Cashmeran. 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. The relative mean tissue viability obtained after 15 minutes treatment with Cashmeran compared to the negative control tissue was 10.8%.

Since the mean relative tissue viability for Cashmeran was below 50% after 15 minutes treatment Cashmeran is considered to be irritant. The positive control had a mean cell viability of 5.5% 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.

Finally, it is concluded that this test is valid and that Cashmeran is irritant in the in vitro skin irritation test under the experimental conditions described in this report.