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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:
11 Spet - 17 Sept 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
(adopted 22 July 2010)
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)
Remarks:
The Department of Health of the Government of the United Kingdom

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-(1-methylethoxy) ethyl actate
- Physical state: liquid
- Analytical purity: 99.9%
- Storage conditions: room temperature, in the dark

Test animals

Species:
human
Strain:
other: EpiSkin; reconstructed three-dimensional human epidermis
Details on test animals and environmental conditions:
TEST SKIN MODEL
- Source: SkinEthic Laboratories, Nice, France

TEST METHOD
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 coated with type IV collagen (EPISKIN Model Kit, SkinEthic Laboratories, Nice, France). A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising the main basal, spinous and granular layers and a functional stratum corneum. The test item is applied topically to the straturm corneum surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The test is based on the experience that irritant chemicals are cytotoxic after exposure to the EPISKIN model. Irritant chemicals are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers after a longer incubation period. Toxicity is determined by the metabolic conversion of the vital dye MTT to formazan by viable cells in the test item treated cultures relative to the negative control. Additionally, IL-1alpha in the culture medium is measured to for test items which are borderline non-irritant based on MTT reduction. This complementary endpoint will be used to either confirm a non-irritant result or to override the non-irritant result.

ADAPTATION TO CELL CULTURE CONDITIONS
Tissues were transferred into 12-well plates containing 2 mL of prewarmed maintenance medium and incubated for 1 day at 5% CO2.

Test system

Type of coverage:
open
Preparation of test site:
other: intact reconstructed human epidermis
Vehicle:
unchanged (no vehicle)
Controls:
other: 106concurrent control tissues treated with 10 µL of DPBS served as negative controls, positive controls were exposed to 5% (w/v) SDS
Amount / concentration applied:
TEST MATERIAL: 10 µL

POSITIVE CONTROL SUBSTANCE: 5% (w/v) SDS; 10 µL; to ensure uniform contact SDS was spread over the whole surface with a pipette tip, after 7 minutes, SDS was re-spread to ensure distribution
Duration of treatment / exposure:
15 minutes followed by 42 hours post-exposure in medium
Observation period:
not applicable
Number of animals:
not applicable
The test was performed in duplicates for each test or control group and treatment period
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: The test item was washed from the skin surface with DPBS.
- Time after start of exposure: 15 min

CELL VIABILITY MEASUREMENTS & IL-1alpha
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 the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30ºC for possible inflammatory mediator determination.
The tissues were transferred to 2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium and incubated for 3 hours at 37°C, 5% CO 2 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 EPISKINTM 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 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°C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. The optical density of 200 µL (duplicate measurements) was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: cell viability (%)
Value:
100
Remarks on result:
other:
Remarks:
Basis: other: mean value of negative controls. Time point: 15 min + 42 hours. Reversibility: other: not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: cell viability (%)
Value:
106.1
Remarks on result:
other:
Remarks:
Basis: other: mean value of the test item. Time point: 15 min + 42 hours. Reversibility: other: not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: cell viability (%)
Value:
9.9
Remarks on result:
other:
Remarks:
Basis: other: mean value of the positive control. Time point: 15 min + 42 hours. Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:
The relative mean viability of the test item treated tissues was 106.1% after a 15-Minute exposure period and is therefore in range with the negative control and no irritating effect was observed. The positive control showed irritation. Additionally, the acceptance criteria are fulfilled.
Other effects:
The MTT solution containing the test item (in a pretest) did not turn blue which indicated that the test item did not directly reduce MTT.

Any other information on results incl. tables

Table 1: Mean OD540Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD540 of tissues

Mean OD 540 of triplicate tissues

±SD of OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

 

Negative Control Item

0.920

0.854

0.060

107.7

100

7.0

0.839

98.2

0.804

94.1

Positive Control Item

0.047

0.085

0.051

5.5

9.9

6.0

0.064

7.5

0.143

16.7

Test Item

0.816

0.906

0.078

95.6

106.1

9.1

0.955

111.8

0.947

110.9

 

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified