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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted in accordance with internationally recognised test guidelines. For read across justification see Section 13.

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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
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): R3
- Physical state: liquid
- Lot/batch No.: 0004
- Expiration date of the lot/batch: 2012-07-01
- Storage conditions: room temperature

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 stratum 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: 20 µL undiluted substance

POSITIVE CONTROL SUBSTANCE: 20 µL 5% (w/v) sodium dodecyl sulphate (SDS)
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 triplicate 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 tubes and stored in a freezer at approximately -20ºC 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% 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 a 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 mixed thoroughly on a vortex mixer then refrigerated at at approximately 4°C for 3 days allowing the extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was centrifuged to eliminate debris and the optical density of duplicate 200 µL aliquots of the extracted solution was measured (quantitative viability analysis) at 595 nm.

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:
87.3
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:
13
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 87.3% 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

Mean OD595Values and Percentage Viabilities for the Negative Control, Positive Control and Tested substance

Item

OD595 of tissues

Mean OD 595 of triplicate tissues

±SD of OD595

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

 

Negative Control

1.032

0.969

0.068

106.5

100

6.5

0.907

93.6

0.968

99.9

Positive Control

0.189

0.169

0.040

19.5

17.4

4.4

0.198

20.4

0.120

12.4

Test

0.897

0.803

0.077

97.5

87.3

8.9

0.763

82.9

0.750

81.5

 

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The potential of the substance to be irritant to the skin has been investigated using a reconstructed human epidermis (RhE) model in accordance with OECD/EU test methods. The tested substance had essentially no effect on the test system (87.3% of mean cell viability when compared to negative controls), indicating that there is little potential for the substance to be a skin irritant.
Executive summary:

. The potential of the substance to be irritant to the skin has been investigated using a reconstructed human epidermis (RhE) model in accordance with OECD/EU test methods. The tested substance had essentially no effect on the test system (87.3% of mean cell viability when compared to negative controls), indicating that there is little potential for the substance to be a skin irritant.