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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:
04Sept - 10Sept 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

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:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
adopted 24 August 2009
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 22 July 2010
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): TMAC
- Physical state: white, crystalline powder
- Expiration date of the lot/batch: 27 July 2013 (allocated by WIL Research Europe B.V., 1 year after receipt of the test substance)
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark, in well-sealed container (due to hygroscopic properties)

Test animals

Species:
human
Details on test animals and environmental conditions:
ENVIRONMENTAL CONDITIONS
- Temperature (°C):36.9 - 37.7
- Humidity (%): 83-96

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amounts applied: 10.5 to 11.8 mg, The test substance was weighed in a small glass weight boat, however when applying the test substance it attracted that many water that the test substance was liquefied. The test substance was applied directly on top of the skin tissue via pipetting and using a cotton swab. Since optimal contact was obtained using this method this deviation has no impact on the study integrity.

NEGATIVE CONTOL:
- Amount applied: 25 µl Phosphate buffered saline.

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) Sodium dodecyl sulphate in PBS.

Before application of the test substance, the positive or the negative control, the skin was moistened with 5 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue.
Duration of treatment / exposure:
15 minutes
Details on study design:
TEST SITE
- EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 12-EKIN-032). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. The EPISKIN human epidermis was obtained from SkinEthic Laboratories, Lyon, France.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
28
Remarks on result:
other:
Remarks:
Basis: other: percentage of control. Time point: 15 minutes. Remarks: Negative control = 100%; Positive control = 5%. (migrated information)

Any other information on results incl. tables

TMAC was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that TMAC did not interact with MTT.

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
An in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that the test substance is irritating in the in vitro skin irritation test.
Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model) according to OECD 439 guideline and GLP principles, the influence of TMAC on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2 cultured skin (10.5 to 11.8 mg, in presence of 5 μl Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 5% whereas the test substance showed cell viability of 28%. Since the mean relative tissue viability after exposure to the test substance was below 50%, it can be concluded that the test substance is irritating in the in vitro skin irritation test.