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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:
December 17, 2012 - December 24, 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:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
(2010)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(2009)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Amiloride Compound 5
IUPAC Name:
Amiloride Compound 5
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Amiloride Compound 5
- CAS Number: 5424-01-1
- Description: Tan powder

Test animals

Species:
human
Details on test animals or test system and environmental conditions:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 36.9 - 37.4
- Humidity (%): 69 - 95

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amounts applied: 10.8 to 12.1 mg, 5 μl Milli-Q water to moisten skin.

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

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) Sodium dodecyl sulphate
Duration of treatment / exposure:
15 minutes
Details on study design:
TEST SITE
- EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Lot no.: 12-EKIN-047). 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.

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: other: tissue viability
Value:
110
Remarks on result:
other:
Remarks:
Basis: other: percentage of control. Time point: 15 minutes. Remarks: Negative control = 100%; Positive control = 31%. (migrated information)

In vivo

Irritant / corrosive response data:
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 Amiloride Compound 5 compared to the negative control tissues was 110%. Since the mean relative tissue viability for Amiloride Compound 5 was above 50% after 15 minutes treatment Amiloride Compound 5 is considered to be non-irritant.

Any other information on results incl. tables

The test substance 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 the test substance did not interact with MTT.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The 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 Amiloride Compound 5 is not irritating in the in vitro skin irritation test.
Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Small Model), the influence of Amiloride Compound 5 on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2 cultured skin (10.8 to 12.1 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 31% whereas the test substance showed cell viability of 110%. Since the mean relative tissue viability after exposure to Amiloride Compound 5 was above 50%, it can be concluded that Amiloride Compound 5 is not irritating in the in vitro skin irritation test.