Registration Dossier

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
other: in vitro (validated and accepted)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2013
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:
other: EU method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test), adopted 24 August 2009
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, adopted 22 July 2010
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
- Name of test material (as cited in study report): EXP1200078
- Physical state: dark brown viscous liquid
- Storage condition of test material: at room temperature in the dark

- Analytical purity: 100%
- Lot/batch No.: E00275-350
- Expiration date of the lot/batch: 31 Dec 2013
- Stability at higher temperatures: yes, maximum temperature 60 °C; maximum duration 1 hour

Test animals

Species:
human
Details on test animals and environmental conditions:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 36.9-37.5
- Humidity (%): 74-97

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amounts applied: 25 µl

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
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
TEST SITE
- EPISKIN Standard ModelTM(EPISKIN-SMTM, 0.38 cm2, Lot no.: 13-EKIN-009).This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates 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 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (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:
% tissue viability
Value:
33
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The relative mean tissue viability obtained after 15 minutes treatment with EXP1200078 compared to the negative control tissues was 33%. Although the test substance shows interaction with MTT, which can result in a false negative (non-irritant) conclusion, the outcome of the mean relative tissue viability for EXP1200078 was below 50% after 15 minutes treatment. Therefore the substance is considered to be 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 a colour change was observed it was concluded that the test substance did interact with MTT. In addition to the normal procedure, three killed tissues treated with test substance and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by EXP1200078 was 49% of the negative control tissues.

The non-specific MTT (NSMTT) was outside the acceptability criteria of the assay. A high NSMTT indicates that the test substance shows a high interaction with MTT. When a high response of MTT is observed, a high OD570 will be the consequence. However, although the test substance interacts with MTT, the test substance showed a very low OD570 without correction for the NSMTT. Correcting for the NSMTT would result in negative values. Therefore, the NSMTT was not taken into account for the determination of the viability of the test substance treated skin tissues.

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The in vitro skin irritation test was conducted according to OECD 439 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), the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2 cultured skin (25 µl). 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 19% whereas the test substance showed a cell viability of 33%. 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.