Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to internationally recognised guideline; under GLP conditions.

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:
In Vitro Skin Irritation: Reconstructed human Epidermis Test Method, 22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 761/2009, L 220, Annex III Part B.46. “ In vitro Skin Irritation: Reconstructed Human Epidermis Model Test”. 23-Jul-2009
Deviations:
no
Qualifier:
according to
Guideline:
other: L’Oréal Standard Operating Procedure: “EpiSkin Skin Irritation Test Method“; -ECVAM Skin Irritation Validation Study - Validation of the EpiSkin Test Method for the Prediction of Acute Skin 15 min - 42 hours Irritation of Chemicals. Version 1.8, Feb-2009
Deviations:
no
Qualifier:
according to
Guideline:
other: ECVAM Performance Standards for in vitro Skin Irritation Test Methods based on Reconstructed human Epidermis (RhE). Updated Version 24-Aug-2009
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO/FDIS 10993-10, Annex D: 2009(E), “Biological evaluation of medical devices - Part 10: Tests for irritation and skin sensitization”
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): 11-HN (11 alpha-hydroxynorandrostenedione)
- Physical state: Solid
- Expiration date of the lot/batch: 19/11/2013
- Storage condition of test material: room temperature

Test animals

Species:
other: in vitro: EPISKIN Standard model
Strain:
other: n/a
Details on test animals and environmental conditions:
This test uses the EPISKIN-SM recontructed human epidermis model (SKinEthic) which consists of human keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closey mimics the biochemical and physiological properties of the upper parts of the human skin i.e. the epidermis.

Test system

Type of coverage:
open
Preparation of test site:
other: No preparation of the test site.
Vehicle:
water
Controls:
other: additional tissues in triplicate were available for the positive and negative control groups.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (with 5ul distilled water)

VEHICLE
- name: distilled water
- - Amount(s) applied for moistening (volume or weight with unit): 5 ul

NEGATIVE CONTROL
- name: Phosphate buffered saline (PBS)
- Amount(s) applied (volume or weight with unit): 10 ul

POSITIVE CONTROL
- name: sodium dodecyl sulfate (SDS)
- Amount(s) applied (volume or weight with unit): 10 ul of 5% SDS solution
Duration of treatment / exposure:
15 minutes
Details on study design:
Pre-test: to check the MTT reducing ability of the test material, 10mg of the test material was mixed with 2 ml of MTT medium and incubated for 3 hours at 37 degrees C in the dark.

Main test: The test was performed with triplicate tissues for the test substance, positive and control groups. The test material was applied to directly on top of the skin tissue. After the exposure period of 15 minutes at room temperature, the tissues were washed with PBS to remove residual test substance. The skin tissues were transferred to new well plates containing maintainance medium incubated at 37 +/-1 degrees C, 5% CO2 for 42 hours. After the 42 hour incubation period, determination of cytotoxic effect was performed. Tissues were transferred into an MTT assay plate (containing prewarmed MTT solution) and incubated for 3 hours at 37 +/- 1 degrees C, 5% CO2. After incubation, a biopsy of each tissue were transferred to tube containing isopropanol and kept over the weekend in the dark at 2-8 degrees C. The OD of the extracts were measured using a spectrophotometer.

Results and discussion

Any other information on results incl. tables

Pre-test: The test material did not cause the MTT to change colour to blue/purple indicating that the test material does not have MTT-reducing capability.

Results of the main test are shown in the following table:

Name Negative Control Positive Control Test Material
1 2 3 1 2 3 1 2 3
Absolute OD 550nm 1.325 1.338 1.225 0.144 0.135 0.138 1.267 1.398 1.161
1.260 1.311 1.196 0.143 0.131 0.144 1.233 1.379 1.111
Blank-corrected OD 550nm 1.282 1.295 1.182 0.102 0.092 0.095 1.224 1.355 1.118
1.217 1.268 1.153 0.100 0.089 0.101 1.190 1.336 1.068
Mean OD 550nm (blank corrected) 1.250 1.282 1.168 0.101 0.090 0.098 1.207 1.346 1.093
Total mean OD 550nm of 3 replicate tissues (blank-corrected) 1.233 0.096 1.215
SD OD 550nm 0.06 0.01 0.11
Relative tissue viabilities (%) 101.4 103.9 94.7 8.2 7.3 8.0 97.9 109.1 88.7
Mean tissue viability (%) 100 7.8 98.6
SD tissue viability (%) 4.8 0.4 10.3

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test material showed no irritant effects producing a mean relative tissue viability of 98.6%. Therefore the test material is considered to be a non-irritant. The positive and negative control groups achieved the expected result and confirm the validity of this study.
Executive summary:

This study protocol is based on OECD 439 guideline; Commission Regulation (EC) No. 761/2009, L 220, Annex III Part B.46; L’Oréal Standard Operating Procedure: “EpiSkin Skin Irritation Test Method“; -ECVAM Skin Irritation Validation Study - Validation of the EpiSkin Test Method for the Prediction of Acute Skin 15 min - 42 hours Irritation of Chemicals. Version 1.8; ECVAM Performance Standards for in vitro Skin Irritation Test Methods based on Reconstructed human Epidermis (RhE); ISO/FDIS 10993-10, Annex D: 2009(E); and was performed under GLP conditions. EPISKIN tissue were exposed to 10 mg of test material (applied directly to the tissue with 5ul distilled water) for 15 minutes. After exposure tissues were rinsed and further incubated for a 42 hours at 37 degrees C. After this incubation period, determination of the cytotoxic effect was performed by transferring tissues to plates containing MTT medium and incubated for 3 hours. Tissue biopsies were taken and extraction of contents were performed by placing tissues in isopropanol. The OD of the extracts were measured using a spectrophotometer to determine cytotoxicity. Under the conditions of this study, the test material showed no irritant effects producing a mean relative tissue viability of 98.6%. The test material is considered to be a non-irritant. The positive and negative control groups achieved the expected result and confirm the validity of this study.