Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Draft Proposal for a New Guideline, Vers. 4, 11. December 2009
Deviations:
no
Qualifier:
according to
Guideline:
other: ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EpiSkin and EpiDerm assays and on the Skin Integrity Function Test (Altern lab Anim. 2007 Dec; 35 (6):559-601)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
other: human-derived
Cell type:
other: epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SITE
- Area of exposure: tissue surface 0.38 cm²

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Using a wash bottle the tissues were gently rinsed with PBS trying to remove any residual test material. Due to the test item’s highly viscous consistency, it could not be removed completely. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.
- Time after start of exposure: 15 min

SCORING SYSTEM: measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt

TEST SYSTEM
EpiSkin™ Kit Components Needed for the Assay
- 1 Sealed 12-well plate contains 12 inserts with EpiSkin™ tissues on agarose
- 1 12-well plate for MTT viability assay
- 1 bottle Assay Medium (Basic medium for use in MTT assays)
- 1 bottle EpiSkin™ Maintenance Medium (Basic medium for incubations)

MTT-Solution
3 mg MTT Formazan salt were dissolved in 1 mL PBS. Before treatment of the tissues the MTT solution was diluted with assay medium to reach a final concentration of 0.3 mg/mL.

Cell Culture
The EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues will be shipped on medium-supplemented agarose gels in a 12-well plate. Including time in transit, tissues may be stored in the refrigerator at 2 - 8 °C for up to 6 days prior to use. On day of receipt, EpiSkin™ tissues will be transferred to cell culture plates with maintenance medium.
EpiSkin™ tissues were shipped with ice packs on medium-supplemented agarose gels in a 12-well plate. On day of experiment EpiSkin™ tissues were transferred to 12-well plates with maintenance medium.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Amount(s) applied (volume or weight with unit): approximately 10 μL (due to its highly viscous consistency, the test item could not be weighed exactly)
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h postincubation + 3 h incubation with MTT
Number of replicates:
in total, 3 12-well plates were used

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other:
Run / experiment:
mean (test item)
Value:
62.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: value is well above the threshold for irritancy of ≤ 50%
Irritation / corrosion parameter:
other:
Run / experiment:
mean (positive control)
Value:
22.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: value is well below the threshold for irritancy of ≤ 50% ensuring the validity of the test system

In vivo

Irritant / corrosive response data:
The relative absorbance values were reduced to 62.9 % after the treatment. This value is above the threshold for skin irritation (≤ 50%). Therefore, the test item is considered as non-irritant to skin. For details see "Any other information on results incl. tables"

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study and under the experimental conditions reported, the test item is non irritant to skin.
Executive summary:

This in vitro GLP study according to OECD Guideline 439 was performed to assess the irritation potential of the test substance by means of the Human Skin Model Test. Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes. 10 μL of the test item were applied to each tissue, spread to match the tissue size. 10 μL of either the negative control (deionised water) or the positive control (5% sodium lauryl sulfate) were applied to each tissue. After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 for the 15 minutes treatment interval thus showing the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 15 minutes treatment interval thus ensuring the validity of the test system. The standard deviations between the % variabilities of the test item, the positive and negative controls were below 8% and therefore, below the threshold of 18% of the OECD Guideline, thus ensuring the validity of the study. After treatment with the test item the relative absorbance values decreased to 62.9%. This value is well above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is non irritant to skin.