[Name confidential or not available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 5, 2008 to February 5, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Principles of method if other than guideline:

The study procedures were based on the following documents:
- L'Oreal Standard Operating Procedure, ECVAM Skin Irritation Validation Study, Validation of the Episkin Skin Irritation Test (42 hours) assay for the prediction of acute skin irritation of chemicals, January 2005.
- The ESAC statement (ECVAM Scientific Advisory Committee) on the validity of in vitro tests for skin irritation, April 2007

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: White powder
- Analytical purity: 99%
- Lot/batch No.: 08-018
- Expiration date of the lot/batch: 30 April 2009
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature protected from light

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

water

Details on test system:

TEST SYSTEM:
Name: EPISKIN Standard model (EPISKIN-SM(TM), 0.38 cm2, human three dimensional epidermal model)
Lot No.: 09-EKIN-001
Source: SkinEthic Laboratories, Nice, France
Rationale: Recommended test system by the Institute for Health & Consumer Protection, European Centre for the Validation of Alternative Methods (ECVAM).

TREATMENT:
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. 10mg of the test substance with 10 μL of Milli-Q water was applied directly on top of the skin tissue. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate saline to remove residual test substance. The skin tissues were kept in new well plates on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed.
After a 42 hour (at 37C) incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.
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 the test substance compared to the negative control tissue was calculated

NEGATIVE CONTROL
- Name: Phosphate buffered saline (PBS, Invitrogen Corporation, Breda, The Netherlands)
- Amount(s) applied (volume or weight with unit): 10 μL

POSITIVE CONTROL
- Name: Sodium dodecyl sulphate (SDS, Sigma Aldrich, Zwijndrecht, The Netherlands)
- Amount(s) applied (volume or weight with unit): 10 μL
- Concentration: 5% (aqueous solution)

SCORING SYSTEM:
Cell viability measurement: reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT. The amount of extracted formazan was determined spectrophotometrically at 570 nm induplicate with Multiskan Spetrum (Thermo Labsystems).

ACCEPTABILITY OF THE ASSAY:
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the three tissues of the negative control should be >=0.6 and the Standard Deviation value (SD) of the % viability should be <=18.
b) The mean relative tissue viability of the positive control should be <=40% relative to the negative control and the Standard Deviation value (SD) of the % vailbility shoukd be <=18.
c) The SD calculated from individual percentual tissues viabilities of the three identically treated replicates should be <=18.

DATA EVALUATION:
A test substance is considered irritant if:
The relative mean tissue viability of three individual tissues after 15 min of exposure to the test substance and 42 hours of post incubation is <=50% of the mean viability of the negative controls.
A test substance is considered non-irritant if:
The relative mean tissue viability of three individual tissues after 15 min of exposure to the test substance and 42 hours of post incubation is >50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg
- Concentration (if solution):

VEHICLE
- Name: water (Milli-Q water)
- Amount(s) applied for moistening (volume or weight with unit): 10 μL

Duration of treatment / exposure:

15 minutes

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

101

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1 Mean absorption in the in vitro skin irritation with the test substance

 

Performance of study from 6 January 2009 to 12 January 2009

 

	A (OD570)	B (OD570)	C (OD570)	Mean   SD (OD570)
Negative control	0.695	0.737	0.693	0.708 + 0.025
Test substance	0.703	0.713	0.727	0.714 + 0.012
Positive control	0.038	0.051	0.046	0.045 + 0.006

OD = optical density

SD = Standard deviation

Triplicate exposure are indicated by A, B and C. 

In this table with the values are corrected for background absorption. Isopropanol was used to mesure the background absorption.

Table 2 Mean tissue viability in the in vitro skin irritation test with the test substance

 

	Mean tissue viability (percentage of control)
Negative control	100
Test substance	101
Positive control	6

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

It is concluded that this test is valid and that the test substance is non-irritant in the in vitro skin irritation test under the experimental conditions described in the report.