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:
other: GLP guideline study, according to OECD guideline

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
other: Three dimensional human epidermis model: EpiDermTM model consists of normal, human-derived epidermal keratinocytes.
Details on test animals and environmental conditions:
TEST SYSTEM
- Source: MatTek Corporation, Ashland MA, USA
- Tissue model: Epi-200
- Culture form: to form a multi layered, highly differentiated
- Composition: organized 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
- Tissue surface: 0.6 cm²
- Culture plates: specially prepared cell culture inserts (MILLICELLs®, 10 mm ∅), containing 24 tissues on shipping agarose

Test system

Vehicle:
physiological saline
Remarks:
phosphat buffered saline (PBS)
Controls:
other: Negative control (NC): PBS, sterile; Positive control (PC): 5 % (w/v) sodium dodecyl sulfate (SDS) in highly de-ionized water, sterile
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μL bulk volume (about 10 mg)
Duration of treatment / exposure:
1-hour topical exposure
Observation period:
about 42-hours post-incubation period
Number of animals:
irritation test: three EpiDerm™ tissue samples with the test substance, the PC and NC
Details on study design:
BASIC PROCEDURE:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.
Three tissues were treated with the test substance, the PC and NC, respectively.
For the solid test substance, 25 μL sterile PBS was applied first. Thereafter, a bulk volume of 25 μL of the test material was applied with a sharp spoon and homogeneously distributed together with the fluid.
Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5 % SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards.
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab.
Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours.
After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.
After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation.
The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol for at least 2 hours at room temperature on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Results and discussion

Any other information on results incl. tables

The EpiDerm skin irritation test showed the following results:

The test substance is not able to reduce MTT directly.

The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 119%.

Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show a skin irritation potential in the EpiDermTM skin irritation test under the test conditions chosen.

Irritation Test results:

Test-Article

 

Tissue 1

Tissue 2

Tissue 3

Mean

SD

NC

Mean OD570

1.8343

1.5913

1.8213

1.749

 

Viability [% of NC]

104.9

91.0

104.1

100

7.82

09/0503

 -1

Mean OD570

2.1678

1.9638

2.0938

2.0752

 

Viability [% of NC]

123.9

112.3

119.7

119

5.9

PC

Mean OD570

0.1428

0.1698

0.1383

0.1503

 

Viability [% of NC]

8.2

9.7

7.9

9

0.97

NC: Negative control

PC: Positive control

OD570: Optical density (wavelength 570 nm)

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: EU-GHS