Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline for Testing of Chemicals No. 431, April 13, 2004 (“In vitro Skin Corrosion: Human Skin Model Test”)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
the study follows the test strategy for determination of a corrosive property as given in the following guideline: OECD Guideline for Testing of Chemicals No. 404, April 24, 2002 (“Acute Dermal Irritation/Corrosion”)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Gelb LD 6259 (No 08/0646-1)
- Physical state: solid/ sun yellow
- Analytical purity: Main component Gelb LD 6259: HPLC fingerprint at 230 and 400 nm: 87.6% and 89.1 area% , respectively. Free acid content: about 61.3 g/100g
- batch No.: 0817 VP 01
- Stability under test conditions: the stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility
- Storage condition of test material: room temperature
- Other: the test substance was homogeneous by visual inspection, pH value: ca. 5 (undiluted test substance, moistened with water)

Test animals

Species:
other: in vitro test with EpiDerm™ 200 kit
Strain:
other: not applicable
Details on test animals and environmental conditions:
- EpiDerm™ 200 kit: MatTek Corp., Ashland MA, USA containing 24 Epi-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® 1 cm diameter

The test system was a three dimensional human epidermis model: the EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of 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. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

- Tissue model: Epi-200
- Origin: MatTek Corporation, Ashland MA, USA
- Tissue for MTT reduction control: Epi-200 tissue that is killed by twice freezing at –20°C and thawing at room temperature
- Assay medium: Dulbecco's modified eagle's medium (DMEM); for the assay and for diluting MTT (MatTek Corp., Ashland MA, USA / Sigma, Germany)
- Wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca 2+, Mg 2+ (MatTek Corp., Ashland MA, USA and Biochrom, Germany)
- Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (MatTek Corp., Ashland MA, USA / Sigma,Germany), 1.0 mg / mL assay medium
- Extracting agent: Isopropanol p.a.

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
other: not applicable
Controls:
other: not applicable
Amount / concentration applied:
25 μL highly deionized water was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water. Control tissues were concurrently applied with 50 μL of highly deionized water (negative control, NC) or with 50 μL of 8 n potassium hydroxide (positive control, PC).
Duration of treatment / exposure:
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used.
Observation period:
The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours.
After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with isopropanol over night at room temperature. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 3 microtiter wells filled with Isopropanol for each microtiter plate.
Number of animals:
not applicable
Details on study design:
On day of receipt EpiDermTM tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-substance application, tissues were transferred to 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium was replaced with fresh medium immediately before application .

Results and discussion

In vivo

Results
Irritation parameter:
other: in vitro test
Basis:
other: see above
Time point:
other: see Tables 1&2
Remarks on result:
other: test substance is not corrosive
Irritant / corrosive response data:
CONCLUSION
Based on the observed results and applying the evaluation criteria cited above it was concluded, that Gelb LD 6259 does not show a corrosive potential in the EpiDerm skin corrosivity test under the test conditions chosen.
The test method does not yet allow for the evaluation of skin irritation. The result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from an in vivo study would be needed.

Any other information on results incl. tables

Table 1: Findings after 3 min exposure

 

Test compound

Exposure: 3 min

OD570 tissue 1

OD570 tissue 2

OD570 KC

Mean OD570

Mean OD570 KC corrected

Viability [% of NC]

Negative control

1.994

1.813

-

1.878

-

100

Test substance

1.790

1.984

-

1.887

-

100

Positive control

0.448

0.442

-

0.435

-

24

 

Table 2: Findings after 1 hour exposure

 

Test compound

Exposure: 3 min

OD570 tissue 1

OD570 tissue 2

OD570 KC

Mean OD570

Mean OD570 KC corrected

Viability [% of NC]

Negative control

1.888

1.759

-

1.823

-

100

Test substance

2.120

2.117

-

2.118

-

116

Positive control

0.108

0.148

-

0.128

-

7

 

Applicant's summary and conclusion