Registration Dossier

Diss Factsheets

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-11-25 to 2010-11-26
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Diethyl oxalate
EC Number:
202-464-1
EC Name:
Diethyl oxalate
Cas Number:
95-92-1
Molecular formula:
C6H10O4
IUPAC Name:
diethyl oxalate
Test material form:
other: colourless oily liquid
Details on test material:
- Name of test material: Diethyloxalate

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: reconstructed human epidermal model EpiDerm™
Details on animal used as source of test system:
TEST MATERIAL
Reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Ashland, USA); lot No. 13885, Kit C

Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Ashland, USA) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm™ System is manufactured according to defined quality assurance procedures.
The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing 24 tissues on shipping agarose together with the necessary amount of culture media.

PRINCIPLE OF THE ASSAY
The test consisted of a topical exposure of the neat test chemical to a reconstructed human epidermis (RhE) model followed by a cell viability test. Cell viability was measured by conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazolium-bromide] into a Blue formazan salt, caused by dehydrogenase present in cell mitochondria. The conversion was quantitatively measured after extraction from tissues. The reduction of the viability of tissues exposed to chemicals in comparison to negative controls (treated with sterile water for injection) was used to predict the skin corrosion potential. Effect of a substance was determined by measuring of optical density of the formazan extracts using a spectrophotometer at 570 nm. Relative cell viability was calculated für each triplet of tissues as % of the mean of the negative control tissues.

DIRECT MTT REDUCTION:
Some test substances, which are able to reduce the MTT directly, may interfere with the MTT endpoint. Therefore, before exposure, functional checks were performed as follows: 50 µL of the test substance were added to 1 mL MTT medium (red) and incubated in an incubator (37±1°C, 5±1 % CO2, moistened) for 60 min. At the end of the exposure time, the presence and intensity of the staining (if any) was observed. If the solution changes colour from red to blue, other steps to correction have to be done.

PROCEDURE:
On the day of experiment, EpiDerm tissues were conditioned by incubation to release transport stress related compounds and debris. After pre-incubation, tissues were topically exposed to the test chemicals for 3 and 60 minutes. In each time interval three tissues were used per test chemical, three for the positive control (PC) and three for the negative control (NC). After exposition, tissues are thoroughly rinsed and blotted to remove the test substance (controls).
After that, tissues were transferred to 24-well plates containing MTT medium (1 mg/mL). After a 3 hour MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 mL/tissue of isopropanol and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm.
Control samples:
other: sterile water for injection as negative control and 8N KOH in H20 as positve control
Amount/concentration applied:
The test substance (50 µL, undiluted) was placed directly on top of the tissue and was spread on the tissue surface. There were no problems with spreading the test substance on the skin surface, therefore a mesh was not used.
Duration of treatment / exposure:
3 min and 60 min

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min treatment duration
Value:
89.5
Remarks on result:
no indication of irritation
Remarks:
OD570=1.638; Max. score=1.884
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min treatment duration
Value:
9.4
Remarks on result:
positive indication of irritation
Remarks:
OD570=0.158; Max. score=0.181

Any other information on results incl. tables

MTT test results (viable tissues)

time

treatment

OD570 values in experiment:

mean

SD

interval of values allowed

%NC

 

 

 

1

2

3

 

 

 

 

 

 

NC

water

1.834

1.813

1.845

1.831

0.013

1.556

2.105

100.0

3 min

C1

Diethyloxalate

1.450

1.702

1.763

1.638

0.135

1.393

1.884

89.5

 

PC

8N KOH

0.278

0.390

0.286

0.318

0.051

0.270

0.366

17.4

 

NC

water

1.754

1.532

1.744

1.677

0.102

1.425

1.928

100.0

60 min

C1

Diethyloxalate

0.254*

0.174

0.141

0.158

0.017

0.134

0.181

9.4

 

PC

8N KOH

0.230

0.152

0.177

0.186

0.033

0.158

0.214

11.1

Notes:
0.254* number out of the acceptable SD range
NC negative control
PC positive control
C1 test substance
mean arithmetic mean
% NC viability of single tissues compared with negative control
SD standard deviation calculated from individual % tissue viabilities

Applicant's summary and conclusion

Interpretation of results:
Category 1B (corrosive) based on GHS criteria