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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Dec 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
adopted in 2004
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Landesinstitut für Arbeitsschutz und Produktsicherheit, München, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm, reconstructed three-dimensional human epidermis (EPI-200)
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SKIN MODEL
- Source: MatTek Corporation, Ashland MA, USA

TEST METHOD
EpiDermTMSCIT (EPI-200):
The model represents a reconstructed three-dimensional skin model based on normal human-derived epidermal keratinocytes which have been cultured to form a multilayered epidermis including basal, spinous and granular layers, and a multi-layered stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS
Upon receipt, tissues were transferred into 6-well plates containing 900 µL prewarmed assay medium per well and preincubated in a humidified incubator for at least 1 h (37 ± 1 °C, 5% CO2) before use.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1
- CO2 gas concentration (%): 5
- Humidity: maximum

REMOVAL OF TEST SUBSTANCE
- Washing: The test item was washed from the skin surface with phosphate buffered saline.
- Time after start of exposure: 3 and 60 min

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, tissues were incubated in 300 µL prewarmed MTT solution for 3 h at 37 ± 1 °C and 5% CO2. After aspiration of the MTT solution, tissues were washed 3 times in phosphate buffered saline followed by tissue drying. Extraction of the formazan product was carried out in 2 mL isopropanol. The optical density was measured at 550 nm wave length in a plate spectrophotometer.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 mg
- Concentration: 100% (moistened with 25 µL water)

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: Potassium hydroxide (8 N, CAS 1310-58-3, Lot 10357004)
Duration of treatment / exposure:
3 and 60 min
Number of replicates:
The test was performed in duplicates for each test or control group and treatment period (3 and 60 min).

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min of exposure
Value:
93
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min of exposure
Value:
88
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
After treatment with the test item the viability was 93% and 88% after treatment for 3 and 60 min, respectively. Therefore the test item is considered to be not corrosive.

Any other information on results incl. tables

Table 1: MTT assay after 3 min exposure

 

Negative control

Positive control

Test item

Tissue sample

1

2

1

2

1

2

OD550

2.088

2.085

2.082

1.947

1.985

1.978

0.309

0.315

0.312

0.269

0.271

0.269

2.001

1.907

1.878

1.803

1.859

1.818

OD550(mean)

2.085

1.970

0.312

0.270

1.929

1.827

SD

0.003

0.020

0.003

0.001

0.064

0.029

OD550(mean values of replicates)

2.028

0.291

1.878

Viability (%)

100

14

93

Mean inter tissue viability difference (%)

5.7

14.5

5.4

 

Table 2: MTT assays after 60 min exposure

 

Negative control

Positive control

Test item

Tissue sample

1

2

1

2

1

2

OD550

2.032

1.954

1.976

2.014

1.992

1.974

0.114

0.117

0.115

0.119

0.129

0.121

1.857

1.754

1.762

1.742

1.717

1.736

OD550(mean)

1.987

1.993

0.115

0.123

1.791

1.732

SD

0.040

0.020

0.002

0.005

0.057

0.013

OD550(mean values of replicates)

1.990

0.119

1.761

Viability (%)

100

6

88

Mean inter tissue viability difference (%)

0.3

6.4

3.4

 

Table 3: Historical data

 

mean

SD

n

OD550of Negative Controls (3 min)

1.731

0.176

25

OD550of Negative Controls (60 min)

1.748

0.192

25

Relative tissue viability (%) of positive controls (3 min)

19.0

5.958

24

Max. inter tissue viability difference (%)

13.9

6.627

144

 

Applicant's summary and conclusion

Interpretation of results:
other: non-corrosive (Skin Irrit. 2 or not classified according to Regulation (EC) No 1272/2008)
Conclusions:
There is regulatory acceptance in the EU that a substance can be considered corrosive based on a positive result in the human epidermis model test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant and shall therefore be subject to further evaluation.