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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:
11 - 13 Sep 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
(2004)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: No data given
Source strain:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C and 5% CO2 in air
- Temperature of post-treatment incubation: 37 °C and 5% CO2 in air

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Duplicate tissues were treated with the test item and the concurrent positive and negative controls for an exposure period of 3, 60 and 240 min. At the end of the exposure period, each tissue was removed from the well and rinsed with Dulbecco´s Phosphate Buffered Saline (DPBS) containing calcium and magnesium.
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: microplate reader
- Wavelength: 540 nm

CELL VIABILITY MEASUREMENTS
- For determining alterations in cell viability, MTT reduction assays were performed immediately after the exposure period. The rinsed tissues were transferred to other wells of the same 12-well plate containing 2.2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium in each well. The tissues were incubated for 3 h at room temperature protected from light. At the end of the 3 h incubation period each tissue was placed onto absorbent paper to dry. The epidermis was carefully separated from the collagen matrix and both parts (epidermis and collagen) placed into labeled 1.5 mL tubes containing 850 μL of acidified isopropanol. The tubes were plugged, mixed thoroughly and stored overnight at room temperature, protected from light, allowing the extraction of formazan crystals out of the MTT-loaded tissues. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a 96-well plate. The optical density was measured at 540 nm wave length in a microplate reader.
Control samples:
other: concurrent control tissues treated with sodium chloride solution served as negative controls, positive controls were exposed to glacial acetic acid
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 20 mg (moistened with 100 µL 0.9% (w/v) NaCl solution)

NEGATIVE CONTROL
- Amount(s) applied: 50 μL NaCl solution, 0.9% (w/v)

POSITIVE CONTROL
- Amount(s) applied: 50 μL glacial acetic acid
Duration of treatment / exposure:
3, 60 and 240 min
Duration of post-treatment incubation (if applicable):
not applicable
Number of replicates:
The test was performed in duplicates for each treatment and control group.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 min exposure
Value:
90.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Remarks:
60 min exposure
Value:
97.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Remarks:
240 min exposure
Value:
98.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Other effects / acceptance of results:
The relative mean viability of the test item treated reconstructed human-derived keratinocyte tissues after a 3, 60 and 240 min exposure period was 90.7, 97.7 and 98.1%, respectively compared to the negative control. Therefore, the test item is considered to be non-corrosive.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The assay establishes the acceptance criterion for an acceptable test if the mean optical density for the negative control treated tissues is ≥ 0.115 and ≤ 0.400.
- Acceptance criteria met for positive control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was 0 to 20% relative to the negative control treated tissues following the 240 min exposure period.




Any other information on results incl. tables

Table 1: Mean OD540 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

Exposure period

(min)

Mean OD540 of

duplicated tissues

Relative mean

viability (%)

Negative Control

Item

240

0.214

100*

Positive Control

Item

240

0.019

8.9

Test Item

240

0.210

98.1

60

0.209

97.7

3

0.194

90.7

*: the mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:
other: non-corrosive
Conclusions:
CLP: not classified