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:
8 May 2012 - 13 July 2012
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium periodate
EC Number:
232-197-6
EC Name:
Sodium periodate
Cas Number:
7790-28-5
Molecular formula:
HIO4.Na
IUPAC Name:
sodium periodate
Test material form:
solid: crystalline
Details on test material:
- Physical state: Solid, white
- Storage conditions of test material: Room temperature in the dark

In vitro test system

Test system:
human skin model
Remarks:
Reconstructed human epidermis model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on test system:
The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

EPISKIN Model Kit 0.38 cm2
Supplier: SkinEthic Laboratories, Nice, France

MATERIAL PREPARATION
Test material: the substance was ground before use.
MTT: a 3 mg/mL stock solution was prepared in DPBS. This was diluted to 03 mg/mL with assay medium when required.
Acidified isopropanol: 0.04 N concentration of hydrochloric acid in isopropanol.

CONTROLS
0.9 % w/v sodium chloride solution was used as the negative control.
Glacial acetic acid was used as the positive control.

PRE-TEST
Assessment of Direct Test Item Reduction of MTT

MTT Dye Metabolism, Cell Viability Assay
The MTT assay, a colourimetric method of determining cell viability, is based on the reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.

One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction
The test item was checked for the ability to directly reduce MTT according to the following procedure:

20 mg of the test item was added to 2.2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at room temperature for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turned blue relative to the control, the test item was presumed to have reduced the MTT. In this case, the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

PRE-INCUBATION (Day 0: tissue arrival)
2.2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into two wells of the first column of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12-well plate was used for each test item, control and time point. The tissues were incubated at 37 °C, 5 % CO2 in air overnight. After 24 hours the medium underneath the tissues was refreshed and the tissues were returned to the incubator for a further 24 hours.

MAIN TEST
APPLICATION OF TEST ITEM AND RINSING (Day 2)
2.2 mL of assay medium, warmed to approximately 37 °C, was pipetted into 2 wells of the second column of the 12-well plate. The tissues were transferred into the second column.
Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control items for an exposure period of 240 minutes. 20 mg of the solid test item was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. 100 µL of 0.9 % w/v sodium chloride solution was added for wetting of the test item. Duplicate tissues, treated with 50 µL of 0.9 % w/v sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 µL of glacial acetic acid served as positive controls. The treated tissues were kept in a biological safety cabinet at room temperature for the appropriate exposure period.

At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item. Each tissue was then placed in a 6 well plate prefilled with culture media (post soak) for 20 minutes. The purpose of the post soak was to assist removal of absorbed test item from within the tissue and to adjust the pH to normal conditions where the MTT conversion to formazan occurs efficiently.

2.2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12 well plate. The tissues were blotted and transferred into the MTT filled wells. The tissues were incubated for 3 hours ± 5 minutes at room temperature in a biological safety cabinet ensuring that the plates were protected from light. At the end of the 3 hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was taken using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 mL micro tubes containing 850 µL of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT-loaded tissues.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (Day 3)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.

For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as `blanks'. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

INTERPRETATION OF RESULTS
Quantitative MTT Assessment (percentage tissue viability)

The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240 minute exposure periods, compared to the mean of the negative control tissues (n = 2) treated with 0.9 % w/v sodium chloride solution. The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD540 of test item / mean OD540 of negative control) x 100

Classification of corrosivity potential was based on relative viabilities for each exposure time according to table 1.

QUALITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
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.
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 minute exposure period.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
20 mg
Duration of treatment / exposure:
Exposure periods of 3, 60 and 240 minutes.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: OD540
Value:
0.018
Remarks on result:
other:
Remarks:
Time point: 240 minutes exposure. Max. score: 0.14. Reversibility: other: not applicable. Remarks: Viability 12.9 %. (migrated information)
Irritation / corrosion parameter:
other: other: OD540
Value:
0.078
Remarks on result:
other:
Remarks:
Time point: 60 minutes exposure. Max. score: 0.14. Reversibility: other: not applicable. Remarks: Viability: 55.7 %. (migrated information)
Irritation / corrosion parameter:
other: other: OD540
Value:
0.181
Remarks on result:
other:
Remarks:
Time point: 3 minutes exposure. Max. score: 0.14. Reversibility: other: not applicable. Remarks: Viability: 129.3. (migrated information)
Other effects / acceptance of results:
Test Material and Controls
Mean OD540 values and viabilities are given in Table 2.

The relative mean viability of the test item treated tissues was as follows:
240 minutes exposure: 12.9 %
60 minutes exposure: 55.7 %
3 minutes exposure: 129.3 %

-The 240 minute exposure induced tissue viability less than 35 %. Therefore according to the prediction model detailed above, a corrosive classification was triggered.
-The 3 minute and 60 minute exposures were greater than 35 % and therefore the test item did not trigger a corrosive classification after the 3 or 60 minute exposure.

Direct MTT Reduction
The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT. Quantitative correction of the results was therefore unnecessary.

Quality Criteria
-The relative mean tissue viability for the positive control treated tissues was 8.6 % relative to the negative control treated tissues following the 240 minute exposure period. The positive control acceptance criterion was therefore satisfied.
-The mean OD540 for the negative control treated tissues was 0.140. The negative control acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 2: Mean OD540 Values and Viabilities

Item

Exposure Period

(Minutes)

Mean OD540 of Duplicate Tissues

Relative Mean Viablity (%)

Negative Control

240

0.140

100*

Positive Control

240

0.012

8.6

 

Test Material

240

0.018

12.9

60

0.078

55.7

3

0.181

129.3

*The mean viability of the negative control is set at 100 %.

Applicant's summary and conclusion

Interpretation of results:
other: Classified as Category 1C (corrosive) in accordance with EU criteria
Conclusions:
The test item was classified as corrosive to the skin. The following classification criteria apply:
-EU CLP (1272/2008/EC) and UN GHS Hazard statement H314 "Causes severe skin burns and eye damage" Category 1C.
Executive summary:

The corrosivity potential of the test material was investigated in the EPISKIN Reconstructed Human Epidermis Model in accordance with OECD Guideline 431 and EU Method B.40.

The relative mean viability of the tissues treated with test material were:

240 minutes exposure: 12.9 %

60 minutes exposure: 55.7 %

3 minutes exposure: 129.3 %

As a result of this, the test item was classified as corrosive to the skin. The following classification criteria apply:

-EU CLP (1272/2008/EC) and UN GHS Hazard statement H314 "Causes severe skin burns and eye damage" Category 1C.

Categories Display