Registration Dossier

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:
2013-02-26 to 2013-02-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Sodium selenite
EC Number:
233-267-9
EC Name:
Sodium selenite
Cas Number:
10102-18-8
IUPAC Name:
disodium selenite
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Sodium selenite
- Substance type: inorganic

Test animals

Details on test animals or test system and environmental conditions:
Commercially available Epi-200-Kit. (MatTek In Vitro Life Science Laboratories. Day of delivery: 26. Feb. 2013; Batch EPI-200-CORR: 16880)
The EpiDerm™ tissue 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 EpiDerm™ tissues are cultured on specially
prepared cell cultures inserts.

Test system

Details on study design:
Pre-Tests
The test item Sodium selenite was tested for the ability of direct formazan reduction. To test for this ability, 23.4 mg were added t01 mL of MTT
reagent and the mixture was incubated in the dark at 37°C ± 1°C and 5 % CO2 for 60 minutes. Untreated MTT reagent was used as control.
The MTT reagent didn't change its colour within one hour, therefore, direct MTT reduction had not taken place, and no data correction was necessary.

Preparations
On the day of the start of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the MTT solvent and the solution was
stored at 4°C in the dark. The tissue plate was brought out of the fridge one hour before the treatment. The assay medium was warmed in the water
bath to 37°C.

Description of the Method
Four 6-well-plates were prepared with 0.9 mL assay medium in each well. The inserts containing the tissues were transferred to the wells using
sterile forceps and the 6-well-plates were set into the incubator at 37°C ± 1°C and 5 % CO2for one hour (pre-incubation).
For each experiment ("three minutes" and "one hour"), one 24-well-plate was prepared as holding plate. 12 wells of each plate were filled with 300 µL assay medium, the other 12 with 300 µL MTT reagent. One additional plate was left empty. The plates were stored in the incubator.
For each experiment ("three minutes" and "one hour"), two 6-well-plates were used. After pre-incubation, the assay medium was replaced by fresh assay medium and the test was started, using two wells as negative control with 50 IJL H20 demin., two wells as positive controls with 50 µL potassium
hydroxide solution and two other wells for testing the test item.
25 mg ± 2.5 mg of the solid test item were applied together with 25 µL H20.
At the start of each experiment (application of negative controls), a stop watch was started.

Amounts of Test Item
Incubation 3 minutes 1 hour
Tissue 1 25.5 mg 24.8 mg
Tissue 2 23.3 mg 23.6 mg

After the respective incubation time (three minutes ± 10 sec and one hour), the inserts were removed from the plates using sterile forceps. The
inserts were thoroughly rinsed with PBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium. After transfer of all inserts, they were immediately moved to the wells containing MTT reagent, blotting the bottom with cellulose tissue again
before setting the insert into the MTT well. The tissues were incubated with MTT reagent for three hours. After this time, the MTT reagent was
aspirated and replaced by PBS buffer.
This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate.
Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then covered with Parafilm® and left to
stand over night at room temperature.
On the next day, the inserts in which formazan had been produced over night were pierced with an injection needle, taking care that all colour was
extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, three replicates with 200 IJL solution (each) were pipetted into a 96-wellplate which was read in a plate spectral photometer at 570 nm.



Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
after three minutes treatment
Value:
83.9
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
after 1 hour treatment
Value:
55.4
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The test item is considered not corrosive.
After three minutes treatment, the relative absorbance values were decreased to 83.9 %.
This value is well above the threshold for corrosivity (50 %). After one hour treatment relative absorbance values were reduced to 55.4 %. This value is well
above the threshold for corrosivity (15 %). In the guideline, values greater or equal to the threshold are considered as "non-corrosive to skin".
The values of the negative control were well above the required acceptability criterion of mean OD > 0.8 for both treatment intervals thus showing the quality
of the tissues.
The value for the positive control for the one hour experiment was not within the range of historical data of the test facility. This is considered as uncritical for the following reason: Variation of biological systems within this order of magnitude is not unusual.
Moreover the positive control showed very clearly corrosive effects.
The positive control induced a decrease in the relative absorbance as compared to the negative control to 36.4 % for the three minutes treatment interval and
8.9 % for the one hour treatment interval thus ensuring the validity of the test system.
For these reasons, the result of the test is considered valid.

Any other information on results incl. tables

Comparison of Formazan Production:

For the test item and the positive control, the following percentage values of mean formazan production were calculated in comparison to the mean of the negative controls:

Test Item

Positive Control

Incubation

83.9 %

36.4 % 

3 min   

55.4 %

8.9 %

1 hour 

Applicant's summary and conclusion

Interpretation of results:
other: non-corrosive to skin
Remarks:
Criteria used for interpretation of results: other: CLP; EU GHS (Regulation (EC) No 1272/2008)
Conclusions:
Sodium selenite is considered as not corrosive in the Human Skin Model Test.
Executive summary:

Two tissues of the human skin model EpiDerm™ were treated with Sodium selenite for three minutes and one hour, respectively. In average, 25 mg of the solid test item were applied to each tissue and spread to match the tissue size. One valid experiment was performed.

Deionised water was used as negative control, 8m KOH was used as positive control. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT which can be reduced to a blue formazan. Formazan production was measured by measuring the optical density (OD) of the resulting solution. After treatment with the negative control, the absorbance values were well above the required acceptability criterion of mean OD > 0.8 for both treatment intervals thus showing the quality of the tissues. The positive control showed clear corrosive effects for both treatment intervals.

After three minutes treatment with the test item, the relative absorbance values were reduced to 83.9 %. This value is well above the threshold for corrosion potential (50 %). After one hour treatment, relative absorbance values were reduced to 55.4 %. This value, too, is well above the threshold for corrosion potential (15 %).

In the guideline, values greater or equal to the threshold are considered as "non-corrosive to skin".

Therefore, Sodium selenite is considered as not corrosive in the Human Skin Model Test.