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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2018 - 18 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Reconstructed Human Epidermis Test method
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Ruthenium dioxide (hydrate)
Cas Number:
32740-79-7
IUPAC Name:
Ruthenium dioxide (hydrate)
Constituent 2
Chemical structure
Reference substance name:
Ruthenium (IV) oxide
EC Number:
234-840-6
EC Name:
Ruthenium (IV) oxide
Cas Number:
12036-10-1
Molecular formula:
O2Ru
IUPAC Name:
ruthenium (IV) oxide
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Ruthenium(IV)oxid hydrat
- Physical state: Powder
Specific details on test material used for the study:
purity: 75.08% Ru content (calculated after loss on reduction = 100% - 24.92%)

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDermTM (EPI-200, Lot no. 28611) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic
Source strain:
other: Reconstructed human epidermis model (see below)
Details on animal used as source of test system:
Not applicable
Justification for test system used:
The reconstructed human epidermis model system is suitable to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water.
Vehicle:
unchanged (no vehicle)
Details on test system:
The test method is based on reconstructed human epidermis models, which in their overall design (the use of human derived epidermal keratinocytes as cell source, representative tissue and cyto-architecture) closely mimic the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The procedure described under this test method allows the hazard identification of irritant substances in accordance with UN GHS Category 1 or Category 2.

EpiDerm is a three-dimensional reconstructed human epidermis model EpiDermTM, comprised of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

The principle of the reconstructed human epidermis model test is based on the premise that irritant substances are able to penetrate the stratum corneum by diffusion and are cytotoxic to the cells in the underlying layers. Cell viability was measured by dehydrogenase conversion of the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue), into a blue formazan salt that was quantitatively measured after extraction from tissues. Irritant substances were identified by their ability to decrease cell viability below defined threshold levels (i.e.≤ 50% for UN GHS Category 1 or Category 2).

The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDerm lot. The ET50 must fall within a range established based on a historical database of results.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of test item were applied to the skin model and uniformly covered the skin surface with an area of 0.63 cm2. The test item is a fine powder. For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline (D-PBS). Three tissue replicates were used.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL D-PBS was added to each of the three negative control skin units.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL SDS was added to each of the three positive control skin units.
- Concentration (if solution): 5% aqueous solution
Duration of treatment / exposure:
Exposure for 60 minutes: 35 minutes at 37°C, 5% CO2 and 95% relative humidity followed by 25 minutes at room temperature under sterile hood.
Duration of post-treatment incubation (if applicable):
The post-treatment incubation period of the rinsed tissues in fresh assay medium was 42 hours.
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
Score is a percentage of the negative control
Run / experiment:
mean (42 hour time point)
Value:
95.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The mean optical density (OD) of the negative control of 3 tissues was 1.542 and was well within the acceptable range of ≥ 0.8 to ≤ 2.8.
Positive controls validity:
valid
Remarks:
The viability of cells treated with the positive reference item was 6.6% of the negative control and fulfilled the acceptance criterion of <=20%.
Remarks on result:
no indication of irritation
Remarks:
Mean viability of cells exposed to Ruthenium(IV) oxide was 95.5% of the negative control and was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. Ruthenium(IV) oxide was considered to be non-cytotoxic and predicted to be non-irritant to skin.
Other effects / acceptance of results:
Assay acceptability criteria:
1: The mean optical density (OD) of 3 negative control tissues was 1.542 and was well within the acceptable range of ≥0.8 to ≤2.8.
2: The viability of cells treated with the positive reference item, 5% SDS, was 6.6% of the negative control and fulfilled the acceptance criterion of ≤20%.
3: The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤ 18%.

Any other information on results incl. tables

The mean viability of cells exposed to Ruthenium(IV) oxide was 95.5% of the negative control and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of >50%. Ruthenium(IV) oxide was considered to be non-cytotoxic and predicted to be non-irritant to skin.

The mean optical density (OD) of 3 negative control tissues was 1.542 and was well within the acceptable range of ≥0.8 to ≤2.8. The viability of cells treated with the positive reference item, 5% SDS, was 6.6% of the negative control and fulfilled the acceptance criterion of ≤20%.

The summary of the results is given below:

   Optical density (n = 3 tissues) CV (%)

 

 viability (%)

 

 SD

 Negative control (D-PBS)

 1.542

 10.5

 

 100

 

 10.6

 Ruthenium(IV) oxide

 1.473

 16.4

 95.5

 

 15.7

 Positive control (5% SDS)

 0.102

 16.9

 6.6

 

 1.1

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro skin irritation test, using a reconstructed human epidermis model (EpiDermTM), Ruthenium(IV) oxide tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, did not show irritant properties and is therefore not classified as irritant (UN GHS no category).
Executive summary:

The cytotoxic properties of Ruthenium( IV) oxide to skin cells, which might lead to irritation of human skin, were determined by using an artificial three dimensional model of human skin (EpiDerm TM model). Three replicates were used for each treatment and concurrent control group. The

optical density (OD) was determined by using the MTT reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.

Ruthenium( IV) oxide was applied as solid test item to the model skin surface, which was moistened with Dulbecco's phosphate buffered saline (D-PBS). D-PBS was used as the negative control. 5% aqueous sodium dodecyl sulphate (SOS) was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium.

The mean viability of cells exposed to Ruthenium(IV) oxide was 95.5% of the negative control and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. Ruthenium(IV) oxide was considered to be non-cytotoxic and predicted to be non-irritant to skin.

All acceptance criteria required were fulfilled and the study can be considered as valid.

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