Tetraammonium decachloro-μ-oxodiruthenate(4-)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22-24 February 2012

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:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

OECD Guidelines for Testing of Chemicals, Section 4, No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” adopted 22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals. Available at: [http://ecvam.jrc.ec.europa.eu]

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS MODEL TEST in relation to Regulation (EC) No 440/2008 (as amended) and Regulation (EC) No 1907/2006 on REACH (Annex III, B.46).

Deviations:

no

Principles of method if other than guideline:

The test is designed to predict and classify the skin irritant potential of chemicals according to chemical safety regulations, using the reconstructed human epidermis model EPISKIN-SM and parameters related to skin irritation.
EPISKIN-SM is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability. Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of cell viability in treated tissues is compared to negative controls and expressed as a %. The % reduction in viability is used to predict the irritation potential.

The EPISKIN-SM has been found scientifically valid for reliably predicting no label and R38 (irritant) substances in respect to the previous EU classification scheme and has been confirmed in April 2009 by ESAC for use under the UN GHS system as "applicable to all authorities". It is approved by international regulatory agencies as a replacement for the identification of irritants/corrosives in the in vivo rabbit skin assay(OECD 404).

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Diammonium hexachlororuthenateTetraammonium decachloro-μ- oxodiruthenate

IUPAC Name:

Diammonium hexachlororuthenateTetraammonium decachloro-μ- oxodiruthenate

Constituent 2

Reference substance name:

Tetraammonium decachloro-μ- oxodiruthenate (IV)

IUPAC Name:

Tetraammonium decachloro-μ- oxodiruthenate (IV)

Constituent 3

Reference substance name:

Tetraammonium decachloro-μ-oxodiruthenate(4-)

EC Number:

286-924-7

EC Name:

Tetraammonium decachloro-μ-oxodiruthenate(4-)

Cas Number:

85392-65-0

Molecular formula:

Cl10ORu2.4H4N

IUPAC Name:

tetraammonium decachloro-μ-oxodiruthenate(4-)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Tetraammonium decachloro-μ-oxodiruthenate
- Substance type: Technical product
- Physical state: solid (brown powder)
- Analytical purity: >95%
- Composition of test material, percentage of components: 28.73% ruthenium
-Impurities: Ammonium chloride (0.7%), water (4.0%) and 'metallic impurities' (0.3%).
- Purity test date: 20 July 2015
- Lot/batch No.: 10111
- Expiration date of the lot/batch: 31 July 2012
- Storage condition of test material: Room temperature
- pH: 2.54

The purity analysis was performed in 2015 (after the experimental period, and after the expiration date of the test article). The analytical purity of the substance seems to have in that time decreased from 99% to 95%. This has had no influence on the results of this study, and the compound is still valid with regards to its chemical specifications.

Test animals

Species:

human

Strain:

other: Reconstructed human epidermis model (see details below)

Details on test animals or test system and environmental conditions:

EPISKIN-SM (Source: SkinEthic, France, Batch No.:12-EKIN-008, Expiry date: 27 February 2012) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are 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 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Test system

Type of coverage:

other: Applied evenly to the epidermal surface following the application of 10 ul distilled water to this surface

Preparation of test site:

other: in vitro cell culture

Vehicle:

water

Remarks:

distilled

Controls:

other: negative control skin unit tested in triplicate

Amount / concentration applied:

20 mg to each of three test skin units.
20 µl PBS (phosphate buffered saline) was added to each of the three negative control skin units and 20 µl SDS (sodium dodecyl sulfate, 5% aqueous solution) was added to each of the three positive control skin units.
For additional control for staining effects of the test item, 10 µl distilled water was applied to the epidermal surface of a single skin unit to ensure good contact with the epidermis, then 20 mg of the test item was applied evenly to the epidermal surface.

Duration of treatment / exposure:

Exposure for 15 minutes (± 0.5 min) at room temperature (20-37°C).
Then incubated with fresh “maintenance medium” for 42 hours (± 1h) at 37°C.

Observation period:

Not applicable to this test system

Number of animals:

Not applicable to this test system

Details on study design:

EPISKIN-SM assay plate contained reconstructed epidermis units (area: 0.38 cm2); each was attached to the base of a tissue culture vessel and maintained on nutritive agar.

After test substance exposure and subsequent incubation, preparations for cell viability determination were: incubation with MTT solution (at 37 degrees C for 3 hours) followed by incubation with acidified isopropanol for formazan extraction (around two hours at room temperature with gentle agitation).

For cell viability measurements, the OD (Absorbance / Optical Density) of the samples in a spectrophotometer was read at 540 nm using acidified isopropanol solution blank (6×200 µL). (The validity of the microplate reader was verified with a standard verification plate daily before use. The standard plate was calibrated yearly by the manufacturer.)

For each treated tissue, OD (as adjusted for colouring potential of the test substance) was calculated and the tissue viability was expressed as a % relative to negative control.

Criteria for classification as irritant/non-irritant: If the resulting mean relative viability (as adjusted for intrinsic colour) is less than or equal to 50% of the negative control, the test substance is considered to be irritant to skin.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

Time point: 42 hours

Run / experiment:

mean of 3 replicates

Value:

86

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Mean cell viability (as adjusted for intrinsic colour) was 86% of the negative control (range 85%-86%).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro reconstructed human epidermis (EpiSkin) assay, conducted in accordance with OECD Test Guideline 439 and to GLP, tetraammonium decachloro-μ-oxodiruthenate was considered to be non-irritating to skin.

Executive summary:

Tetraammonium decachloro-μ-oxodiruthenate was tested for skin irritation potential in an in vitro reconstructed human epidermis model (EpiSkin assay) conducted in accordance with OECD Test Guideline 439, and to GLP.

 

EpiSkin is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability. Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of cell viability in treated tissues is compared to negative controls and expressed as a %. If the resulting mean relative viability (as adjusted for intrinsic colour) is less than or equal to 50% of the negative control, the test substance is considered to be irritant to skin.

 

Following a 15-minute exposure to the test substance, the test system skin cell viability was calculated to be greater than 50% (the average was 86% and the range was 85 -86%), and it was therefore considered to be non-irritating to skin.

 

Under the conditions of this assay, tetraammonium decachloro-μ-oxodiruthenate does not require classification for skin irritation according to EU CLP criteria (EC 1272/2008).