Registration Dossier

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:
other: Good-quality, well-documented study in accordance with OECD guideline.

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:
other: 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
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
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

Constituent 1
Chemical structure
Reference substance name:
Tetraammineplatinum dinitrate
EC Number:
243-929-9
EC Name:
Tetraammineplatinum dinitrate
Cas Number:
20634-12-2
Molecular formula:
H12N4Pt.2NO3
IUPAC Name:
platinum(2+) ion tetraamine dinitrate
Details on test material:
- Name of test material (as cited in study report): Tetraammineplatinum dinitrate
- Substance type: Technical product
- Physical state: Liquid (colourless)
- Analytical purity:100% (platinum content 3.320%)
- Impurities (identity and concentrations): no data (trace element analysis briefly reported in Appendix 3 of attached full study report)
- Purity test date: 13 January 2012
- Lot/batch No.: CPI-14954
- Expiration date of the lot/batch: 07 December 2012
- Storage condition of test material: Room Temperature (not further specified)

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: 20 ul was applied evenly to the epidermal surface
Preparation of test site:
other: in vitro cell culture
Vehicle:
unchanged (no vehicle)
Controls:
other: negative control skin unit tested in triplicate
Amount / concentration applied:
20 ul 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.
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 was calculated and the tissue viability was expressed as a % relative to negative control. No adjustment to OD results was necessary because the test substance was colourless.

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:
other: other:
Value:
95
Remarks on result:
other:
Remarks:
Basis: mean Cell/tissue viability. Time point: 42 hours. Reversibility: other: Not applicable. Remarks: Score is a percentage (%) of negative control. Mean relative viability <=50% the test substance is considered to be irritant to skin.. (migrated information)
Other effects / acceptance of results:
Mean cell viability was 95% of the negative control (range 85%-100%), see attached table.

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In an in vitro reconstructed human epidermis (EpiSkin) assay, conducted in accordance with OECD Test Guideline 439 and to GLP, tetraammineplatinum dinitrate was considered to be non-irritating to skin.
Executive summary:

Tetraammineplatinum dinitrate was tested for skin irritation potential i n 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 95% and the range was 85-100%), and it was therefore considered to be non-irritating to skin.

 

Under the conditions of this assay, tetraammineplatinum dinitrate does not require classification for skin irritation according to EU CLP criteria (EC 1272/2008).