Iridium

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3-19 September 2019

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:

2020

Report date:

2020

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:

Reconstructed Human Epidermis Test method

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Iridium

EC Number:

231-095-9

EC Name:

Iridium

Cas Number:

7439-88-5

Molecular formula:

Ir

IUPAC Name:

iridium(3+)

Details on test material:

Iridium

Specific details on test material used for the study:

purity: >99.9% ASTM powder

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. 30830) 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): 25 mg D-PBS .

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 mg SDS
- 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:

105.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The mean optical density (OD) of the negative control of 3 tissues was 1.453 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 8.7% of the negative control and fulfilled the acceptance criterion of <=20%.

Remarks on result:

no indication of irritation

Remarks:

The mean viability of cells exposed to Iridium was 105.4% of the negative control and, hence, was above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. Iridium 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 within the acceptable range of ≥0.8 to ≤2.8.
2: The viability of cells treated with the positive reference item 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%.

The standard deviation calculated from individual % tissue viabilities of the 3 identically treated replicates was slightly above (18.1%) the limit of acceptance of 18%. This slight elevation is not considered critical, as each individual tissue viability is clearly above the evaluation cut off (> 50%) and hence, not irritant.

Any other information on results incl. tables

The mean viability of cells exposed to Iridium was 105.4% of the negative control and, hence, was above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. Iridium was considered to be non-cytotoxic and predicted to be non-irritant to skin.

The summary of the results is given below:

	Optical density (n = 3 tissues)	CV (%)	viability (%)		SD
Negative control (D-PBS)	1.453	8.2	100		8.2
Iridium	1.531	17.1	105.4		18.1
Positive control (5% SDS)	0.126	6.8	8.7		0.6

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), iridium (applied as a powder) 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 purpose of this study was to determine whether Iridium possesses cytotoxic properties to skin cells, which might lead to irritation of human skin, by using an artificial three-dimensional model of human skin (EpiDermTM model).

Three tissues were used for each treatment and concurrent control group. Cell viability was determined by using the MTT reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.

Iridium was applied topically as solid test item to the model skin surface, which was moistened with Dulbecco's phosphate buffered saline (DPBS). DPBS was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) 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 cell viability observed in treated tissues, when compared with that of the concurrent negative control, was above the cut-off cell viability of 50% that distinguishes irritant from non-irritant test items. Iridium is therefore predicted to be non-irritant to skin.

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

The standard deviation calculated from individual % tissue viabilities of the 3 identically treated replicates was slightly above (18.1%) the limit of acceptance of 18%. This slight elevation is not considered critical, as each individual tissue viability is clearly above the evaluation cut off ( > 50%) and hence, not irritant.