Potassium dicyanoaurate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 October 2013 to 04 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline test, available as an unpublished report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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)

Deviations:

yes

Remarks:

The control groups were shared with study 41302738. The control raw data were filled with study 41302738. This deviation was considered not to affect the purpose or integrity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

yes

Remarks:

The control groups were shared with study 41302738. The control raw data were filled with study 41302738. This deviation was considered not to affect the purpose or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Potassium dicyanoaurate

EC Number:

237-748-4

EC Name:

Potassium dicyanoaurate

Cas Number:

13967-50-5

Molecular formula:

C2AuN2.K

IUPAC Name:

potassium dicyanoaurate

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Potassium dicyanoaurate
- Physical state: soild
- Colour: White
- pH: 10.2 (50 g/L)
- Stability under test conditions: Stable under test conditions.
- Storage condition of test material: Room temperature in the dark.

Test animals

Species:

other: EPISKIN (TM) reconstructed human epidermis model

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

Not applicable.

Test system

Type of coverage:

other: Topical

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: Not applicable.

Amount / concentration applied:

- Treatment group: Test carried out in triplicate. Approximately 10 mg of the test item was applied topically, ensuring an even covering, to the epidermis surface which had previously been moistened with 5 µL sterile, distilled water to improve contact between the solid test item and the epidermis.
- Negative control: Triplicate tissues treated with 10 µL Dulbecco's Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
- Positive control: Triplicate tissues. 10 µL Sodium Dodecyl Sulphate (SDS) at 5% w/v aqueous solution spread over entire surface of the epidermis using a pipette tip with the process being repeated after 7 minutes.

Duration of treatment / exposure:

- Treatment period: 15 minutes
- Post-exposure incubation: At the end of the exposure period, tissues were rinsed with DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to a second column of three wells, each containing 2 mL of maintenance medium, and incubated for 42 hours, at 37°C and 5% CO2 in air.

Observation period:

Not applicable.

Number of animals:

Not applicable.

Details on study design:

Preincubation
2 mL of maintance medium, warmed to approximately 37°C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epiderimis unit was transferred into the maintance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37°C, 5% Co2 in air overnight.

Application of test Item and rinsing (Day 1)
2 mL of maintenance medium, warmed to approx. 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the coresponding tissues ensuring uniform covering. 5 µL sterile distilled water was topically applied to the epidermal surface in order to improve further contact between the test item and the epidermis. 10 mg of the test item was then applied to the epidermal surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. The plates were kept in the biological safety cabnet at room temperature for 15 minutes. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca ++ and Mg++. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissue were incubated at 37°C., 5% CO2 in air for 42 hours.


The measurement of tissue viability (cytotoxicity) was measured by means of the colourimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (by the mitochondrial succinate dehdroganase in viable cells) in the test item treated tissue relative to the negative control using the following procedure. After incubation, each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30°C for possible inflamatory mediator determination. 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plate(s). The tissues were transferred to the MTT filled wells, and were incubated for 3 hours at 37°C, 5% CO2 in air. Then the epidermis was carefully separated from the collagen matrix using forceps and both parts (epiderimis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10° C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: % relative viability

Value:

7.1

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes exposure followed by the 42-hour post-exposure incubation period. Reversibility: no data. Remarks: SD of relative mean vaibility (%) = 1.1. (migrated information)

Other effects / acceptance of results:

The relative mean viability of the test item treated tissues after 15-minute exposure period and 42 hours post-exposure incubation period was 7.1% with standard deviation of 1.1%.
The mean optical density measured at 562 nm was 0.067 with standard deviation of 0.011.
The test item is considered to be irritant using the EPISKIN (TM) human epidermis model.

- Positive control: The relative mean tissue viability was 10.4 % relative to the negative control and the standard deviation was 4.5 %.
- Negative control: The mean optical density was 0.950 and the standard deviation was 0.037.

Any other information on results incl. tables

The acceptance criteria were satisfied according to the protocol criteria for both the positive control (relative mean viability ≤ 40% and standard deviation ≤ 18%) and negative control (OD562≥ 0.6 and standard deviation of individual tissue viability ≤18%). The standard deviation of the triplicate treated tissues was 3.9 % and hence the test item acceptance criterion (≤ 18%) was also satisfied.

Table 1. Mean OD562 values and percentage viabilities for the negative control item, positive control item and test item.

Item	OD562of tisues	Mean OD562 of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of relative mean viability (%)
Negative control item#	0.931	0.950	0.037	98.0	100*	3.9
	0.927			97.6
	0.993			104.5
Positive control item#	0.102	0.099	0.043	10.7	10.4	4.5
	0.054			5.7
	0.140			14.7
Test item	0.055	0.067	0.011	5.8	7.1	1.1
	0.071			7.5
	0.075			7.9

SD – standard deviation

*The mean viability of the negqative control tissues is set at 100%

OD₅₆₂– optical density

# - control group shared with Harlan study number 41302738

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Based on the relative mean viability of 7.1% the test item was classified as irritant. The following classification criteria apply: EU DSD (67/548/EEC) Irritant requires symbol 'Xi' risk phrase R38 'Irritant to skin'. EU CLP and UN GHS Hazard statement H315 'Causes Skin Irritation' Category 2.

Executive summary:

The purpose of the test was to evaluate the skin irritation potentail of the potassium dicyanoaurate using the EPISKIN reconstructed human epiderimis model according to the OECD 439 guideline. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. Results were the relative mean vaibility of the test item treated tissues was 7.1%. The test item was classified as irritant. The following classification criteria apply: EU DSD (67/548/EEC) Irritant requires symbol 'Xi' risk phrase R38 'Irritant to skin'. EU CLP and UN GHS Hazard statement H315 'Causes Skin Irritation' Category 2.