Potassium dicyanoaurate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 06 November 2013 to 26 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline test, available as an unpublished report. Reliable without restriction.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

yes

Remarks:

The control group and other common data incl. eye receipt and preparation, technical documentation, reagent record and preparation, initial opacity readings, group allocation and optical density print out were shared with study 413027735.

Principles of method if other than guideline:

The 'open-chamber' method was adopted. The window-locking ring and glass were removed from the chamber prior to treatment. The 'open-chamber' method was also followed for the control groups although solutions were applied. Following application the ring and glass were replaced during the exposure period until rinsing.

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
- Stability under test conditions: Stable under test conditions
- Storage condition of test material: Room temperature in the dark
- Other:

Test animals / tissue source

Species:

other: Not applicable

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: Not applicable

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: Approximately 0.873 g of the solid test item was applied to the cornea. It covered adequately the corneal surface.

Duration of treatment / exposure:

240 minutes at 32±1°C

Number of animals or in vitro replicates:

Three corneas were allocated to the test item, three to the negative control and three to the positive control.

Details on study design:

Treatment of corneas
Approximately 0.873 g of the solid test item was found to adequately cover the corneal surface. 750 uL of each control item was applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32± 1°C for 240 minutes. At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete minimum essential medium (MEM) containing phenol red before a final rinse with complete MEM. the anterior and posterior chambers were refilled with fresh complete MEM. A post treatment opacity reading was taken and each cornea was visually observed.

Application of sodium fluorescein
Following the opacity measurement the permability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32± 1°C for 90 minutes.

Permeability determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 uL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Opacity measurement
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permeability measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

SCORING SYSTEM:
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
In Vitro Irritancy Score = mean opacity value + (15 x mean OD 492 value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induces a response through only one of the two endpoints.
A test item that induces an In Vitro Irritancy Score ≥55.1 is defined as an ocular corrosive or severe irritant and will be labelled EU DSD (67/548/EEC) R41 and EU CLP/UN GHS Category 1.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

240 minutes

Value:

171.5

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The test item was considered to be an ocular corrosive or severe irritant.

Any other information on results incl. tables

The corneas treated with the test item were opaque post incubation. The corneas treated with the negative control item were clear post incubation. The corneas treated with positive control item were cloudy post incubation.

The positive control In Vitro Irritancy Score was 82.1 and the negative control In Vitro Irritancy Score was 3.7.

Criterion for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 65.9 to 140.8. The positive control acceptance criterion was therefore satisfied.

The In Vitro irritancy scores are summarized as follows:

 

Treatment	In Vitro Irritancy Score
Test Item	171.5
Negative Control	3.7
Positive Control	82.1

 

Table 1. Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD)		In VitroIrritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment-Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	1	1	4	3		0.035
	2	1	4	3		0.067
	3	2	5	3		0.028
				3.0*		0.043¨		3.7
Positive Control	4	2	69	67	64.0	1.590	1.547
	5	2	58	56	53.0	0.759	0.716
	6	1	78	77	74.0	1.477	1.434
					63.7·		1.232·	82.1
Test Item	10	1	169	168	165.0	1.899	1.856
	11	2	144	142	139.0	2.360	2.317
	12	1	118	117	114.0	2.312	2.269
					139.3·		2.147·	171.5

OD= Optical density; * = Mean of the post-treatment -pre‑treatment values; ¨= Mean permeability; ·= Mean corrected value

Table 2. Corneal Epithelium Condition Post Treatment

 

Treatment	Cornea Number	Observation
		Post Treatment
Negative Control	1	clear
	2	clear
	3	clear
Positive Control	4	cloudy
	5	cloudy
	6	cloudy
Test Item	10	opaque
	11	opaque
	12	opaque

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The test item was considered to be an ocular corrosive or severe irritant.

Executive summary:

Potassium dicyanoaurate was tested in vitro for its ocular irritancy potential to the isolated bovine cornea. The test item was applied neat for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Potassium dicyanoaurate induces an In Vitro Irritancy Score of 171.5 and therefore was considered to be an ocular corrosive or severe irritant.