Dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol (1:2)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

5 August - 9 September 2015

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:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage, adopted July 26, 2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol (1:2)

EC Number:

268-717-3

EC Name:

Dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol (1:2)

Cas Number:

68133-90-4

Molecular formula:

C2H7NO.1/2H6O6Pt.H

IUPAC Name:

dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol (1:2)

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Dihydrogen hexahydroxyplatinate/2-aminoethanol (1:2)
- Substance type: No data
- Physical state: yellow/orange coloured liquid
- Analytical purity: No data
- Impurities (identity and concentrations in pmm): Ag (<0.5), Al (12), As (<10), Au (<10), Bi (<5), Ca (36), Cr (<2), Cu (<5), Fe (34.0), Ir (<3), K (<5), Mg (17.0), Mn (<1), Mo (<5), Na (3096), Ni (<5), Pd (<5), Rh (40), Ru (<7), Sb, (<15), Si (172), Sn (<5), Zn (<0.5), Cl- (1096)
- Composition of test material, percentage of components: Platinum content 17.15% (w/w)
- Isomers composition: Not applicable
- Purity test date: 26 May 2015
- Lot/batch No.: 15135C1AES
- Expiration date of the lot/batch: 25 May 2018
- Stability under test conditions: No data
- Storage condition of test material: At +10°C to +25°C, kept in a tightly closed container and stored in a dry place, protected from light.

Test animals / tissue source

Species:

other: Bovine eyes from cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source of eyes: Eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse. To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing 1% Penicillin/Streptomycin.

Preparation of corneas: Only corneas from eyes free of defects were used. The corneas were dissected with a 2-3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.

After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer. A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment, solvent and positive control groups (three corneas/group).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: negative control corneas tested in triplicate

Amount / concentration applied:

0.75 mL

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Not applicable

Details on study design:

TREATMENT
Negative control: 0.9% sodium chloride solution
Positive control: 1% sodium hydroxide solution in in aqua ad iniectabilia

750 μL of the test or control items as recommended as suitable test volume according to OECD TG 437 were added to completely cover the cornea’s epithelium in the anterior chamber.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the exposure period the exposure solution was removed from each chamber and the epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. No discolouration of phenol red was visible after exposure of the test item. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.
- Time after start of exposure: 10 minutes

After rinsing, the corneas were incubated at 32±1°C for two hours prior to examination.

EXAMINATION
Corneal injury was assessed by evaluating the opacity and permeability of the cornea. Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.

To determine the corneal permeability 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32±1°C for 90±5 minutes. The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader (Tecan Sunrise Magellan Version 6.4). Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer (Tecan Sunrise) using a standard 1 cm path length.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (10 minute time point)

Value:

4.585

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The negative control displayed a mean opacity value of 1.155 ± 0.261 and a mean permeability value of 0.029 ± 0.015. The calculated IVIS value of 1.585 ± 0.264 was well below the cut-off value of 3 (UN GHS no category).

Positive controls validity:

valid

Remarks:

The positive control displayed a mean opacity value of 66.879 ± 6.538 and a mean permeability value of 2.1201 ± 0.106 compared to the solvent control. The calculated IVIS value of 98.674 ± 5.107 was well above the cut-off value of 55.

Remarks on result:

not determinable

Remarks:

A score of >55 is considered to be an ocular corrosive or severe irritant; a score of ≤3 is not considered to require a classification for eye irritation or serious eye damage; a score in between these values indicates that no prediction can be made

In vivo

Irritant / corrosive response data:

The IVIS was 4.585 ± 1.181 for dihydrogen hexahydroxyplatinate/2-aminoethanol (1:2)

Other effects:

No data

Any other information on results incl. tables

The corneas treated with the negative control item 0.9% sodium chloride solution revealed a mean opacity value of 1.155 ± 0.261 and a mean permeability value of 0.029 ± 0.015. The calculated IVIS value of 1.585 ± 0.264 was well below the cut-off value of 3 (UN GHS no category).

The corneas treated with the positive control item 1% NaOH in aqua ad iniectabilia revealed a mean opacity value of 66.879 ± 6.538 and a mean permeability value of 2.1201 ± 0.106 compared to the solvent control. The calculated IVIS value of 98.674 ± 5.107 was within two standard deviations of the current historical mean and well above the cut-off value of 55. Hence, the acceptance criteria for the test were fulfilled.

Following treatment with Dihydrogen hexahydroxyplatinate/2-aminoethanol (1:2) a mean opacity of 1.620 ± 0.977 and a mean permeability value of 0.198 ± 0.076 compared to the negative control were determined. The calculated IVIS of 4.585 ± 1.181 is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55, identifying test substances as inducing serious eye damage (UN GHS Category 1). Consequently no prediction concerning irritant or corrosive potential of the test item can be made.

The opacity and permeability values are given in Tables 1 and 2. The IVIS values are given in the table below:

	Cornea No.	Opacity	Permeability	IVIS
				Per Cornea	Per Group
					Mean	SD
0.9% NaCl	1	0.916	0.044	1.576	1.585	0.264
	2	1.116	0.014	1.326
	3	1.434	0.028	1.854
1% NaOH	4	60.040	2.242	93.670	98.674	5.107
	5	73.068	2.054	103.878
	6	67.530	2.063	98.475
Dihydrogen hexahydroxyplatinate/2-aminoethanol (1:2)	7	1.554	0.119	3.339	4.585	1.181
	8	2.629	0.204	5.689
	9	0.678	0.270	4.728

Applicant's summary and conclusion

Interpretation of results:

other: No prediction can be made from the available IVIS.

Conclusions:

In an in vitro bovine corneal opacity and permeability assay conducted in accordance with OECD guideline 437, the IVIS for dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) was calculated to be 4.585, indicating that no prediction concerning irritant or corrosive potential of the test item can be made.

Executive summary:

In an in vitro bovine corneal opacity and permeability assay conducted in accordance with OECD guideline 437 and to GLP, dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) was applied to isolated bovine corneas for 10 minutes, followed by an incubation period of 120 minutes.

The In Vitro Irritancy Score (IVIS) calculated from individual scores for induced opacity (decreased light transmission through the cornea) and permeability (passage of sodium fluorescein dye through the cornea) was 4.585, which is above the cut-off value of 3 (no category) and below the cut-off value of 55 (identifying test substances as inducing serious eye damage). Hence, no prediction concerning irritant or corrosive potential of the test item can be made.