Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 12 March 2014; Experimental Completion Date: 12 March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

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:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

EC Number:

289-056-7

EC Name:

Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

Cas Number:

85959-61-1

Molecular formula:

C162H180N12O18Cl6+O3.P2O5.xWO3.yMoO3

IUPAC Name:

heptakis(N-[(3E)-6-(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-3H-xanthen-3-ylidene]ethan-1-aminium) dioxomolybdenumbis(olate) dioxotungstenbis(olate) phosphate

Test material form:

other: granula solid

Details on test material:

Identification: Pigement Red 81:4
Batch: S-3101
Physical state / Appearance: magenta colored granula solid
Expiry Date: 01 June 2014
Storage Conditions: Room temperature in the dark

Test animals / tissue source

Species:

other: Eyes from adult cattle

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks' Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated on arrival and used within 24 hours of receipt.

Test system

Vehicle:

other: test item was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution.

Controls:

no

Amount / concentration applied:

0.75 mL of the test item preparation or control items were applied to the appropriate corneas.

Duration of treatment / exposure:

Exposure of 240 minutes.

Observation period (in vivo):

Not applicable.

Number of animals or in vitro replicates:

Not applicable.

Details on study design:

TEST ITEM FORMULATION AND EXPERIMENTAL PREPARATION:
For the purpose of this study the test item was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution.
The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation.

PREPARATION OF NEGATIVE AND POSITIVE CONTROLS:
The negative control item, 0.9% w/v sodium chloride solution, was used as supplied.

The positive control item, Imidazole, was used as a 20% w/v solution in 0.9% w/v sodium chloride solution.

PREPARATION OF CORNEAS:
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.

The anterior and posterior chambers of each BCOP holder were filled with complete Eagle's minimum essential medium (MEM) and plugged. The holders were incubated at 32 ± 1°C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

SELECTION OF CORNEAS AND OPACITY READING:
The medium from both chambers of each holder was replaced with fresh complete MEM.

A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.

Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

TREATMENT OF CORNEAS:
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were 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 preparation and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete 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 permeability 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 (5 mg/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 µL 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-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

EVALUATION OF RESULTS:
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity Measurement:
The change in opacity for each cornea (including the negative control) 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.

In Vitro Irritancy Score:
The following formula was used to determine the In Vitro irritancy score:

In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

Visual Observation:
The condition ofthe cornea was visually assessed immediately after rinsing.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

0.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Corneal Epithelium Condition:
The corneas treated with the test item were clear post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

Any other information on results incl. tables

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in the table below.

Treatment	Cornea Number	Opacity				Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment - Pre-Treatment	Corrected Value		Corrected value
Negative Control	1	3	6	3		0.052
	5	3	3	0		0.015
	18	3	4	1		0.112
				1.3		0.06		2.2
Positive Control	2	3	70	67	65.7	1.091	1.031
	4	2	71	69	67.7	1.235	1.175
	6	2	72	70	68.7	1.231	1.171
					67.3		1.126	84.2
Test Item	3	1	2	1	0	0.061	0.001
	7	3	3	0	0	0.074	0.014
	9	3	4	1	0	0.093	0.033
					0		0.016	0.2

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was considered not to be an ocular corrosive or severe irritant and does not require classification for eye corrosion/irritation according to the BCOP test method.