4,11-diamino-2-(3-methoxypropyl)-1H-naphth[2,3-f]isoindol-1,3,5,10(2H)-tetrone

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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

Principles of method if other than guideline:

None

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4,11-diamino-2-(3-methoxypropyl)-1H-naphth[2,3-f]isoindol-1,3,5,10(2H)-tetrone

EC Number:

235-402-7

EC Name:

4,11-diamino-2-(3-methoxypropyl)-1H-naphth[2,3-f]isoindol-1,3,5,10(2H)-tetrone

Cas Number:

12217-80-0

Molecular formula:

C20H17N3O5

IUPAC Name:

4,11-diamino-2-(3-methoxypropyl)-1H-naphtho[2,3-f]isoindole-1,3,5,10(2H)-tetrone

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Test animals / tissue source

Species:

cattle

Strain:

not specified

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 prepared immediately on arrival.

Test system

Vehicle:

other: 0.9% w/v sodium chloride solution

Controls:

yes

Amount / concentration applied:

20 % w/v

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

None

Number of animals or in vitro replicates:

Three corneas were allocated to the test item

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 2 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. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.


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) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC 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 ºC 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 MEM containing phenol red before a final rinse with complete MEM without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

The negative and positive control data was shared with Harlan Laboratories Ltd. study numbers 41500304 and 41500337.


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 ºC 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 of the cornea was visually assessed post treatment.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

main test

Value:

0.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

In vivo

Irritant / corrosive response data:

The In Vitro irritancy scores are summarized as follows:
The test item In Vitro Irritancy Score was 0.9.
The negative control In Vitro Irritancy Score was 0.8.
The positive control In Vitro Irritancy Score was 73.6.

Other effects:

Corneal Epithelium Condition
The corneas treated with the test item or 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

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity of ≤4.1 and permeability ≤0.105. The negative control acceptance criteria were therefore satisfied.

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†	5	5	5	0		0.004
	17	4	4	0		0.018
	20	3	5	2		0.007
				0.7*		0.010♦		0.8
Positive Control†	12	3	71	68	67.3	0.679	0.669
	27	4	62	58	57.3	0.754	0.744
	28	5	70	65	64.3	0.721	0.711
					63.0●		0.708●	73.6
Test Item	16	3	3	0	0.0	0.024	0.014
	19	3	2	-1	0.0	0.006	0.000
	26	3	6	3	2.3	0.014	0.004
					0.8●		0.006●	0.9

OD= Optical density            

* = Mean of the post-treatment -pre‑treatment values            

♦ = Mean permeability                      

● = Mean corrected value

† = Control data was shared with Harlan Laboratories Ltd. study numbers 41500304 and 41500337

Corneal Epithelium Condition Post Treatment

Treatment	Cornea Number	Observation Post Treatment
Negative Control†	5	clear
	17	clear
	20	clear
Positive Control†	12	cloudy
	27	cloudy
	28	cloudy
Test Item	16	clear
	19	clear
	26	clear

† Control data was shared with Harlan Laboratories Ltd. study numbers 41500304 and 41500337

Opacitometer Calibration

The opacitometer was calibrated on the day of the test.

 

Calibration of Opacitometer
Balance dial = 0	Target	Reading displayed	Acceptable
Calibrator number 1	75	75	Yes
Calibrator number 2	150 ±2%	150	Yes
Calibrator number 3	225 ±2%	225	Yes

 

Calibration of the opacitometer was considered to be acceptable.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 36152/M requires no classification to UN GHS or EU CLP.

Executive summary:

A supporting test was performed to identify whether FAT 36152/M can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/ EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

The test item was applied at a concentration of 20 % w/v in 0.9 % w/v sodium chloride solution 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). The negative and positive control data was shared with Harlan Laboratories Ltd. study numbers 41500304 and 41500337.

Interpretation

The test item is classified according to the prediction model below:

IVIS	UN GHS	European Regulation (EC) 1272/2008
≤3	No category	Not classified for irritation
>3;≤ 55	No prediction can be made	No prediction can be made
> 55	Category 1	Category 1 H318: Causes serious eye damage

The In Vitro irritancy scores are summarized as follows:

Treatment	In vitro irritancy score
Test item	0.9
Negative control	0.8
Positive control	73.6

In conclusion, FAT 36152/M requires no classification to UN GHS or EU CLP.