N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase start and completion: 07 July 2020.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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)

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

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-N-{6-[(2-aminophenyl)formamido]-2-{3-[(2-aminophenyl)formamido]propyl}hexyl}benzamide

EC Number:

951-985-7

Molecular formula:

C30H38N6O3

IUPAC Name:

2-amino-N-{6-[(2-aminophenyl)formamido]-2-{3-[(2-aminophenyl)formamido]propyl}hexyl}benzamide

Test material form:

solid: crystalline

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Source of Bovine Eyes
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.


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 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 (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 70 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Test system

Vehicle:

physiological saline

Remarks:

For the purpose of this study the test item was prepared as a 20% w/v solution in sodium chloride 0.9% w/v.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

.TEST MATERIAL
- Concentration (if solution): 0% w/v solution in sodium chloride 0.9% w/v

NEGATIVE CONTROL
The negative control item, sodium chloride 0.9% w/v, was used as supplied.

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

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

Three replicates (corneas) per treatment (test substance, negative control, positive control).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS

QUALITY CHECK OF THE ISOLATED CORNEAS : Yes

NUMBER OF REPLICATES: Three per treatment

NEGATIVE CONTROL USED : Sodium Chloirde 0.9% w/v

SOLVENT CONTROL USED (if applicable): No

POSITIVE CONTROL USED: Imidiazole

APPLICATION DOSE AND EXPOSURE TIME : 240 minutes

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 washings with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red
- POST-EXPOSURE INCUBATION: No

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer (calibrated on the day of the test)
- Corneal permeability: passage of sodium fluorescein dye measured using microtiter plate reader at 492nm (without a reference filter)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: As specified in the test guideline.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Substance (mean of three replicates)

Value:

1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

in vitro irritancy score = 0.3

Positive controls validity:

valid

Remarks:

in vitro irritancy score = 92.4

Any other information on results incl. tables

The test item is classified according to the following criteria:

IVIS	UN GHS
≤ 3	No Category
>3 ≤ 55	No prediction can be made
> 55	Category 1

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the test, the test item is given No Category classification according to the UN GHS classification scheme.

Executive summary:

Introduction

The ability of the test item to induce eye damage was assessed using a method designed to meet the requirements of the following guidelines:

 

• OECD Guideline for the Testing of Chemicals No. 437 (updated 09 October 2017) “Bovine Corneal Opacity and Permeability Assay”


•  Method B.47 of Commission Regulation (EC) No. 440/2008  

Method

The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v 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).  Three corneas were used per treatment group.

 

Data Interpretation

The test item is classified according to the followign criteria:

 

IVIS	UN GHS
≤ 3	No Category
>3 ≤ 55	No prediction can be made
> 55	Category 1

 

Results

The In Vitro irritancy scores are summarised as follows:

 

Treatment	In Vitro Irritancy Score
Test Item	1.0
Negative Control	0.3
Positive Control	92.4

 

Conclusion

Under the conditions of the test, the test item is given No Category classification according to the UN GHS classification scheme.