2,2'-[[4-[(2-hydroxyethyl)amino]-3-nitrophenyl]imino]bisethanol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-07-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with OECD guideline 437 (2009) : Bovine Corneal Opacity and Permeability Assay.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 437 : Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (September 2009)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[[4-[(2-hydroxyethyl)amino]-3-nitrophenyl]imino]bisethanol

EC Number:

251-410-3

EC Name:

2,2'-[[4-[(2-hydroxyethyl)amino]-3-nitrophenyl]imino]bisethanol

Cas Number:

33229-34-4

Molecular formula:

C12H19N3O5

IUPAC Name:

2-({4-[bis(2-hydroxyethyl)amino]-2-nitrophenyl}amino)ethan-1-ol

Details on test material:

Name : Jarocol Blue 2
Batch number : JBT03110003
Purity : 99.4%
CAS number : 33229-34-4
EC number : 251-410-3
Name : 2-[(4-N,N-bis-(2'-hydroxyethyl)amino-2-nitrophenyl)amino]ethanol

Test animals / tissue source

Species:

other: In-vitro assessment using fresh bovine corneae

Strain:

other: In-vitro assessment using fresh bovine corneae

Test system

Vehicle:

other: 0.9% (w/v) NaCl in deionised water)

Controls:

other: In-vitro assessment using fresh bovine corneae

Amount / concentration applied:

20% (w/v) preparation in 0.9% (w/v) sodium chloride solution of the test item

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

Not applicable

Details on study design:

This study should provide a rational basis for risk assessment in humans. An ocular contact is one of the probable routes of human exposure. This test is designed to measure the opacity of the cornea by quantifying the ability of light to pass through it. The permeability, as a result of the irritation potential of the test item, is determined using Na-fluorescein solution. Comparison of the opacity before and afterexposure to test item and determination of permeability after treatment provide an indication of the irritant of the test item. For this purpose the inflammatory in vitro changes of the bovine cornea after application of the test item is measured with regard to the opacity and permeability of dye. The results of both criteria are combined. The resulting in vitro irritation factor is compared with a criterion scale, produced prior to the experiment. The changes are classified into four grades : mild, moderate, severe, very severe.

Results and discussion

In vivo

Results

Irritation parameter:

other: In-vitro irritation score

Basis:

other: Test item corneae

Time point:

other: 240 minutes

Score:

5.4

Any other information on results incl. tables

Evaluation of Results

Opacity

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 corneae. 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

The corrected OD₄₉₀ was calculated by subtracting the mean OD₄₉₀ of the negative control corneae from the OD₄₉₀ value of each treated cornea. The OD ₄₉₀ value of each treatment group was calculated by averaging the corrected OD₄₉₀ values of the treated corneae for the treatment group.

In-Vitro Score Calculation

The following formula was used to determine the in-vitro irritancy score :

In-vitro Irritancy Score = mean opacity value + (15 X mean OD₄₉₀ value)

Results

Corneal Opacity and Permeability Measurements

Test Group	Cornea Number	Opacity Value = Difference (t240 -t0) of Opacity	Permeability at 490 nm(OD490nm)	In-Vitro Irritancy Score
Negative Control	1	0	0.055
Negative Control	2	2	0.033
Negative Control	3	3	0.063
Negative Control		1.7*	0.050**	2.4
Positive Control	4	64.3	2.745
Positive Control	5	71.3	2.260
Positive Control	6	54.3	2.670
Positive Control		63.3***	2.558***	101.7
Test Item	7	7.3	0.024
Test Item	8	3.3	0.041
Test Item	9	4.3	0.019
Test Item		5.0***	0.028***	5.4

* : Mean of the post treatment

** : Mean permeability

*** : Mean corrected value

Corneal Epithelium Condition Post Treatment

Treatment	Cornea Number	Observation Post Treatment
Negative Control	1	clear
Negative Control	2	clear
Negative Control	3	clear
Positive Control	4	cloudy
Positive Control	5	cloudy
Positive Control	6	cloudy
Test Item	7	clear
Test Item	8	clear
Test Item	9	clear

Applicant's summary and conclusion

Interpretation of results:

other: Not an ocular corrosive or severe irritant

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item was considered not to be an ocular corrosive or severe irritant.

Executive summary:

The in-vitro study was performed to assess the corneal irritation and damage potential of 2-[(4-N,N-bis-(2'-hydroxyethyl)amino-2-nitrophenyl)amino]ethanol (HC Blue 2) by means of the BCOP assay using fresh bovine corneae. Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an in-vitro irritancy score. 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. The positive control (20% w/v imidazole) showed clear opacity and distinctive permeability of the corneae and therefore is classified as very severe eye irritant. The test item 2-[(4-N,N-bis-(2'-hydroxyethyl)amino-2-nitrophenyl)amino]ethanol (HC Blue 2) is considered not to be an ocular corrosive or severe irritant.