m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Principles of method if other than guideline:

This study was performed to assess the corneal damage potential of the solid test item Bayscript Yellow GGN with the Bovine Corneal Opacity and Permeability test (BCOP) using fresh bovine cornea.
The study was conducted in accordance with international accepted Guidelines (e.g. OECD TG 437).

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

EC Number:

275-602-1

EC Name:

m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

Cas Number:

71550-21-5

Molecular formula:

C27H24N6O9S2.2C4H11NO2

IUPAC Name:

m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

Details on test material:

Test item: Bayscript Gelb GGN
Test item identity (including alternative names): Bayscript Gelb GGN Benzenesulfonic acid, 3,3’-(carbonylbis(imino(3-methoxy- 4,1-phenylene)-2,1-diazenediyl))bis-, compd. with 2,2’-iminobis(ethanol) (1:2)
CAS Number: 71550-21-5
Appearance: Russet colored crystaline solid

Specific details on test material used for the study:

Test item: Bayscript Yellow GGN
Chemical name: Benzenesulfonic acid, 3,3' -( carbonylbis(imino(3-methoxy-4, 1-
phenylene)-2, 1-diazenediyl)bis-, compd. with 2,2'iminobis(ethanol) (1 :2)
CAS number: 71550-21-5
Molecular mass: 850,9 g/mol
pH-value, diluted 1:9 in water: 4.6
Content: 89.6%
Appearance: solid, crystalline, sorrel

Test animals / tissue source

Species:

other: Bovine Corneal Opacity and Permeability test (BCOP) using fresh bovine cornea.

Strain:

other: Bovine Corneal Opacity and Permeability test (BCOP) using fresh bovine cornea.

Details on test animals or tissues and environmental conditions:

Test System
Test system: isolated cornea from eyes of slaughtered cattle
Source: Slaughterhouse Laame, Buchenhofen 26, 42329 Wuppertal, Germany
Extraction: Staff of the slaughterhouse
Transport: 1 L containers with 500 mL HSS and 1 % penicillin I streptomycin solution, transport of the containers in coolers on ice. The test system matches with the guideline.

Test system

Vehicle:

other: The test item was suspended prior to application in physiologic saline solution to achieve a concentration of 20 % (w/v).

Controls:

other: Positive control: Imidaole; Negative control: Saline solution (0.9% NaCl).

Amount / concentration applied:

750 µL of the test material formulations (20% (w/v) in physiological saline) were applied to the corneas, each.

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

None

Number of animals or in vitro replicates:

20% (w/v) concentration ofthe test item and the positive control imidazole in physiologic saline solution were tested on 3 bovine corneas each in comparison to the negative control (physiologic saline solution).

Details on study design:

Methods and Parameters
Preparation of Corneas
Eyes were examined after delivery to the laboratory for any damage (like opacity, scratches or neovascularization) on the day of slaughter (1 day before the experiment). Eyes without any visible defects were transferred into new containers with fresh HSS solution supplemented with 1 %penicillin I streptomycin solution and 1 % FBS and stored overnight at refrigerator temperature (2-8 °C). Eyes with defects were discarded.
On the next day (day of experiment) the containers with the eyes were transferred in an incubator at 32°C (± 1 °C) for about 2 hours.
For the preparation of the cornea the sclera of each eye was incised with a scalpel and cut by scissors. A 2-3 mm scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 %penicillin I streptomycin solution and 1 % FBS. Each cornea was placed into a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders so prepared were transferred for at least 1 hour in the incubator at 32°C (± 1 °C).

Selection of corneas for application
After approximately 1 hour, the MEM medium was aspirated and the chambers were filled with fresh MEM medium. For each cornea the reference opacity value was measured then. The mean and standard deviation of the measured values were calculated by using Microsoft Excel. The corneas with values within the range of mean ± 1 standard deviation were selected for the actual test and assigned to the test groups.
The numbers of the selected corneas selected were documented in an appropriate protocol. The holders were labeled with a new serial number for the further testing.

Positive control and Test item Formulation
The positive control imidazole was formulated (20 % imidazole in physiologic saline solution (wlv)) immediately before administration. The formulation was visually described as solution. The test item was suspended with a glass rod shortly prior to application in physiologic saline solution to achieve a concentration of 20 % (w/v). The formulation was visually described as suspension. Before each application the suspension was mixed thoroughly.

Application of the test material and incubation
Immediately before application, the medium was aspirated from the anterior chamber. 750 µL of the test material formulations were applied to the corneas, each through the holes of the anterior chamber. The holes of both chambers were sealed with adhesive tape and the holders were kept with the front side up, so that the formulations covered the cornea sufficiently. The holders were transferred into the incubator at 32°C (± 1 °C) for the exposure time of 4 hours.
After the exposure, the formulations were aspirated from the anterior chamber and the corneas were rinsed at least 3 times with phenol red containing MEM to show effectiveness of test substance removal. During the final rinse cycle the corneas were rinsed again with pure MEM medium in order to remove residues of the dye. The anterior chamber was then filled again with MEM medium to avoid drying out of the cornea. Before measuring opacity, fresh MEM medium was filled in the chambers.

Determination of Opacity
The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer BASF OP3.0 (with integrated light meter testo 545 and Comfort V3.4 SP6 software from Testo AG, Lenzkirch). Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.

Determination of Permeability
The medium in anterior chamber of each holder was replaced by 1 mL of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32°C (± 1 °C) for about 90 minutes.
After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 µL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluorescein solution was prepared and also filled into the 96-well plate, in triplicates.
The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA- Reader.

Results and discussion

In vitro

Results

Irritation parameter:

other: In vitro irritancy score (IVIS)

Run / experiment:

Test item 20% Bayscript Yellow GGN

Value:

-4.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No classification

In vivo

Irritant / corrosive response data:

Based on OECD TG 437 and the experimental conditions reported the test item is classified as not seriously damaging the eye (no Category).

Any other information on results incl. tables

Based on OECD TG 437 and the experimental conditions reported the test item is classified as not seriously damaging the eye (no Category).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on OECD TG 437 and the experimental conditions reported the test item is classified as not seriously damaging the eye (no Category).

Executive summary:

This study was performed to assess the corneal damage potential of the solid test item Bayscript Yellow GGN with the Bovine Corneal Opacity and Permeability test (BCOP) using fresh bovine cornea. The study was conducted in accordance with international accepted Guidelines (e.g. OECD TG 437).

20% (w/v) concentration ofthe test item and the positive control imidazole in physiologic saline solution were tested on 3 bovine corneas each in comparison to the negative control (physiologic saline solution). For determination of corneal damage opacity as well as tissue permeability was measured after a 4 hour treatment time.

Test items were applied to the epithelial surface of the cornea in a special corneal holder. Corneal opacity was measured quantitatively as the amount of light transmission through the cornea before and after treatment with the test item. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea after treatment. The In Vitro Irritancy Score (IVIS) value was calculated based on these data.

In accordance with OECD TG 437 and the study results Bayscript Yellow GGN was characterized by having no potential to seriously damage the eye.