2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08-05-2017 to 24-05-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 July 2013

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

EC Number:

287-719-5

EC Name:

2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

Cas Number:

85567-07-3

Molecular formula:

C36H42Cl2KN6NaO28S8

IUPAC Name:

2,2'-[[3-chloro-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]phenyl]imino]bisethyl bis(hydrogen sulphate), potassium sodium salt

Test material form:

solid

Details on test material:

Batch: 1283067001

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test system:
Freshly isolated bovine cornea obtained as a by-product from animals freshly slaughtered
- Source: A. Moksel AG, Buchloe, Germany

Test system

Vehicle:

physiological saline

Remarks:

final concentration 20%

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of the test item, the negative or positive control

Duration of treatment / exposure:

4 hours ± 5 minutes incubation at 32 ± 1 °C

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

After the illuminance measurement the corneas were incubated for 90 minutes at 32 ± 1 °C

Number of animals or in vitro replicates:

3 replicates (corneas)

Details on study design:

Preparation of the corneas:
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated. The eyes were carefully examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws.
The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

Treatment of the corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control. 750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded.
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Evaluation of results:
The following formula was used to determine the in vitro irritation score (IVIS):

IVIS = mean opacity value + (15 x mean permeability OD490 value)

Study acceptance criteria:
The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean. The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of 3 corneas

Value:

30.04

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

Any other information on results incl. tables

In vitro irritation score:

Cornea no.	Test item	Corrected opacity	Corrected OD490 value	IVIS
1	Negative control	-0.04	0.006	0.67
2		0.15	0.015
3		1.35	0.015
MV		0.49	0.012
4	Positive control	76.72	0.951	97.7
5		82.82	0.932
6		88.91	1.094
MV		82.82	0.992
7	Test item	28.06	0.304	30.04
8		27.55	0.334
9		20.91	0.269
MV		25.51	0.302

Applicant's summary and conclusion

Interpretation of results:

other: no prediction can be made

Conclusions:

The mean in vitro irritation score (IVIS) was 30.04. Therefore, no prediction can be made regarding the classification of the test substance according to the evaluation criteria.

Executive summary:

The eye irritancy potential of the test substance was investigated in the bovine corneal opacity and permeability assay (BCOP), according to OECD 437. The test item was suspended with physiological saline 0.9% NaCl to obtain a 20% concentration.

All 3 corneas treated with the test substance showed an intense orange coloration of the tissue with a strong redness at the edge. The following mean in vitro irritation score was calculated: 30.04. No prediction can be made regarding the classification of the test substance according to the evaluation criteria. Further testing in another suitable method is required.

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. Furthermore, the negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.