Octasodium 7,7'-[(2,2'-disulphonato[1,1'-biphenyl]-4,4'-diyl)bis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino[2-(carbamoylamino)]-4,1-phenylene]azo]]bis(naphthalene-1,3,6-trisulphonate)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 14 to 15, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2017

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)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reactive Yellow 084

IUPAC Name:

Reactive Yellow 084

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes source: breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic.
Eyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death. No detergent was used. Only healthy animals (12 to 30 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test. The risk of contamination was minimised (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/ml and streptomycin at 100 μg/ml).
The time interval between collection of the eyes and use of corneas in the BCOP was minimised (typically collected and used on the same day). The results were based on the selection criteria for the eyes, as well as the positive and negative control responses. All eyes used in the assay were from the same group of eyes collected on a specific day.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Suspension prepared test substance at 20 % concentration in a 0.9 % sodium chloride solution. 2 g of test substance was suspended in 10 ml of 0.9 % sodium chloride solution.
750 µl of test item, to cover the epithelial side of cornea.

Duration of treatment / exposure:

4 h

Duration of post- treatment incubation (in vitro):

Incubation for 1.5 h at 32 ± 1 °C with 5mg/ml of sodium fluorescein, before measurement of absorbance at 490 nm.

Number of animals or in vitro replicates:

3 corneas per negative control
3 corneas per positive control
3 corneas per test substance

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes, once they arrive at the laboratory, were carefully examined for defects including scratched, and neovascularisation. Only corneas from eyes free of such defects were used.
The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded.
From 19 eyes, 2 eyes were eliminated after inductive incubation, because baseline opacity values were >7 and one cornea was eliminated by reason of damage of cornea during mounting in holders. Nine corneas were used for the study (corneas no. 1, 2, 3, 4, 5, 7, 8, 9 and 10), 4 eyes was superfluous and 3 corneas were used for testing of other substances.

NEGATIVE CONTROL USED: 0.9 % NaCl

POSITIVE CONTROL USED: 20 % imidazole

APPLICATION DOSE AND EXPOSURE TIME: 750 µl of a 20 % concnetration in 0.9 % NaCl solution, for 4 h.

TREATMENT METHOD: closed chamber, the test item was applicable by micropipette. Test item (750 µl) to cover the epithelial side of the cornea is introduced into the anterior chamber through the dosing holes on the top surface of the chamber, and the holes were subsequently sealed with the chamber plugs during the exposure.

POST-EXPOSURE:
After the exposure period, negative control and positive control substance was removed from the anterior chamber with EMEM (containing phenol red); corneas were given a final rinse with EMEM (without phenol red). EMEM (without phenol red) was used as a final rinse to ensure removal of phenol red from the anterior chamber prior to opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. Opacity and permeability of each cornea were recorded.
Test item was removed from the anterior chamber with EMEM – more repeatedly, because test item is coloured . Corneas were also rinsed with EMEM (containing phenol red). Lastly EMEM (without phenol red) was used for final rinsing. Test item was completely removed. The anterior chamber was then refilled with fresh EMEM without phenol red. Opacity and permeability of each cornea were recorded.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYSTM 10 UV/VIS Scanning). The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.
- Mean opacity: opacity values of treated corneas were corrected by subtracting individual background opacity values and the mean opacity is calculated.
- Mean permeability: mean OD value of treated corneas was corrected by subtracting the mean OD value of negative control and the mean permeability is calculated.

SCORING SYSTEM:
IVIS calculation: resulting mean opacity and OD490 values for each treatment group was combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:
IVIS = mean opacity value + (15 × mean permeability OD490 value)

DECISION CRITERIA:
The IVIS cut-off value for identifying test item as including serious eye damage (UN GHS Category 1) and test item not requiring classification for eye irritation or serious damage (UN GHS No Category) will be given hereafter:

IVIS UN GHS
≤ 3 no category (UN GHS: not classified)
> 3; ≤ 55 no prediction can be made
> 55 category 1 (UN GHS: “serious eye damage”)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

mean (3 corneas)

Value:

0.013

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (3 corneas)

Value:

2.47

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
Appearance of corneas was observed before and after application of test item, negative and positive control.
No macroscopic damage was observed on corneas before application.
Corneal opacity was observed on corneas treated by positive control. Corneas treated by negative control and test item were without macroscopic damage.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: opacity and permeability values are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control.
- Acceptance criteria met for positive control: IVIS falls within one standard deviations of the current historical mean, which is to be updated at least every three months, or each time an acceptable test is conducted in laboratories where tests are conducted infrequently.

IVIS – historical value for positive control
mean standard deviation lower limit
79.00 11.62 67.38
Notes: lower limit = mean - one standard deviation of the current historical mean
Historical means of IVIS are updated at least every three months.

The value of IVIS for positive control (20 % imidazole) during the study was 99.31, thus the study is considered acceptable.


Opacity and permeability – historical values for negative control
value mean standard deviation upper limit
opacity 1.74 1.00 2.74
permeability 0.0279 0.0148 0.0427
Notes: upper limit = mean + one standard deviation of the current historical mean
Historical means of opacity and permeability are updated at least every three months.

Opacity for negative control (0.9 % NaCl) obtained during the study was 1.72 and value of permeability was 0.013, thus the study is considered acceptable.

Any other information on results incl. tables

Opacity values

group	cornea no.	opacity after treatment	baseline opacity	opacity difference	mean opacity difference	mean opacity (corrected)
NC (0.9 % NaCl)	1	4.99	2.75	2.24	1.72	-
	2	4.55	2.97	1.58
	3	4.06	2.73	1.33
PC (20 % imidazole in 0.9 % NaCl)	4	69.88	2.29	67.59	69.48	67.76
	5	76.93	1.86	75.07
	7	68.01	2.24	65.77
test substance	8	7.65	1.99	5.66	4.19	2.47
	9	6.30	3.01	3.29
	10	5.73	2.10	3.63

Values of permeability (optical density values)

group	cornea no.	values of permeability (optical density at 490 nm)	mean permeability	mean permeability (corrected)
NC (0.9 % NaCl)	1	0.018	0.013	-
	2	0.009
	3	0.012
PC (20 % imidazole in 0.9 % NaCl)	4	2.222	2.116	2.103
	5	2.164
	7	1.963
test substance	8	0.013	0.013	0
	9	0.011
	10	0.015

Applicant's summary and conclusion

Interpretation of results:

other: not irritant according to the CLP Regulation (EC 1272/2008)

Conclusions:

Not irritant.

Executive summary:

Method

Evaluation of the potential for ocular corrosivity or severe irritancy was measured as the ability of test substance to induce opacity and increased permeability in an isolated bovine cornea.

Test was performed according to EU method B.47.  

A total of 9 corneas was used, divided in 3 groups. In particular, a treatment group, a positive control group with 20 % imidazole and negative control group with 0.9 % NaCl.

Test item was tested as suspension prepared from test item at 20 % concentration in a 0.9 % NaCl solution.

The closed-chamber method was used, because test item was applicable by micropipette. The opacity and permeability of each cornea were measured.In Vitro Irritancy Score (IVIS) was calculated from values of opacity and permeability.

Results 

IVIS for test substance was 2.47. Corneas treated by test item were without macroscopic damage.