[x Sodium, y Potassium, z Lithium, x+y+z =2] 4-amino-3-[[[(2,4-diaminophenyl)diazenyl]phenyl]diazenyl]-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

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

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:

9th October 2017

Deviations:

yes

Remarks:

The positive control is 10% (w/v) Benzalkonium chloride (purity not indicated by the producer) in saline since the laboratory historical control data is established with this chemical.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

EC Number:

272-559-0

EC Name:

Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Cas Number:

68877-33-8

Molecular formula:

C28H21N9O7S2.2Na

IUPAC Name:

disodium 4-amino-3-({4-[(2,4-diaminophenyl)diazenyl]phenyl}diazenyl)-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS (Hank’s Buffered Salt Solution) containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes. The corneae were directly used in the BCOP test on the same day.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

NEGATIVE CONTROL USED, Saline (0.9% NaCl in deionised water)
POSITIVE CONTROL USED, 10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl (saline) using sonication for 10 minutes.
TEST ITEM: The test item was tested as a 20% suspension (w/v) in saline using sonication for 10 minutes.

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3 for each Negative control, Positive control and Test item

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments. For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed before basal opacity measurement (t0).
At the end of the incubation period, the basal opacity was determined (t0).
The basal opacity of all corneae was recorded. Only corneae with a value of the basal opacity < 7 were used. Sets of three corneae were used for treatment with the test item and for the negative and positive controls, respectively.

NUMBER OF REPLICATES : 3 for each Negative control, Positive control and Test item

NEGATIVE CONTROL USED : Name: Saline (0.9% NaCl in deionised water)

POSITIVE CONTROL USED : Name: 10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl (saline) using sonication for 10 minutes.

APPLICATION DOSE AND EXPOSURE TIME
The anterior compartment received the test item suspension or the negative or positive controls at a volume of 0.75 mL each on the surface of the corneae, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted 240 minutes.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
After exposure, the test item or the control items, respectively, were each rinsed off from the according application sides with EMEM containing phenol red at least three times or more if phenol red was still discoloured (yellow or purple), or the test item was still visible. Once the medium was free of the test item the corneae were given a final rinse with cMEM without phenol red. Fresh cMEM was added into the anterior compartment and opacity was measured (t240). The opacity measurement is described in chapter 3.5.
The corneae treated with the test item became dyed black after incubation.
In the second step of the assay, permeability of the cornea was determined. The permeability measurement is described in chapter 3.6.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
Measures taken at the end of the incubation period, the medium was changed before basal opacity measurement (t0). After exposure of the corneae to the different test groups and after rinsing the opacity value was determined again (t240).
- Corneal permeability: Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value of permeability determination)
The following formula is used to determine the IVIS of the positive control and the test item: IVIS = (opacity value – mean opacity of the negative control) + (15 x (permeability value – mean permeability of the negative control))
The mean IVIS value of each treated group is calculated from the respective individual IVIS values.
Depending on the IVIS score obtained, the test item is classified into the following Category according to OECD guideline 437:
IVIS ≤ 3 UN GHS / EU CLP No Category
IVIS > 3; ≤ 55 UN GHS / EU CLP No prediction can be made
IVIS > 55 UN GHS / EU CLP Category 1

DATA EVALUATION
Opacity: The change of the opacity value of each treated cornea or of the positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
Permeability: The corrected OD490 value of each cornea treated with positive control or test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea

DECISION CRITERIA:
The test will be acceptable if :
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

A single testing run composed of at least three corneae should be sufficient for a test chemical when the resulting classification is unequivocal. In cases of borderline results in the first testing run, a second testing run will be considered, as well as a third one in case of discordant mean IVIS results between the first two testing runs. A result in the first testing run is considered borderline if the predictions from the 3 corneae are non-concordant, such that:
• 2 of the 3 corneae give discordant predictions from the mean of all corneae, or,
• 1 of the 3 corneae gives a discordant prediction from the mean of all 3 corneae, and the discordant result is >10 IVIS units from the cut-off threshold of 55.
• If the repeat testing run corroborates the prediction of the initial testing run (based upon the mean IVIS value), then a final decision can be taken without further testing.

If the repeat testing run results in a non-concordant prediction from the initial testing run (based upon the mean IVIS value), then a third and final testing run should be conducted to resolve equivocal predictions, and to classify the test chemical. It may be permissible to waive further testing for classification and labelling in the event any testing run results in a UN GHS Category 1 prediction.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

72.08

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

300.41

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Any other information on results incl. tables

Results of the first experiment after 240 Minutes Treatment Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposed in vitro Irritancy Score
		Mean		Mean
Negative Control	0	0.33	0.088	0.080	1.32	1.54	No Category
	1		0.080		2.20
	0		0.073		1.10
Positive Control	77.67*		0.581*		86.38	87.39	Category 1
	78.67*		0.633*		88.16
	82.67*		0.331*		87.63
test item	63.67*		-0.013*		63.47	72.08	Category 1
	71.67*		-0.021*		71.35
	80.67*		0.050*		81.41

*corrected values

Results of the second experiment after 240 Minutes Treatment Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposed in vitro Irritancy Score
		Mean		Mean
Negative Control	0.00	0.33	0.079	0.076	1.19	1.48	No Category
	1.00		0.060		1.90
	0.00		0.090		1.35
Positive Control	96.67*		0.438*		103.23	109.51	Category 1
	107.67*		0.559*		116.05
	101.67*		0.505*		109.24
test item	299.67*^#		0.081*		300.88	300.41	Category 1
	299.67*^#		0.100*		301.16
	297.67*		0.101*		299.18

*corrected values

^#Values were set on 300 to enable the analysis. The measured values were 334 and 423.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item is serious eye damaging (EU CLP/UN GHS Category 1).

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of the substance by means of the BCOP assay using fresh bovine corneae.

The experiment was performed twice on sponsor’s request due to an unexpected result. The result of the first experiment could be confirmed. Both experiments will be stated in this report.

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item as well as the positive and the negative controls were each applied to different corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240).  

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.  

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed in both experiments.

In the first experiment the positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (EU CLP/UN GHS Category 1). The IVIS of the positive control (IVIS of 87.39) was below two standard deviations (SD = 11.82) of the current historical mean (116.38) but within the range of all obtained historical control data for the positive control (80.38—146.55).  

In the second experiment the positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (EU CLP/UN GHS Category 1) with an IVIS of 109.51.

Relative to the negative control, the test item caused an increase of the corneal opacity. In the first experiment the calculated mean in vitro irritancy score was 72.08 and in the second experiment 300.41. According to OECD 437 (see table in chapter 3.8.3) the test item is classified as serious eye damaging (EU CLP/UN GHS Category 1).