Disodium 3(or 5)-[[4-[(7-amino-1-hydroxy-3-sulphonato-2-naphthyl)azo]-1-naphthyl]azo]salicylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 3(or 5)-[[4-[(7-amino-1-hydroxy-3-sulphonato-2-naphthyl)azo]-1-naphthyl]azo]salicylate

EC Number:

252-305-5

EC Name:

Disodium 3(or 5)-[[4-[(7-amino-1-hydroxy-3-sulphonato-2-naphthyl)azo]-1-naphthyl]azo]salicylate

Cas Number:

34977-63-4

Molecular formula:

C27H19N5O7S.2Na

IUPAC Name:

tetrasodium 3-[(1E)-2-{4-[(1E)-2-(7-amino-1-hydroxy-3-sulfonatonaphthalen-2-yl)diazen-1-yl]naphthalen-1-yl}diazen-1-yl]-2-hydroxybenzoate 5-[(1E)-2-{4-[(1E)-2-(7-amino-1-hydroxy-3-sulfonatonaphthalen-2-yl)diazen-1-yl]naphthalen-1-yl}diazen-1-yl]-2-hydroxybenzoate

impurity 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

Test material form:

solid: particulate/powder

Details on test material:

Appearance: black powderTrade name: Saturn Black LNBatch No: 9013CAS No: 34977-63-4

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

TEST SYSTEM:Bovine eyes source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech RepublicEyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death. Only healthy animals (12 to 60 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 minimized (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 minimized (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.SELECTION CRITERIA FOR EYES USED IN BCOP: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 an opacity >7 opacity units were discarded. From 22 eyes the 6 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study, 4 eyes was superfluous and remaining 3 were used for the testing of another substance.NUMBER OF CORNEAS PER GROUP:Exposed group (test substance) - 3 corneasPositive control group (20% Imidazole) – 3 corneasNegative control group (0.9% NaCl) – 3 corneas

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL- Amount(s) applied (volume or weight with unit): 750 µL of the solution in physiological saline solution- Concentration (if solution): 2g of the test substance was dissolved in 10 mL of 0.9% sodium chloride solution.VEHICLE- Amount(s) applied (volume or weight with unit): 2g of the test substance was dissolved in 10 mL of 0.9% sodium chloride solution- Concentration (if solution): 0.9% sodium chloride in water- Lot/batch no. (if required): 145068131

Number of animals or in vitro replicates:

NUMBER OF CORNEAS PER GROUP:Exposed group (test substance) - 3 corneasPositive control group (20% Imidazole) – 3 corneasNegative control group (0.9% NaCl) – 3 corneas

Details on study design:

PROCEDURE SCHEME:Selection of corneas, mounting in holders → incubation with EMEM 1 hour (32 ± 1°C) → removed EMEM, measurement of baseline opacity → treatment by positive and negative control substances and test substance (incubation 4 hour) → washing epithelium, measurement of opacity after application → application of sodium fluorescein (5 mg/ml), incubation 1.5 hour (32 ± 1°C) → measurement of absorbance (490 nm).PREPARATION OF THE EYES:Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium and endothelium. Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM). The device was then equilibrated at 32 ± 1°C for at least one hour in water bath to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded. Each test group (test substance, concurrent negative and positive controls) consisted of the three eyes. The three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups.CONTROL SUBSTANCES: - Positive control: physiological saline solution (0.9% NaCl solution in water)- Negative control: imidazole (CAS: 288-32-4) 20% solutionPOST-EXPOSURE: After the exposure period, the test substance, the negative control, or the positive control substance was removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The test substance was removed multiplicity by reason of colouring of the test substance (black). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded. ENDPOINTS MEASURED:- Opacity: the amount of light transmission through the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale. - Permeability: the amount of sodium fluorescein dye that penetrates all corneal cell layers (i.e., the epithelium on the outer cornea surface through the endothelium on the inner cornea surface) measured indirectly using visible light spectrophotometry. 1 mL sodium fluorescein solution (5 mg/mL) was added to the anterior chamber of the corneal holder, which interfaced with the epithelial side of the cornea, while the posterior chamber, which interfaced with the endothelial side of the cornea, is filled with fresh EMEM. The holder was incubated in horizontal position for 1.5 hours at 32 ± 1 ºC. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively 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.EVALUATION OF RESULTS- Mean opacity:Opacity values of treated corneas were corrected by subtracting individual background opacity values and the mean opacity is calculated.- Mean permeability:Mean optical density value of treated corneas was corrected by subtracting the mean optical density value of negative control and the mean opacity is calculated. - 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 x mean permeability OD490 value)- Decision criteria:A substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant. - Study acceptance criteria: A test is considered acceptable if the positive control gives IVIS that 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. The negative or solvent/vehicle control responses should result in opacity and permeability values that 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.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

48.61

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Tab. No. 1: Permeability and opacity:

Group	Opacity					Permeability
	Baseline	Opacity after treatment	Difference	Mean difference	Mean Opacity (corrected)	OD490^a	Mean Permeability	Mean Permeability(corrected)
negative control (0.9% NaCl)	7	7	0	0.67	-	0.012	0.008	-
	6	7	1			0.011
	7	8	1			0.000
positive control (20% Imidazole)	7	62	55	54.67	54.00 (54.67 – 0.67)	2.068	1.893	1.885 (1.893 – 0.008)
	6	64	58			1.814
	7	58	51			1.796
Direct Black 51	5	40	35	41.00	40.33 (41.00 – 0.67)	0.417	0.560	0.552 (0.560 – 0.008)
	5	48	43			0.551
	6	51	45			0.710

^a - Values of permeability (Optical density at 490nm)

Tab. No. 2: IVIS values

Group	IVIS
	Calculation	Result
negative control (0.9% NaCl)	0.67 + 15 x 0.008	0.79
positive control (20% Imidazole)	54.00 + 15 x 1.885	82.28
Direct Black 51	40.33 + 15 x 0.552	48.61

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive (Category 1)

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The In Vitro Irritancy Score (IVIS) for Direct Black 51 was 48.61. This is less than the limit value of IVIS (55.1), which means that the test substance was not identified as a corrosive or severe irritant.

Executive summary:

The test substance, Direct Black 51, was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea.

The test was performed according to the Method B.47 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants, Council Regulation (EC) No.1152/2010, published in O.J. L 324, 2010.

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used. Closed-chamber method was used, because the test substance was applicable by micropipette. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

The In Vitro Irritancy Score (IVIS) for Direct Black 51 was 48.61. This is less than the limit value of IVIS (55.1), which means that the test substance was not identified as a corrosive or severe irritant.