Benzoic acid, 5-[(4-aminophenyl)azo]-2-hydroxy-, reaction products with 3-[(4-amino-3-methoxyphenyl)azo]benzenesulfonic acid and carbonic dichloride, sodium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22.11. – 23. 11. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Version / remarks:

Adopted 14th Feb 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Benzoic acid, 5-[(4-aminophenyl)azo]-2-hydroxy-, reaction products with 3-[(4-amino-3-methoxyphenyl)azo]benzenesulfonic acid and carbonic dichloride, sodium salts

EC Number:

232-339-7

EC Name:

Benzoic acid, 5-[(4-aminophenyl)azo]-2-hydroxy-, reaction products with 3-[(4-amino-3-methoxyphenyl)azo]benzenesulfonic acid and carbonic dichloride, sodium salts

Cas Number:

8005-52-5

Molecular formula:

C27H22N6O8S.2Na

IUPAC Name:

hexasodium 2-hydroxy-5-[(1E)-2-{4-[({2-methoxy-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}carbamoyl)amino]phenyl}diazen-1-yl]benzoate 3-[(1E)-2-{3-methoxy-4-[({2-methoxy-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}carbamoyl)amino]phenyl}diazen-1-yl]benzene-1-sulfonate 5-[(1E)-2-{4-[({4-[(1E)-2-(3-carboxylato-4-hydroxyphenyl)diazen-1-yl]phenyl}carbamoyl)amino]phenyl}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

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES- Source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic- Characteristics of donor animals (e.g. age, sex, weight): 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. - Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):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).- Time interval prior to initiating testing: 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). All eyes used in the assay were from the same group of eyes collected on a specific day.- indication of any existing defects or lesions in ocular tissue samples: Only corneas from eyes free of defects including scratched, and neovascularisation 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.

Test system

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL - Amount(s) applied (volume or weight with unit): 2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution. VEHICLE Hank`s Balanced Salts Solution (HBSS) - Lot/batch no. (if required): SLBL5152V, Sigma-Aldrich

Duration of treatment / exposure:

4 hrs

Duration of post- treatment incubation (in vitro):

1.5 hr

Number of animals or in vitro replicates:

The results were based on the selection criteria for the eyes, as well as the positive and negative control responses.Number of corneas per group: Exposed group (test substance) - 3 corneas (No. 7, 8, 9) Positive control group (20% Imidazole) – 3 corneas (No. 4, 5, 6) Negative control group (0.9% NaCl) – 3 corneas (No. 1, 2, 3)

Details on study design:

SELECTION AND PREPARATION OF CORNEASSelection criteria for eyes used in BCOP: Only corneas from eyes free of defects including scratched, and neovascularisation 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. Preparation: 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 an 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 a baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline 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. QUALITY CHECK OF THE ISOLATED CORNEASFrom 19 eyes the 2 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study (the corneas No. 1, 2, 3, 4, 5, 6, 7, 8 and 9), 8 eyes were superfluous.NUMBER OF REPLICATESNumber of corneas per group:Exposed group (test substance) - 3 corneas (No. 7, 8, 9) Positive control group (20% Imidazole) – 3 corneas (No. 4, 5, 6) Negative control group (0.9% NaCl) – 3 corneas (No. 1, 2, 3) NEGATIVE CONTROL USED0.9% NaClSOLVENT CONTROL USED (if applicable)0.9% NaClPOSITIVE CONTROL USED20% ImidazoleAPPLICATION DOSE AND EXPOSURE TIME2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution. Open-chamber method was used, because the test substance at 20% concentration was viscous. The test substance (the test substance in quantity enough to completely cover the cornea) was applied directly to the epithelial surface of the cornea using the spatula. After dosing, the glass window was replaced on the anterior chamber to recreate a closed system.Exposure time: 4 hrsTREATMENT METHOD: open-chamber method POST-INCUBATION PERIOD: 1.5 hrREMOVAL OF TEST SUBSTANCE, POST-EXPOSURE INCUBATIONAfter the exposure period, the negative control and 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 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. The test substance was removed from the anterior chamber with EMEM – more repeatedly, because the test substance is coloured (yellow colour). The corneas (applied the test substance) were also rinsed with EMEM (containing phenol red). Lastly EMEM (without phenol red) was used for final rinsing. The test substance was complete removal. The anterior chamber was then refilled with fresh EMEM without phenol red. The 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: 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.SCORING SYSTEM: In Vitro Irritancy Score (IVIS)IVIS = mean opacity value + (15 x mean permeability OD490 value)DECISION CRITERIA: IVISUN GHS ≤ 3 No Category> 3; ≤ 55 No prediction can be made ≥ 55 Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

2.05

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS: Study acceptance criteria were fulfilled.- Acceptance criteria met for negative control: The value of opacity for negative control (0.9% NaCl) obtained during the study was 0.61 and value of permeability was 0.045. The values obtained during this study not exceeded upper limits, so the study is considered acceptable.- Acceptance criteria met for positive control: The value of IVIS for positive control (20% imidazole) obtained during the study was 79.04. This value is within the acceptance limit (one standard deviations of the current historical mean), so the study is considered acceptable.

Any other information on results incl. tables

The In Vitro Irritancy Score (IVIS) was computed according the following formula:

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

Group	IVIS calculation	Result
NC (0.9% NaCl)	0.61 + 15 x 0.045	1.29
PC (20% Imidazole in 0.9% NaCl)	49.91 + 15 x 1.942	79.04
EXP (Direct Yellow 44)	2.05 + 15 x 0.0	2.05

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The In Vitro Irritancy Score (IVIS) for Direct Yellow 44 was 2.05.This value of IVIS is ≤ 3 therefore the classification of test substance effect according to UN GHS criteria for eye irritation or serious eye damage is: No category.

Executive summary:

The test substance, Direct Yellow 44, 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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage, Adopted 14th February 2017

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.

Open-chamber method was used, because the test substance was viscous. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) is to be calculated from the values of opacity and permeability.

The In Vitro Irritancy Score (IVIS) for Direct Yellow 44 was 2.05.

This value of IVIS is ≤ 3 therefore the classification of test substance effect according to UN GHS criteria for eye irritation or serious eye damage is: No category.