Sodium 4-amino-5-hydroxy-3-(4-nitrophenylazo)-6-(phenylazo)naphthalene-2,7-disulphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From October 5 to 6, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted on a Similar Substance. The reliability of the original report is 1. The complete justification for the read across approach is attached at section 13.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

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:

2013

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 5-acetylamino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonate

EC Number:

223-098-9

EC Name:

Disodium 5-acetylamino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonate

IUPAC Name:

Acid Red 001

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

other: Bovine

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: 12 to 30 months old
- Time interval prior to initiating testing: typically collected and used on the same day
- Indication of any existing defects or lesions in ocular tissue samples: 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.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 μg/mL and streptomycin at 100 μg/mL

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
Amount applied: 2 g
Concentration 0.2 g/ml

VEHICLE
Amount applied: 10 ml of 0.9% NaCl solution

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

1.5 hour

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Eyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death. Only healthy animals considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test.

QUALITY CHECK OF THE ISOLATED 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 25 eyes the 9 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study (the corneas No. 6, 7, 9, 10, 11, 14, 15, 16 and 20), 4 eyes was superfluous and remaining 3 were used for the testing of another substance.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
0.9% NaCl

POSITIVE CONTROL USED
20% Imidazole

APPLICATION DOSE AND EXPOSURE TIME
2g of test substance in 10 ml of physiological saline for 4 hours

TREATMENT METHOD
Closed chamber was used, because the test substance was applicable by micropipette. The test substance (750 µL of application form) 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.

REMOVAL OF TEST SUBSTANCE
After 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 – repeatedly, because the test substance is coloured . Subsequently the test substance was removed mechanically using a cotton swab and brush. 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, but corneas stayed coloured by the test substance. 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
- 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. Opacity values of treated corneas were corrected by subtracting individual background opacity values and the mean opacity is calculated.
- 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 GENESYS TM 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 OD value of treated corneas was corrected by subtracting the mean OD value of negative control and the mean permeability is calculated.

SCORING SYSTEM: In Vitro Irritancy Score (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
The IVIS cut-off value for identifying the test substance as including serious eye damage (UN GHS Category 1) and the test substance not requiring classification for eye irritation or serious damage (UN GHS No Category) will be given hereafter:
IVIS:
≤ 3 UN GHS No Category: Chemicals that do not meet the requirements for classification as: UN GHS Category 1 or 2. Interchangeable with “Not Classified”
> 3; ≤ 55 No prediction can be made
> 55 UN GHS Category 1: “Serious eye damage”

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

ca. 7.45

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

other: No prediction can be made on test substance potential to cause eye irritation or serious eye damage based on the OECD Guideline Priciples

Conclusions:

IVIS = 7.45

Executive summary:

Method

The test substance 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 theOECD Test Guideline No. 437 (Adopted 26^thJuly 2013).

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.

Results

The In Vitro Irritancy Score (IVIS) for the test substance was 7.45 but this result could be affected by higher opacity values (mild colouring of corneas after removing the test substance: light-pink touch).