disodium 4-amino-6-((4-((4-(2,4-diaminophenyl)azo)phenylsulfamoyl)phenyl)azo)-5-hydroxy-3-((4-nitrophenyl)azo)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:

February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been conduced in GLP on the registering substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

yes

Remarks:

see below "Any other information..."

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

421-880-6

EC Name:

-

Cas Number:

201792-73-6

Molecular formula:

C34H25N11Na2O11S3

IUPAC Name:

disodium 4-amino-6-{2-[4-({4-[2-(2,4-diaminophenyl)diazen-1-yl]phenyl}sulfamoyl)phenyl]diazen-1-yl}-5-hydroxy-3-[2-(4-nitrophenyl)diazen-1-yl]naphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals / tissue source

Species:

other: Bovine eyes

Details on test animals or tissues and environmental conditions:

Slaughter house: Butcher Service s.r.l. - Mattatoio no. 2067 M St. Teverina Km. 7800 - 01100 - Viterbo.
Age of animals: 6-12 months.
Killing time: From 8:30 to 12:00 in the morning.
Transport condition: Eyes were maintained in transport solution at approximately +4 °C.
Examination: Eyes were examined for the presence of any defects which could render the eye unsuitable for use (opacity, scratches or pitting
of the corneal surface, vascularisation or pigmentation).
Cornea excision: Each cornea with 2-3 mm of surrounding sclera was dissected from the eye using a scalpel, scissors and forceps and placed
into a Petri dish containing HBSS.
Mounting in the chamber: Each cornea was mounted into a prewarmed testing chamber with the endothelial surface of the cornea placed in contact
with the O-ring of the posterior part of the chamber. Care was taken to ensure the correct position of the cornea minimizing the
presence of pigmented area inside the O-ring and avoiding movements which would damage the cornea. The chamber was then
filled with complete EMEM without phenol red maintained at 321 °C (posterior part of the chamber first to maintain convexity).
Equilibration: The corneas in their holders were incubated in a liquid bath at 321 °C at least for 1 hour to permit metabolic stabilisation. At the end of the pre-dose incubation period, the two chambers were drained (anterior first) and re-filled with complete EMEM without
phenol red maintained at approximately 32 °C (posterior first).
Selection: Basal opacity of corneas was determined by means of a calibrated meter, measuring difference in light transmission between the corneas placed into the beam paths versus air (Opacitometer). During measurement, attention was paid to keep corneas away
from the bath for the minimal time needed. Corneas with a basal value7 arbitrary units were excluded from testing. The mean
opacity of the remaining corneas was determined. Corneas were distributed in treatment groups starting from those nearest
to the mean value for the negative control group and trying to approximately maintain opacity values similar to the mean of
the negative control group in the test item and positive control groups.

Test system

Vehicle:

physiological saline

Amount / concentration applied:

In the first experiment the test item was prepared at the concentration of 200 mg/mL in physiological saline (Baxter, batch no. 11D0802), immediately before use without correction for the displacement due to the volume occupied by the test item; thus the concentration obtained was lower than 20% w/v, stated in the study protocol. In the second experiment the final concentration was corrected and according to the protocol and OECD indications exactly at 20 % w/v

Duration of treatment / exposure:

Corneas were exposed in horizontal position for 4 hours+/- 10 minutes, incubated in a liquid bath at 32+/-1 °C.

Details on study design:

Eyes transport solution: Hanks Balanced Salt Solution (HBSS) modified, supplemented with Penicillin sulphate and Streptomycin sulphate both at the
final concentration of 100 IU/mL.
Complete EMEM: EMEM1 (with or without phenol red) supplemented with 1% (v/v) Foetal Bovine Serum. These media were prewarmed in a water
bath at 32 °C during the experimental procedure.
Sodium fluorescein solution: 5 mg/mL solutions of sodium fluorescein in DPBS
Method used: A closed chamber method was used, being the test item a solid non surfactant substance.
Treatment: The medium was completely removed from the anterior chamber. Using the closed chamber method, the treatment was
carried out by pipetting the formulated test item or control items through the dosing holes of the chamber.
Exposure period: Corneas were exposed in horizontal position for 4 hours10 minutes, incubated in a liquid bath at 321 °C.
Washing: After exposure, corneas were rinsed thoroughly with complete EMEM with phenol red. For the test item treated group, the
glass windows of the anterior chambers were removed to allow a more effective removal of the test item from the corneas.
A final wash with prewarmed complete EMEM without phenol red was carried out. Finally, the anterior chamber was re-filled
with prewarmed complete EMEM without phenol red.
Post-exposure period: There was no post-exposure period.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

ca. 16.6

Negative controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

other: The test item could not be clearly classified

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Based on the IVIS of 25.5, according to the OECD Guideline no. 437, the test item is clearly classified as non corrosive, but it could not be clearly classified as not irritant.

Executive summary:

The potential of the test item, Acid Black 210 Na, to cause corrosion/severe irritation was examined by using the Bovine Corneal Opacity and Permeability (BCOP) assay, in agreement with OECD Guideline no. 437 (adopted on 26 July 2013) and the Guidance Document OECD series on testing and assessment no. 160.

Two experiments were performed. In the first experiment, the test item was dissolved at 200 mg/mL in physiological saline and tested on the epithelial surface of three idoneous bovine corneas, for an exposure period of 4 hours. The mean opacity detected with an opacitometer at the end of the test item exposure period was 15.5. However, after the exposure period, the treated corneas appeared green-black coloured and the mean opacity value may have been affected by the colouration. No relevant increases in corneas permeability were observed after treatment with the test item.

Since the test item solution for this experiment was prepared without correction for displacement due to the volume occupied by the test item, in order to comply with the relevant guideline, a second experiment was performed.

In the second experiment the test item was dissolved at 20% (w/v) in physiological saline and tested on the epithelial surface of three idoneous bovine corneas, for an exposure period of 4 hours. The mean opacity detected with an opacitometer at the end of the test item exposure period was 23.7. This was confirmed at the macroscopic observation in which opacity of the treated corneas was noted although the treated corneas appeared green-black coloured. Slight increases in corneas permeability were observed after treatment with the test item.

In both experiments, positive and negative controls (a 20% (w/v) Imidazole solution in physiological saline and physiological saline alone, respectively), were concurrently tested in similar conditions and gave the expected results.