Disodium naphthalene-1,5-disulphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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:

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

Version / remarks:

2013

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium naphthalene-1,5-disulphonate

EC Number:

216-732-0

EC Name:

Disodium naphthalene-1,5-disulphonate

Cas Number:

1655-29-4

Molecular formula:

C10H8O6S2.2Na

IUPAC Name:

disodium naphthalene-1,5-disulphonate

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

dihydrogen oxide

Test material form:

solid: crystalline

Details on test material:

Test item: Armstrongsäure-di-Natriumsalz Monohydrat
CAS name: 1,5-Naphthalenedisulfonic acid, sodium salt (1 :2) Monohydrat
CAS number: 207569-02-6
Empirical formula: C10 H6 O6 S2 * 2Na * H2O
Molecular mass: 350.3 g/mol
Batch number: CHNHFC1602
Content monohydrat-form: 99.8 %
Corresponds to 94,7 % anhydrous form, CAS: 1655-29-4

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

Bovine eyes of slaughtered cattle were extracted and transferred in containers with Hank’s balanced salt solution (HBSS) with penicillin/streptomycin solution. For transportation the containers were ice cooled.
Eyes with defects were sorted out and disposed of, eyes without any defects were transferred into fresh HBSS supplemented with penicillin/streptomycin solution and 1 % FBS and stored overnight at 2-8 °C. On the next day (day of testing) the containers with the eyes were placed in an incubator at 32 ° C (± 1 ° C) for about 2 hours before preparation of the corneas.
For the preparation of the cornea the sclera was incised with a scalpel and cut by scissors. A 2-3 mm wide scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin / streptomycin solution and 1 % FBS. Each cornea was placed into a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders were placed for at least 1 hour in the incubator at 32 °C (± 1 °C).

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
- the test item and the solvent were tested in parallel

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

3 corneas per tested material

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Bovine eyes of slaughtered cattle were extracted and transferred in containers with Hank’s balanced salt solution (HBSS) with penicillin/streptomycin solution. For transportation the containers were ice cooled.
Eyes with defects were sorted out and disposed of, eyes without any defects were transferred into fresh HBSS supplemented with penicillin/streptomycin solution and 1 % FBS and stored overnight at 2-8 °C. On the next day (day of testing) the containers with the eyes were placed in an incubator at 32 ° C (± 1 ° C) for about 2 hours before preparation of the corneas.
For the preparation of the cornea the sclera was incised with a scalpel and cut by scissors. A 2-3 mm wide scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin / streptomycin solution and 1 % FBS. Each cornea was placed into a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders were placed for at least 1 hour in the incubator at 32 °C (± 1 °C).

QUALITY CHECK OF THE ISOLATED CORNEAS
yes

NEGATIVE CONTROL USED: physiological saline

SOLVENT CONTROL USED (if applicable): ethyl acetate

POSITIVE CONTROL USED: NaOH (1%)

APPLICATION DOSE AND EXPOSURE TIME
the test materials were applied pure for 4 hours

TREATMENT METHOD:
closed chamber method

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer (BASF OP3.0). Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- Corneal permeability: The medium in anterior chamber of each holder was replaced by 1ml of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32 °C (± 1 °C) for about 90 minutes. After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 μL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluoresceinsolution was prepared and also filled into the 96-well plate, in triplicates. The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA - Reader).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Test item formulations that cause an IVIS value > 55 are classified as seriously damaging the eye (UN GHS Cat 1). Test item formulations that cause IVIS values of ≤ 55 are considered as not seriously damaging the eye (not UN GHS Cat 1).

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

1.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

	Mean opacity value	Mean permeability value	In vitro irritancy score (IVIS)
Negative control; isotonic saline solution	-0,6	0,011	-0,5
Positive control; 20 % Imidazole	74,8	1,676	99,9
20 % Armstrongsaure-di-Natriumsalz	1,4	0,019	1,7

Applicant's summary and conclusion

Executive summary:

This study was performed to assess the corneal damage potential of the solid test item Armstrongsaure-di-Natriumsalz with the Bovine Corneal Opacity and Permeability test

(BCOP) using fresh bovine cornea. The study was conducted in accordance with international accepted Guidelines (e.g. OECD TG 437).

20% (w/v) concentrations of the test item and the positive control imidazole in isotonic saline solution were tested on 3 bovine corneas each in comparison to the negative control, isotonic

saline solution. For determination of corneal damage opacity as well as tissue permeability was measured after a 4 hour treatment time.

Test items were applied to the epithelial surface ofthe cornea in a special corneal holder. Corneal opacity was measured quantitatively as the amount of light transmission through the cornea before and after treatment with the test item. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea after treatment. The In Vitro Irritancy Score (IVIS) value was calculated based on these data.

In accordance with OECD TG 437 and the study results Armstrongsaure-di-Natriumsalz was characterized by having no potential to seriously damage the eye.