4-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]-N-(3-ethoxypropyl)benzenesulphonamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 June 2016 to 19 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

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:

26 July 2013

Deviations:

no

Test guideline 2

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:

EC 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP

Test material

Test material information

Constituent 1

Reference substance name:

4-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]-N-(3-ethoxypropyl)benzenesulphonamide

EC Number:

276-602-4

EC Name:

4-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]-N-(3-ethoxypropyl)benzenesulphonamide

Cas Number:

72363-26-9

Molecular formula:

C25H24N2O7S

IUPAC Name:

4-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]-N-(3-ethoxypropyl)benzenesulphonamide

Details on test material:

Identification: FAT 40444/B
Lot: PCR92X140707(China)
Appearance: red powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PCR92X140707 (China)
- Expiration date of the lot/batch: 14 October 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4 °C in the dark

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir as a by-product from freshly slaughtered animals
- Characteristics of donor animals (e.g. age, sex, weight): adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): excised eyes were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics
- Time interval prior to initiating testing: They were transported to the test facility over ice packs on the same day of slaughter.
- indication of any existing defects or lesions in ocular tissue samples: The corneas were prepared immediately on arrival.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 µg/mL

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution): 20 % w/v solution in 0.9 % w/v sodium chloride

Duration of treatment / exposure:

240 mins

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

QUALITY CHECK OF THE ISOLATED CORNEAS

NUMBER OF REPLICATES
3 per treatment

NEGATIVE CONTROL USED
0.9 % w/v solution

POSITIVE CONTROL USED
Imidazole, 20 % w/v solution in 0.9 % w/v sodium chloride

APPLICATION DOSE AND EXPOSURE TIME
20 % w/v solution, 240 mins

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD492)
- Others (e.g., pertinent visual observations, histopathology): (please specify): Histopathology.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. Yes

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤4.1 and permeability ≤0.105. The negative control acceptance criteria were therefore satisfied.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	4.0
Negative Control	1.8
Positive Control	98.4

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No prediction of eye irritation can be made.

Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The test was carried out according to OECD guideline 437 and EU method B.47.

The test item was applied at a concentration of 20 % w/v in 0.9 % w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The test item is classified according to the prediction model as follows:

IVIS	Classification
≤ 3	No category. Not requiring classification to UN GHS or EUCLP
> 3; ≤55	No prediction of eye irritation can be made
> 55	Category 1. UN GHS or EU CLP Causes serious eye damage

Results

The in vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	4.0
Negative Control	1.8
Positive Control	98.4

Conclusion

No prediction of eye irritation can be made.