Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted on 09 June 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 guidelineopen allclose all
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
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-dihydro-6-hydroxy-1,4-dimethyl-2-oxo-5-[[3-[(phenylsulphonyl)oxy]phenyl]azo]nicotinonitrile
EC Number:
261-693-5
EC Name:
1,2-dihydro-6-hydroxy-1,4-dimethyl-2-oxo-5-[[3-[(phenylsulphonyl)oxy]phenyl]azo]nicotinonitrile
Cas Number:
59312-61-7
Molecular formula:
C20H16N4O5S
IUPAC Name:
3-[(5-cyano-2-hydroxy-1,4-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)diazenyl]phenyl benzenesulfonate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Specific details on test material used for the study:
Identification: FAT 93460/D TE
Physical state/Appearance: Yellow powder
Batch: 20140721 (China)
Purity 94.6 %
Expiry Date: 14 October 2019
Storage Conditions: 4 °C in the dark

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Eyes from adult cattle (typically 12 to 60 months old) were obtained from a 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): The excised eyes were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter.
- Time interval prior to initiating testing :The corneas were prepared immediately on arrival at the test facility
- Indication of any existing defects or lesions in ocular tissue samples: only corneas free from damage were used.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 µg/mL.

Test system

Vehicle:
Hank's balanced salt solution
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Concentration (if solution): 20% w/v solution in 0.9% w/v sodium chloride solution.
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
Three replicates per treatment
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
A pre-treatment opacity reading was taken for each cornea using a calibrated opacimeter. 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: Only corneas free of damage were used

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Sodium chloride 0.9% w/v solution.

POSITIVE CONTROL USED: The positive control item, Imidazole, was used as a 20% w/v solution in 0.9% w/v sodium chloride solution.

APPLICATION DOSE AND EXPOSURE TIME: 20% w/v solution of test item in 0.9% w/v sodium chloride solution. exposure time = 240 mins.

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3

- POST-EXPOSURE INCUBATION:
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes

METHODS FOR MEASURED ENDPOINTS:
- 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:
<= 2.3
Negative controls validity:
valid
Remarks:
In Vitro Irritancy Score = 1.9
Positive controls validity:
valid
Remarks:
In vitro Irritancy Score = 97.0
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No Damage

DEMONSTRATION OF TECHNICAL PROFICIENCY:
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

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
FAT 93460/D is not an eye irritant.
Executive summary:

An in vitro study was conducted to assess eye corrosion potential of FAT 93460/D according to OECD test guideline 437 and Method B.47 of Commission Regulation (EC) No. 440/2008. 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.

Method

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).

Results

The In Vitro irritancy scores are summarized as follows:

Treatment In Vitro Irritancy Score
 Test Item  2.3
 Negative Control  1.9
 Positive Control 97.0 

Conclusion: No category - Not requiring classification to UN GHS or EU CLP.