Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study Initiation date : September 11, 2017 and Study Completion date : November 22, 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:
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Dark blue solid particulate/powder

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Test System : Isolated Cornea from the eyes of freshly slaughtered cattles
Source : Deonar Abattoir slaughter house, Mumbai, Maharashtra, India
Bovine Age : Between 1 to 5 years (age of the animals was determined based on the teeth count and horn ring count)
Transportation Condition : Transported under cold condition in Hanks’ Balanced Salt Solution containing antibiotics [e.g., penicillin at 100 IU/mL and
streptomycin at 100 μg/mL]

Test system

Vehicle:
other: corn oil
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Not applicable
- Concentration (if solution): 750 μL 20% w/v in corn oil

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): Not applicable
- Lot/batch no. (if required): MKBZ9899V
- Purity: Not applicable
Duration of treatment / exposure:
4 h
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
Not applicable
Number of animals or in vitro replicates:
3 in vitro replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS : Corneas that have opacity lesser than seven opacity
units or equivalent for the opacitometer were used in the study. Corneas, free from defects, were dissected to a 2 to 3 mm rim

QUALITY CHECK OF THE ISOLATED CORNEAS : Eyes were examined prior to use. Corneas from eyes free of visible defects were used. Corneas that have opacity lesser than seven opacity units or equivalent for the opacitometer were used in the study.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Normal saline

SOLVENT CONTROL USED (if applicable): Corn oil

POSITIVE CONTROL USED: Imidazole

APPLICATION DOSE AND EXPOSURE TIME
One set of corneas were treated with 750 μL of the prepared test item [20% (w/v) concentration in corn oil]. One set of corneas served as positive control [750 μL of 20% (w/v) concentration in normal saline] and two set of corneas served as negative controls and were treated with 750 μL of normal saline and corn oil, respectively.
Post application of the test item, the cornea holders were turned to a horizontal position and slightly rotated to ensure uniform covering of the test item over the cornea.
Corneas were exposed for 4 h ± 5 minutes at 32 ± 1 °C.

TREATMENT METHOD: [closed chamber ]

POST-INCUBATION PERIOD: yes/no. : Not applicable

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Not specified
- POST-EXPOSURE INCUBATION: Not applicable

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer BASF-OP3.0 : Duratec, Germany
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
- Others (e.g, pertinent visual observations, histopathology): Not applicable

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Classification (OECD 437)

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
The IVIS score for the corneas treated with 750 μL the test substance (suspension) at 20% (w/v) concentration in corn oil
Value:
7.67
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No prediction can be made
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: Yes

Applicant's summary and conclusion

Interpretation of results:
other: No prediction can be made
Conclusions:
The IVIS score for the corneas treated with 750 μL the test substance (suspension) at 20% (w/v) concentration in corn oil were found to be 7.67.
Based on the results of this study, an indication of the classification for the test substance is : No prediction can be made
Classification (OECD 437)
Executive summary:

This study was conducted to evaluate the ocular irritancy of the test substancein compliance with the OECD test guideline for BOVINE CORNEAL OPACITY AND PERMEABILITY (BCOP) Test. In BCOP test, four sets each consisting of three corneas were tested. First set served as control and was treated with 750 μL normal saline. The second set served as positive control and was treated with 750 μL 20% (w/v) imidazole in normal saline. Test item was found to be insoluble and did not form a homogenous suspension in normal saline. Test item formed a homogenous suspension in corn oil, so the same was selected as vehicle for the treatment. One set was treated with 750 μL corn oil and served as vehicle control while one set was treated with 750 μLthe test substance(suspension) at 20% (w/v) concentration in corn oil. Post application the corneas were incubated for approximately 4 h at 32 ºC. At the end of the incubation period reading for opacity were taken.Post opacity reading permeability was measured by applying 1 mL of fluorescein sodium solution (5 mg/mL) on to the anterior surface of the cornea and was incubated for approximately 90 min at 32 ºC. At the end of the incubation period OD was measured at 490 nm for the fluid collected from the posterior chamber.                                                                                           

The meanIn-VitroIrritancy Score (IVIS) of normal saline, corn oil and 750 μL 20% (w/v) imidazole in normal saline (positive control) treated corneas were found to be 0.56, 0.39 and 135.86, respectively.

The IVIS score for the corneas treated with 750 μLthe test substance(suspension) at 20% (w/v)concentration in corn oil was found to be 7.67.

Based on the results of this study, an indication of the classification forthe test substanceis: No prediction can be made