Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 October 2018 (Study Initiation) to 16 January 2019 (Study Completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Nitika Pharmaceutical Specialities Pvt. Ltd.
- Lot/batch No.of test material: IRST7H112A
- Expiration date of the lot/batch: May 2023
- Purity test (release) date: 18 June 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (ambient), container kept tightly closed in a dry, cool and well ventilated place
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: The test item formed a homogenous suspension in corn oil and was tested at 20% (w/v) in corn oil. The test item was insoluble in normal (physiological) saline.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Formulated as a homogenous suspension in corn oil at 20% (w/v).

FORM AS APPLIED IN THE TEST (if different from that of starting material): The test item was formulated as a homogenous suspension in corn oil and tested at a concentration of 20% (w/v) in corn oil.

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: At the end of exposure period, the test item, positive, negative and vehicle controls were removed from the anterior chamber and the corneal epithelium washed until no visual evidence of test item was observed using EMEM (containing phenol red). pH and osmolality are not specified.

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Deonar Abattoir slaughter house, Mumbai, Maharashtra, India
- Age at study initiation: Between 1 to 5 years (animal age was determined based on the teeth count and horn ring count in addition to the Horizontal Diameter of corneas and central corneal thickness)
- Weight at study initiation: Not specified
- Housing: Transported (in a sealed plastic container) under cold conditions in Hanks’ Balanced Salt Solution containing antibiotics [e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL]. Eyes were used within 24 hours of slaughter after confirmation each was free from defects following pre-test procedures. Corneas that had an opacity less than seven opacity units or equivalent for the opacitometer were used in the study.

- Acclimation period: As part of the pre-test procedure, the corneal holder was equilibrated at 32 ± 1°C for at least one hour to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible.

Test system

Vehicle:
other: Corn oil
Controls:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume with unit): 750 µL
- Concentration (if solution): In suspension at 20% (w/v) in corn oil.

VEHICLE
- Amount(s) applied (volume with unit): 750 µL Corn oil
- Lot/batch no. (if required): MKCC0462
- Purity: Not specified
Duration of treatment / exposure:
Corneas were exposed for 4 h ± 5 minutes at 32 ± 1 °C.4h ± 5 min
Duration of post- treatment incubation (in vitro):
The holders were incubated in a horizontal position for 90 ± 5 min at 32 ± 1 ºC. After incubation the medium in the posterior chamber was transferred into labelled tubes.
Number of animals or in vitro replicates:
Four sets, of three corneas were tested.
Set 1: Control: normal (physiological) saline.
Set 2: Positive control: 20% (w/v) imidazole in normal (physiological) saline.
Set 3: Vehicle: Corn oil
Set 4: Test item: Fatty acids, C16-18 (even numbered), iron(III) salts (in suspension) at 20% (w/v) in corn oil.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: Eyes were used within 24 hours of slaughter. Eyes were examined prior to use and only corneas free from any visible defects were used. Corneas that had an opacity of less than seven opacity units or equivalent for the opacitometer were used.

QUALITY CHECK OF THE ISOLATED CORNEAS: A baseline reading for each cornea holder was taken with the medium prior to loading the cornea onto the cornea holder. Corneas, free from defects, were dissected so they had a 2 to 3 mm rim of sclera and were transferred to a container containing Hanks’ Balanced Salt Solution containing antibiotics [e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL].

Selected corneas were mounted on corneal holders with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was then placed on the top of the cornea and fixed in place. Both chambers were then filled to excess with pre-warmed phenol red free Eagle's Minimum Essential Medium (EMEM) (the posterior chamber filled first to allow the cornea to return to its natural concave position), ensuring no bubbles were present. The device was then equilibrated at 32 ± 1°C for at least one hour to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible.

Following equilibration, the medium was removed from both chambers and fresh pre-warmed phenol red free EMEM added to both chambers. A baseline opacity reading was then taken for each cornea.

NUMBER OF REPLICATES: Four sets, of three corneas were tested.

NEGATIVE CONTROL USED : 750 μL normal (physiological) saline

VEHICLE CONTROL USED: 750 μL corn oil

POSITIVE CONTROL USED : 750 μL 20% (w/v) imidazole in normal (physiological) saline.

APPLICATION DOSE AND EXPOSURE TIME
750 μL of Fatty acids, C16-18 (even numbered), iron(III) salts (in suspension) at 20% (w/v) in corn oil. Corneas were exposed for 4 h ± 5 minutes at 32 ± 1 °C.
The same volume was used for corneal administration for the control (normal (physiological) saline), positive control (20% (w/v) imidazole in normal (physiological) saline), and the vehicle (corn oil).

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: yes, 90 ± 5 min at 32 ± 1 ºC.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of exposure period, the test item, positive and negative controls were removed from the anterior chamber and the corneal epithelium washed until no visual evidence of test item was observed using EMEM (containing phenol red). Once the medium was free of test item, the corneas were given a final rinse with EMEM (without phenol red).

- POST-EXPOSURE INCUBATION: The holders were incubated in a horizontal position for 90 ± 5 min at 32 ± 1 ºC. After incubation the medium in the posterior chamber was transferred into labelled tubes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity is measured quantitatively, as the amount of light transmission through the cornea.
Mean Opacity value =
Step 1. Step 1 value of section 2.11.1 – post treatment opacity reading (calculated for each cornea)
Step 2. Calculate the mean of the control group
Step 3. {[Step 1 value of each cornea (except control) – Step 2 value] – b}/a
Step 4. Calculate Mean of Step 3 value
Where b = 0.9894 and a = 0.0251 (a and b values are empirically derived for the instrument)

- Corneal permeability: Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. The passage of sodium fluorescein dye is measured with the aid of a plate reader set to read at OD490.

- Others (e.g, pertinent visual observations, histopathology): None

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In vitro irritancy score (IVIS) was calculated for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The in vitro irritancy score was calculated for each individual treatment.

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. Yes
The IVIS cut-off values for identifying a test item as inducing serious eye damage (UN GHS Category 1) and a test item not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given below:

IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
1 (Control) Normal (physiological) Saline
Value:
1.91
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
1 (Positive control) Imidazole at 20% (w/v) in normal (physiological) saline
Value:
135.92
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
1 (Vehicle) Corn oil
Value:
2.73
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
1 (Test item) Fatty acids, C16-18 (even numbered), iron(III) salts
Value:
1.3
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None

DEMONSTRATION OF TECHNICAL PROFICIENCY: The study was performed by a GLP laboratory competant in this OECD 437 regulatory method with sufficient historical data to validate observed effects.

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: Not applicable

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The IVIS score for corneas treated with 750 μL Fatty acids, C16-18 (even numbered), iron(III) salts (in suspension) at 20% (w/v) in corn oil was 1.30.

Based on results of this study, the classification for Fatty acids, C16-18 (even numbered), iron(III) salts is as follows: Classification (OECD 437 UN GHS): No Category