[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-07-04 to 2017-12-19

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:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: flakes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Clariant Plastics and Coatings (Deutschland) GmbH, BU Additives

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Local abattoir (Slaughter house), Near Frazer town, Bengaluru.

IN-LIFE DATES: From: 04 July 2017 To: 06 July 2017

Test system

Vehicle:

other: 0.9% w/v Sodium chloride

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The neat test item Licocare RBW 300 FL TP (enough quantity to completely cover the cornea) grounded to fine powder was applied.

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 mL of negative control (i.e. 0.9% NaCl w/v) was applied to each designated cornea.
- Batch no. (if required): C263586

Duration of treatment / exposure:

4 hours exposure

Duration of post- treatment incubation (in vitro):

After final opacity measurement, EMEM medium was removed and replaced by fluorescein solution. After 91 minutes, the fluid from posterior chamber was transferred into labeled glasstubes for further investigations.

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- Procurement of corneas: The bovine eyes were procured from local abattoir and transported in a box containing Hank’s Balanced Salt Solution (HBSS) and penicillin-streptomycin (100 IU & 100 µg/mL) in an ice box. On day 1 of experiment 20 numbers and on day 2 of experiment 30 numbers of corneas were procured.

- Dissection: On both days of experiment, post pretest examination, eyes were dissected to isolate the corneas from surrounding tissue and then placed in a container of fresh HBSS (Hank Balanced Salt Solution).

- Mounting: The isolated corneas were mounted in the cornea holder, which separated holder into anterior and posterior chambers. During mounting, it was ensured that the epithelium of the cornea projected into the anterior chamber. The EMEM medium was added in to both the chambers and kept in an incubator (32°C) for 1 to 2 hours.

QUALITY CHECK OF THE ISOLATED CORNEAS
- Pretest examination: On both days of experiment, immediately after receiving the eyes in the lab, all eyes were observed for any evidence of vascularization, pigmentation, opacity or scratches.
- Initial opacity: Corneas exhibited initial opacity < 7 were considered for experiment

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% w/v Sodium Chloride (NaCl)

POSITIVE CONTROL USED: Dibenzoyl-L-tartaric acid

APPLICATION DOSE AND EXPOSURE TIME: Enough of the neat test item (grounded to powder) to cover the cornea; 0.75 mL of the positive control

TREATMENT METHOD: open chamber; 4 hour exposure

POST-INCUBATION PERIOD: yes. If YES please specify duration: 91 minutes with 1 mL of fluorescein solution (500 mg of Sodium fluorescein was weighed and added to 100
mL of Dulbecco’s Phosphate-Buffered Saline to obtain 5mg/mL concentration)

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Using OP3.0 Opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [microtiter plate reader] (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS): IVIS = Mean Opacity Score + (15 × Mean Optical Density Score)

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

IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1
Test item of IVIS score, ≤ 3 would be considered as UN GHS No Category.
Test item of IVIS score, >3 and ≤ 55 would be considered as “No prediction can be made”, subsequently testing with any other adequate method remains at the discretion of the sponsor.
Test item of IVIS score, > 55 would be considered as severe irritant causing serious eye damage and classified as Category 1.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

The corneas treated with Licocare RBW 300 FL TP exhibited IVIS score as 0.1 which is classified as “No Catagory”.

Value:

<= 3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative control/mean

Value:

2.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

positive control/ mean

Value:

187.5

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

Test item /mean

Value:

0.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

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:

The test item Licocare RBW 300 FL TP, negative control 0.9% w/v NaCl and positive control 20% w/v Dibenzoyl-L-Tartaric acid were tested for their occular irritancy score using bovine corneas as test system in Bovine Corneal Opacity and Permeability assay (3 corneas per compound) using Opacitometer and Microplate reader/Spectrophotometer to measure opacity and permeability respectively.
All the test groups were tested in triplicates. The mean of all three corneas treated with test item Licocare RBW 300 FL TP exhibited mean opacity -1.0 and mean permeability 0.0716. Under similar conditions, the positive control (20% w/v Dibenzoyl-L-Tartaric acid) exhibited a mean opacity score of 179.6 and mean permeability 0.5279. Whereas negative control (0.9% w/v NaCl) exhibited a mean opacity score 0.7 and mean permeability 0.1423, confirming the sensitivity of the test system to occular irritancy.
The IVIS for Licocare RBW 300 FL TP (Solid) was found to be 0.1 while the positive control 20% w/v Dibenzoyl-L-Tartaric acid scored 187.5. The negative control, 0.9% w/v NaCl scored 2.8.
Based on the In Vitro Irritancy Score (IVIS), it is concluded that the test item Licocare RBW 300 FL TP (Solid) is an occular non-irritant classified under ‘No Category’ as per UN GHS. Whereas the positive control proved to be severe/corossive eye irritant classified under ‘Category I’. The negative control is classified as ‘No Category’ based on its IVIS.

Executive summary:

The occular irritancy of the test item “Licocare RBW 300 FL TP (Solid)” was carried out using Bovine Corneal Opacity and Permeability assay. The Licocare RBW 300 FL TP was applied directly (neat) to the corneas by means of open chamber method. The positive control i.e. Dibenzoyl-L-Tartaric Acid was applied to the corneas as 20% w/v in 0.9% NaCl solution by open chamber method whereas negative control i.e. 0.9% w/v Sodium Chloride solution was applied to the corneas by means of closed chamber method. The control and treated corneas (n=3)were subjected to the opacity and permeability testing. 

All the test groups were tested in triplicates. The IVIS for Licocare RBW 300 FL TP (Solid) was found to be 0.1 while the positive control 20% w/v Dibenzoyl-L-Tartaric acid scored 187.5. The negative control, 0.9% w/v NaCl scored 2.8, confirming the sensitivity of the test system to occular irritancy.

Based on theIn VitroIrritancy Score (IVIS), it is concluded that the test item Licocare RBW 300 FL TP (Solid) is an occular non-irritant classified under ‘No Category’ as per UN GHS. Whereas the positive control proved to be severe/corossive eye irritant classified under ‘Category I’. Similarly, ‘No Category’ for negative control based on its IVIS.