AVITERA ROSE SE

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

Bovine Corneal Opacity and Permeability (BCOP) Test Method OECD Guideline 437

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 July 2021 to 11 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification / Name: FAT 40879/B TE
Batch Number: BOP 05-20 (MC- 0065114700/800 1:1)
Appearance: Dark red powder
Degree of Purity (% (w/w)): 80.5 % all colored organic constituents; main constituent 66:9 %
Manufacture Date: June 26, 2020
Expiry Date: September 08, 2025
Storage conditions: Freezer (-15 to -20 °C)

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughtered house
- Number of animals: 10
- Characteristics of donor animals (e.g. age, sex, weight): 12 to 60 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):HBSS Buffer with Pen-Strep antibiotic in cold condition (2-8 °C)
- Time interval prior to initiating testing: NA
- Indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: NO
- Selection and preparation of corneas: The eyes arrived at the laboratory were carefully examined for defects of including increased opacity, scratches and neovascularization. The opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period were discarded. Three corneas were used for each group.

Quality check of the isolated corneas: The eyes arrived at the laboratory were carefully examined for defects of including increased opacity, scratches and neovascularization.

Test system

Controls:

yes

yes, concurrent positive control

Amount / concentration applied:

20 % w/v in 0.9 % sodium chloride solution

Duration of treatment / exposure:

4 hour’s incubation at 32 °C

Duration of post- treatment incubation (in vitro):

32 °C for 90 minutes in an incubator

Number of animals or in vitro replicates:

03 Replicates

Details on study design:

NUMBER OF REPLICATES: 03
NEGATIVE CONTROL USED: Normal saline
POSITIVE CONTROL USED: Imidazole
APPLICATION DOSE AND EXPOSURE TIME:a) 750 µL of (20 % w/v) 4 hours
TREATMENT METHOD: [closed chamber / open chamber]: open chamber method

POST-INCUBATION PERIOD: yes/no. If YES please specify duration: Solid Test item Not Required

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 times
- POST-EXPOSURE INCUBATION:90 minutes
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The mean opacity value of negative control, test item (FAT 40879/B) and positive control was found to be 2.00, 122.00 and 48.33, respectively.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490): Permeability: The mean Optical Density (OD) of negative control, test item (FAT 40879/B) and positive control was found to be -0.0003, 0.0713 and 1.3730, respectively.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

after 4 h

Value:

123.07

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Any other information on results incl. tables

Opacimeter readings values for each cornea: Negative control after 4 hour

OPACITOMETER
Replicate	BK	NC 4^thhour reading	Corrected Readings	Mean (Corrected Readings)
1	CH-01	CH-01	CH-01	2.00
	124	122	2
2	CH-02	CH-02	CH-02
	138	136	2
3	CH-03	CH-03	CH-03
	127	125	2

Opacimeter readings values for each cornea: Test item after 4 hour

OPACITOMETER
Replicate	BK	Test Item 4^thhour reading	Corrected Readings	Mean (Corrected Readings)
1	CH-04	CH-04	CH-04	122
	133	7	126
2	CH-05	CH-05	CH-05
	125	5	120
3	CH-06	CH-06	CH-06
	128	8	120

      

Opacimeter readings values for each cornea: Positive control after 4 hour

OPACITOMETER
Replicate	BK	PC 4^thhour reading	Corrected Readings	Mean
1	CH-07	CH-07	CH-07	48.33
	132	71	61
2	CH-08	CH-08	CH-08
	131	90	41
3	CH-09	CH-09	CH-09
	127	84	43

Key: BK = Blank; PC = Positive control; CH = Cornea Holder, OD = Optical Density

Permeability readings (OD) values for each cornea – Negative control

SPECTROPHOTOMETER
Replicate	NC	Mean
Rep1	CH-01	0.0003
	-0.0020
Rep2	CH-02
	0.0010
Rep3	CH-03
	0.0000

Key: NC = Negative control; CH = Cornea Holder

IVIS= Mean Opacity Value (NC) + (15 X Mean Permeability OD₄₉₀Value NC)

In vitro Irritancy Score (IVIS) = 2 + (15*-0.0003) = 1.995

Permeability readings (OD) values for each cornea – Test Item

SPECTROPHOTOMETER
Replicate	Test Item	Corrected OD	Mean (OD)
1	CH-04	CH-04	0.0713
	0.0780	0.0783
2	CH-05	CH-05
	0.0700	0.0703
3	CH-06	CH-06
	0.0650	0.0653

Key: CH = Cornea Holder

IVIS= Mean Opacity Value + (15 X Mean Permeability OD₄₉₀Value)

In vitro Irritancy Score (IVIS) = 122.00 + (15*0.0713)=123.07

Permeability readings (OD) values for each cornea – Positive control

SPECTROPHOTOMETER
Replicate	PC	Corrected OD	Mean (OD)
1	CH-07	CH-07	1.3730
	1.3740	1.3743
2	CH-08	CH-08
	1.3720	1.3723
3	CH-09	CH-09
	1.3720	1.3723

Key: CH = Cornea Holder, PC = Positive Control, OD = Optical Density

IVIS= Mean Opacity Value + (15 X Mean Permeability OD₄₉₀Value)

In vitro Irritancy Score (IVIS) = 48.33 + (15*1.3730) = 68.928

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

FAT 40879/B is considered as irritating to eyes.

Executive summary:

In a GLP-compliant study FAT 40879/B was evaluated in the Bovine Corneal Opacity and Permeability (BCOP) Test carried out according to OECD Guideline 437 and EU Method B.47. Cornea which were free of any defects were used in the experiment. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm rim of sclera and dissected carefully without causing any damage to corneal epithelium and endothelium. Isolated each cornea were mounted in a clean corneal holder that consists of anterior and posterior compartments. Corneal epithelial side interface was towards the anterior side and endothelial side was towards the posterior side of the chamber. Both chambers were filled with pre-warmed phenol red free complete Minimum Essential Medium (cMEM). To achieve normal metabolic activity, the corneal holders were incubated for 1 hour at 32 ^oC ± 1 ^oC with the medium. Post incubation, the medium was changed with fresh pre-warmed phenol red free complete cMEM in both chambers and the baseline opacity for each cornea was recorded. FAT 40879/B (20 % w/v), negative control and positive controlwas instilled into the anterior compartment of the cornea holder. Three corneas for each group was treated with FAT 40879/B (20 % w/v), negative control (NC) and positive control, respectively, and incubated at 32 ^oC ± 1 ^oC for 4 hours ±5 minutes. Post exposure to the test item, negative control and positive control, the cornea(s) were removed from the respective holder and the epithelium was washed (3 times) with pre-warmed MEM with phenol red. Post three washes the cornea(s) were finally washed with MEM without phenol red. The anterior and posterior compartment(s) of the cornea holders were refilled with fresh pre-warmed MEM without phenol red. The compartments were plugged and the opacity was assessed using an opacitometer. For the determination of permeability the MEM medium from the anterior compartment was removed and replaced with 1 mL of a 5 mg/mL sodium fluorescein solution and incubated at 32 ^oC ± 1 ^oC for 90 ± 5 minutes in an incubator. Post incubation the medium was removed from the posterior chamber and transferred into labelled tubes. Permeability determinations were recorded using a spectrophotometer at 490 nm.The mean opacity value of negative control, test item and positive control was found to be 2, 123.07 and 68.33, respectively. While the mean Optical Density (OD) of negative control, test item and positive control was found to be -0.0003, 0.0713 and 1.3730,  respectively. The test system (Bovine Corneas) showed response to the positive control used in this study which in turn validates the sensitivity of the test system employed and validity of the study being performed. Based on the results, FAT 40879/B was considered as irritating to eyes and classified under Category- I as per UN Globally Harmonised System (GHS) and OECD Guideline for the testing of Chemicals.