Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-18 to 2016-08-18
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 guidelineopen allclose all
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
Remarks:
No deviations from the study plan were observed.
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
Remarks:
No deviations from the study plan were observed.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (20 ± 5°C), Keep away from humidity
- Stability under test conditions: H2O: unstable; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
- Solubility and stability of the test substance in the solvent/vehicle: Test material was tested directly, without dilution or preparation of a solution.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : white powder

Test animals / tissue source

Species:
cattle
Strain:
other: Bos primigenius Taurus
Remarks:
fresh bovine corneas
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH (Enzstr. 2-4, 75217 Birkenfeld, Germany)
- Age at study initiation: cattle were between 12 and 60 months old.
- Transportation: The eyes were transported to the test facility in Hank’s balanced salt solution (supplemented with 0.01% streptomycin and 0.01% penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 h.

Test system

Vehicle:
unchanged (no vehicle)
Remarks:
Test material tested directly, without dilution or preparation of a solution.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Tissue 1: 98.7 mg
Tissue 2: 106.0
Tissue 3: 99.6
Duration of treatment / exposure:
Exposure time on the corneas was 3 hours and 55 minutes at 32 ± 1°C
Duration of post- treatment incubation (in vitro):
85 min at 32 ± 1°C
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1°C. On the day of the assay, the Minimum Essential Medium (MEM) without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1°C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the arrival of the corneas, they were examined and only corneas which were free from damage were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1°C.


NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
HBSS- solution: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10), added Penicillin 100 IU/mL-Streptomycin 100µg/mL- solution (1%), batch no.: 20160818

POSITIVE CONTROL USED
Imidazole solution: 20% C3H4N2 (CAS-No. 288-32-4), dissolved in HBSS-solution., batch no.: 20160818

APPLICATION DOSE AND EXPOSURE TIME
The following amounts of the test item were tested neat and applied directly on the cornea using a weighing funnel:
1: 98.7 mg
2: 106.0 mg
3: 99.6 mg

Exposure time on the corneas was 3 hours and 55 minutes at 32 ± 1°C.

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: yes (85 min at 32 ± 1°C)

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2 (After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded. The cMEM without phenol red was then removed from the front chamber, and 1 mL sodium fluorescein solution (concentration: 5 mg/mL) was added to the front chamber.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change of opacity value of each treated cornea with the test material, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea. The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test material and positive control to obtain a corrected opacity.

Opacity = ((I0 / I) – b) / a)

Where:
a = 0.0251 and b = 0.9894 being Opacitometer-specific empirically determined variables
I0 = the empirically determined illuminance through a cornea holder with windows and medium, here: Io= 1063.9
I = the measured illuminance (unit: LUX)

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)
The corrected OD492 value of each cornea treated with test item and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea. The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The IVIS of each replicate of the negative control was calculated from the following equation: IVIS = opacity difference + (15 x corrected OD 492 ± 2 value).

The IVIS of each replicate of the positive control and of the test item were calculated from the following equation: IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD 492 ± 2 – mean OD 492 ± 2 of the negative control)]

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

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Test Material Mean IVIS
Value:
ca. 27.41
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Values for negative controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.
- Acceptance criteria met for positive control: Values for positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.
- Range of historical values if different from the ones specified in the test guideline: No

Any other information on results incl. tables

Table 2. Illuminance Values

Parameter

Negative Control

Test Item

Positive Control

(I) Measured

values before

exposition

1020

961

1001

1050

1017

1012

1020

978

1006

(I) Measured

values after

exposition

966

907

948

704

597

679

397

330

370

 

Table 3. Opacity Values

Parameter

Negative Control

Test Item

Positive Control

Opacity before exposition

2.14

4.69

2.93

0.95

2.26

2.47

2.14

3.92

2.72

Opacity after exposition

4.46

7.31

5.29

20.79

31.58

23.01

67.35

89.03

75.14

Opacity Difference

2.32

2.63

2.37

19.84

29.32

20.54

65.21

85.10

72.42

Mean Opacity Difference

2.44

-

-

Opacity Difference

corrected

-

17.40

26.88

18.10

62.77

82.67

69.99

 

Table 5. Optical density at 492 nm

Parameter

Negative Control

Test Item

Positive Control

1. Measurement

0.073

0.049

0.052

0.639

0.480

0.374

0.462

0.530

0.860

2. Measurement

0.073

0.045

0.056

0.644

0.478

0.371

0.461

0.531

0.865

3. Measurement

0.075

0.044

0.049

0.643

0.479

0.377

0.467

0.531

0.850

1. Measurement - Blank

0.0393

0.0153

0.0183

0.6053

0.4463

0.3403

0.4283

0.4963

0.8263

2. Measurement - Blank

0.0393

0.0113

0.0223

0.6103

0.4443

0.3373

0.4273

0.4973

0.8313

3. Measurement - Blank

0.0413

0.0103

0.0153

0.6093

0.4453

0.3433

0.4333

0.4973

0.8163

Mean of each

replicate

0.0400

0.0123

0.0187

0.6083

0.4453

0.3403

0.4297

0.4970

0.8247

Mean of the

three replicates

0.0237

-

-

Corrected

-

0.5847

0.4217

0.3167

2.1247*

2.4613*

4.0997*

* Note: The three values for the positive control were obtained by measurement of a fivefold diluted

solution and multiplication of the absorbances with factor 5.

 

Table 6. IVIS Values

Test Group

IVIS

Mean IVIS

Relative Standard

Deviation IVIS

Negative Control

HBSS-solution

2.92

2.79

4.96%

2.81

2.65

 

Test Item

INTERCURE®1

26.17

27.41

19.29%

33.21

22.85

 

Positive Control

20% imidazole solution

94.64

115.24

16.31%

119.59

131.48

Applicant's summary and conclusion

Interpretation of results:
other: Category 2 (irritating to eyes) based on CLP criteria
Conclusions:
The calculated IVIS for the test material was 27.41. Under the conditions of this study, the test material showed effects on the cornea of the bovine eye in vitro but does not meet the GHS classification criteria for eye damage.
Executive summary:

A key Guideline (OECD 437) in vitro eye irritation/corrosion study was performed to assess the potential of the test material (INTERCURE®1) to cause corneal damage by quantitatively measuring changes in opacity and permeability in bovine cornea post exposure.

 

The test material was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1°C for 1 hour and whose opacity had been measured. The test material incubated on the cornea for 3 hours and 55 minutes at 32 ± 1°C. After removal of the test material, opacity and permeability values were measured. HBSS-solution was used as the negative control while 20% Imidazole solution was used as the positive control in this study.

 

The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) was 2.79. The positive control induced serious eye damage on the cornea and was within two standard deviations of the historical mean. The calculated IVIS for the positive control was 115.24. The calculated IVIS for the test material was 27.41.

 

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in a UN GHS Category for eye damage. Therefore, under the conditions of this study, the test material showed effects on the cornea of the bovine eye in vitro but does not meet the GHS classification criteria for eye damage.