Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 November 2018 - 13 February 2019
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 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)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Test material: Balsalazide acid
Batch No.: 0400
Description: solid, deep orange, crystalline powder
Storage conditions: Room temperature

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
- Tissue source: The assay used isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL of the test item preparation or the control substance
- Concentration: The test item was suspended with physiological saline 0.9% NaCI to give a 20% concentration.
Duration of treatment / exposure:
4 hours ± 5 minutes incubation at 32 ± °C
Duration of post- treatment incubation (in vitro):
90 minutes at 32 ± 1°C
Number of animals or in vitro replicates:
Three corneas were tested for the test item, negative control and positive control.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS.

QUALITY CHECK OF THE ISOLATED CORNEAS
Before the corneas were mounted in corneal holders with the endothelial side against the 0-ring of the posterior chamber, they were visually examined for defects and any defective cornea were discarded.

NUMBER OF REPLICATES
Three replicates per test substance or control.

NEGATIVE CONTROL USED
Physiological saline 0.9% NaCI

POSITIVE CONTROL USED
Imidazole 20% in physiological saline 0.9% NaCI

APPLICATION DOSE AND EXPOSURE TIME
750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed.

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
The epithelium was washed at least three times with MEM (minimum essential medium) (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (Roswell Park Memorial Institute) 1640 medium (without phenol red).

POST-EXPOSURE INCUBATION: The posterior chamber was refilled with fresh complete RPMI 1640 medium. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1°C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity : The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)
- Others: After the initial incubation period, each cornea was observed visually and pertinent observations were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA: As indicated in the guideline

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
0.27
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The mean in vitro irritation score for Balsalazide acid was 0.27.

OTHER EFFECTS:
- Visible damage on test system: All 3 corneas treated with Balsalazide acid showed a slight yellowish discolouration of the edges of the tissue.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control responses in the 2nd experiment resulted in opacity and permeability values that were less than the established upper limits for background bovine corneas treated with the respective negative control.
- Acceptance criteria met for positive control: In the 2nd experiment the in vitro Irritation score obtained with the positive ontrol fell within the two Standard deviations of the current historical mean and therefore this assay was considered to be valid.
- Range of historical values if different from the ones specified in the test guideline: Mean IVIS value (MV) = 122.15; Standard deviation (SD) = 18.00; MV-2xSD = 86.16; MV+2xSD = 158.14 (number of measurements = 40)

Due to the fact that the IVIS of the positive control did not fall within the two Standard deviations of the current historical mean in the 1st experiment, this experiment was considered to be not valid according to the study acceptance criteria stated in the guideline. Therefore, the experiment was repeated and all data reported here refers to the second experiment.

Any other information on results incl. tables

Table 1: Opacity results

Cornea No.

Test item

Initial opacity

Final opacity

Change of opacity value

Corrected opacity value

1

Negative control

2.21

3.16

0.95

 

2

2.06

2.35

0.29

 

3

2.31

2.68

0.36

 

MV

2.19

2.73

0.53

 

4

Positive control

2.97

88.54

85.56

85.03

5

3.16

99.17

96.01

95.48

6

2.79

90.25

87.46

86.93

MV

2.97

92.65

89.68

89.15

7

Test item

0.79

1.40

0.61

0.08

8

2.71

3.50

0.79

0.25

9

0.65

1.67

1.02

0.49

MV

1.38

2.19

0.81

0.27

MV = mean value

 

Table 2: Permeability results:

Cornea No.

Test item

OD490

Corrected OD490 value

1

Negative control

0.009

 

2

0.014

 

3

0.023

 

MV

0.015

 

4

Positive control

1.378

1.363

5

2.000

1.985

6

2.185

2.170

MV

1.854

1.839

7

Test item

0.010

-0.005

8

0.027

0.012

9

0.009

-0.006

MV

0.015

0.000

MV = mean value

 

Table 3: In vitro irritation score

Cornea No.

Test item

Corrected opacity value

Corrected OD490 value

IVIS

1

Negative control

0.95

0.009

 

2

0.29

0.014

 

3

0.36

0.023

 

MV

0.53

0.015

0.76

4

Positive control

85.03

1.363

 

5

95.48

1.985

 

6

86.93

2.170

 

MV

89.15

1.839

116.73

7

Test item

0.08

-0.005

 

8

0.25

0.012

 

9

0.49

-0.006

 

MV

0.27

0.000

0.27

MV = mean value

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
According to the evaluation criteria of the test guideline, the test item, Balsalazide acid, does not require classification as corrosive to eyes or as an eye irritant.