Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-July 2018 - 04-July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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
Specific details on test material used for the study:
Batch no: 12/18

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
750 mg
Duration of treatment / exposure:
4 hours ± 5 minutes incubation at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
90 minutes at 32 ± 1°C
Number of animals or in vitro replicates:
3 corneas for the test item
3 corneas as negative controls
3 corneas as positive controls
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The assay uses isolated corneas obtained äs a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany

QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were carefully examined for defects and any defective eyes were discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
3 corneas as negative controls treated with physiological saline 0.9% NaCI

POSITIVE CONTROL USED
3 corneas as positive controls treated with imidazole 20% in physioloaical saline 0.9% NaCI

APPLICATION DOSE AND EXPOSURE TIME
750 mg of the test item (open-chamber method) or 750 µL of the control substance

TREATMENT METHOD: Closed chamber

POST EXPOSURE PERIOD: 4 hours ± 5 minutes incubation at 32 ± 1 °C

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Washed at least 3 times with MEM (containing phenol red).

POST-INCUBATION PERIOD: 90 minutes at 32 ± 1 °C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry optical density at 490 nm

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

DECISION CRITERIA: The IVIS cut-off values for identifying test substances as inducing serious eye damage UN GHS Category 1) and test substances not requiring classification for eye Irritation or serious eye damage (UN GHS No Category) was used (see table 1 in materials and methods)

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
4.17
Negative controls validity:
valid
Positive controls validity:
valid

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

0.69

1.02

0.34

 

2

1.06

0.95

-0.10

 

3

1.50

1.33

-0.17

 

MV

1.08

1.10

0.02

 

4

Positive control

2.03

71.26

69.23

69.21

5

1.96

82.72

80.76

80.74

6

           2.42

65.05

62.62

62.60

MV

2.13

73.01

70.87

70.85

7

Test item

-2.07

0.92

2.99

2.97

8

-1.48

2.57

4.05

4.03

9

1.57

7.13

5.46

5.44

MV

-0.63

3.54

4.17

4.15

MV = mean value

 

Table 2: Permeability results:

Cornea No.

Test item

OD490

Corrected OD490 value

1

Negative control

0.004

 

2

0.001

 

3

0.010

 

MV

0.005

 

4

Positive control

1.132

1.127

5

0.912

0.907

6

1.374

1.369

MV

1.139

1.134

7

Test item

0.013

0.008

8

0.004

-0.001

9

0.002

-0.003

MV

0.006

0.001

MV = mean value

 

Table 3: In vitro irritation score

Cornea No.

Test item

Corrected opacity value

Corrected OD490 value

IVIS

1

Negative control

0.34

0.004

 

2

-0.10

0.001

 

3

-0.17

0.010

 

MV

0.02

0.005

0.10

4

Positive control

69.21

1.127

 

5

80.74

0.907

 

6

62.60

1.369

 

MV

70.85

1.134

87.87

7

Test item

2.97

0.008

 

8

4.03

-0.001

 

9

5.44

-0.003

 

MV

4.15

0.001

4.17

MV = mean value

 

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
No prediction can be made regarding the classification of the test substance 4-Aminobenzoyl-b-alanine according to the evaluation criteria. However, it can be concluded that 4-aminobenzoyl-b-alanine is not corrosive to eyes.