Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-03 to 2018-01-03
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)
Version / remarks:
adopted 09 October 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Number of animals: not applicable
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): transported in HBSS + Pen/Strep on ice
- Time interval prior to initiating testing: 1h at 32 ± 1°C

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% solution in physiological saline

VEHICLE
- Concentration (if solution): 0.9% NaCl solution
- Lot/batch no. (if required): 17296405
Duration of treatment / exposure:
4h ± 5 min
Observation period (in vivo):
90 min
Number of animals or in vitro replicates:
3 corneae per treatment
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
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. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded.

NUMBER OF REPLICATES
3 corneae for the test item
3 corneae as negative controls treated with physiological saline 0.9% NaCl
3 corneae as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl


APPLICATION DOSE AND EXPOSURE TIME
750 µl of the test item preparation or the control substance was introduced into the anterior chamber

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes, 90 min

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times with MEM containing phenol red and once rinsed with RPMI without phenol red.

- POST-EXPOSURE INCUBATION: After the washing steps the anterior chamber was filled with RPMI and an illuminance measurement was performed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The corneal opacity was determined by an opacimeter (BASF-OP3.0, Duratec GmbH). Three glass filter were calibrated for the measurement. F2, readout 540-560 lux, F3, readout 300-310 lux and F4 readout 95-105 lux.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (Jenway 6405 UV/VIS; OD490)
- Others: each cornea was observed visually and pertinent observations were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) . The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA: The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
20% solution in physiological saline
Value:
1.05
Vehicle controls validity:
valid
Remarks:
IVIS = 0.35
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
IVIS = 102.44
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
- Acceptance criteria met for positive control: yes, the negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Any other information on results incl. tables

Table 2: Opacity

Cornea No.

Test Item

Initial

Opacity

Final

Opacity

Change of Opacity Value

Corrected Opacity Value

1

Negative control

1.26

1.29

0.04

 

2

1.40

1.43

0.04

3

1.26

1.72

0.47

MV

1.30

1.48

0.18

4

Positive control

2.20

72.73

70.53

70.35

5

2.76

81.45

78.68

78.50

6

2.57

94.26

91.69

91.51

MV

2.51

82.81

80.30

80.12

7

Test item

0.70

1.72

1.03

0.85

8

1.87

2.96

1.09

0.91.

9

1.01

2.46

1.45

1.27

MV

1.19

2.38

1.19

1.01

Table 3: Permeability

Cornea No.

Test Item

OD490

Corrected OD490 Value

1

Negative control

0.010

 

2

0.013

3

0.012

MV

0.012

4

Positive control

1.296

1.284

5

1.363

1.351

6

1.840

1.828

MV

1.500

1.488

7

Test item

0.013

0.001

8

0.024

0.012

9

0.006

-0.006

MV

0.014

0.003

Table 4: In Vitro Irritation Score

Cornea No.

Test Item

Corrected Opacity

Corrected OD490 Value

IVIS

1

Negative control

0.04

0.01

0.35

2

0.04

0.013

3

0.47

0.012

MV

0.18

0.012

4

Positive control

70.35

1.284

102.44

5

78.50

1.351

6

91.51

1.828

MV

80.12

1.488

7

Test item

0.85

0.001

1.05

8

0.91

0.012

9

1.27

-0.006

MV

1.01

0.003

   

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The eye irritancy potential of Tetrahydrofolic acid (THFA) was investigated in the bovine corneal opacity and permeability assay. According to the evaluation criteria the test item Tetrahydrofolic acid (THFA) is classified into UN GHS No Category.