Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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:
2017-10-09
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-09-14

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- State of aggregation: white solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature. Keep container tightly sealed. Protect from humidity and water

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: corneae were isolated and used on the same day after delivery of the eyes

Test system

Vehicle:
other: 0.9 % (w/v) NaCl in deionised water
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration: 20 % suspension (w/v) in vehicle
Duration of treatment / exposure:
240 minutes
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
not required
Number of animals or in vitro replicates:
Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates
Details on study design:
PREPARATION OF CORNEAS
- each isolated cornea was mounted according to the description given in OEDC guideline 437, i.e. in a specially designed cornea holder that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. Both compartments of the holder were filled with incubation medium cMEM (MEM, supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin and 1 % fetal calf serum).
- for equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

QUALITY CHECK OF THE ISOLATED CORNEAS
- all eyes were carefully examined macroscopically for defects before removing the cornea. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- at the end of the equilibration period of the corneae in the holder, the basal opacity was determined (t0).
- corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one-hour equilibration period were discarded.

APPLICATION DOSE AND EXPOSURE TIME
- the anterior compartment received the test item suspension or negative or positive control at a volume of 0.75 mL each on the surface of the corneae.
- corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath (incubation time: 240 minutes).
- after exposure of the test item or control items to the corneae, they were rinsed off from the application sides with EMEM containing phenol red at least three times or more if phenol red is still discoloured (yellow or purple), or the test item is still visible.
- once the medium was free of the test item the corneas were give a final rinse with cMEM without phenol red.
- fresh cMEM was added into the anterior compartment and opacity was measured (t240).
- permeability of the corneae was determined.
- no tissue peeling, residual test chemical and non-uniform opacity patterns were observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: the opacitometer (OP_KiT opacitometer (Electro Design)) was calibrated and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
Evaluation of opacity:
- the change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
- the average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (Versamax® Molecular Devices)(OD490).
- after the final opacity measurement was performed, the incubation medium was removed from both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 0.5% (w/v) sodium fluorescein solution in HBSS.
- corneae were incubated in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C.
- incubation medium from the posterior compartment was removed, mixed and the optical density at 490 nm was determined with a microplate reader.
Evaluation permeability:
- the corrected OD490 value of each cornea treated with positive control or test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – corrected opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values of each individual treatment and positive control cornea.
Depending on the IVIS score obtained, the test item is classified into the following category according to OECD guideline 437 (please refer to table 1 in the field "Any other information on material and methods incl. tables" below).

DECISION CRITERIA:
The test will be acceptable if:
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- a single testing run composed of at least three corneas should be sufficient for a test chemical when the resulting classification is unequivocal. However, in cases of borderline results in the first testing run as described in the OECD guideline 437, a second testing run should be considered as well as a third one in case of discordant mean IVIS results between the first two testing runs.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
10.21
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- after exposure to the negative control (physiological saline) an increase of opacity or permeability of the corneae was not observed (mean IVIS = 1.52).
- exposure to the positive control (10% (w/v) benzalkonium chloride in saline) resulted in clear opacity and distinctive permeability of the corneae (mean IVIS =121.96) corresponding to the classification serious eye damaging (EU CLP/ UN GHS (Category 1)).
- the test is valid since the IVIS of the positive control falls within two standard deviations of the current historical mean. Further, opacity and permeability of the negative control are less than the respective established upper limits for background opacity and permeability.

Please refer to the field "Any other information on results incl. tables" below.

Any other information on results incl. tables

Table 1: Results after 240 Minutes Treatment Time


Test Group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposed in vitro Irritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

0

0.33

0.053

0.079

0.80

1.52

No Category

-1

0.084

0.26

2

0.101

3.52

Positive Control

98.67*

0.101**

100.18

121.96

Category 1

104.67*

0.092**

106.04

158.67*

0.066**

159.65

Zinc fluoride

6.67*

-0.030**

6.21

10.21

No prediction can be made

9.67*

-0.029**

9.23

15.67*

-0.032**

15.18

* final corrected opacity ((t240 - t0) - average-corrected negative control opacity value)

** final corrected OD490 (OD490 - average-corrected negative control OD490 value)

Table 2: Historical Data

 

Positive Control

Negative Control

Mean IVIS (MV)

115.47

1.31

Standard Deviation of IVIS (SD)

8.96

0.21

Range of IVIS

98.30—130.61

0.86—1.64

95 % Control limits of IVISpos

(MV ± 2xSD)

97.55—133.39

 

Mean Opacity t240min

114.26

0.22

Standard Deviation of
Opacity t240min

12.06

0.21

Range of Opacity t240min

82.00—135.00

0.00—0.67

Mean Permeability

0.08

0.07

Standard Deviation of Permeability

0.11

0.01

Range of Permeability

-0.01—0.52

0.06—0.09

Values of 45 studies with solid test items sharing 24 sets of controls, performed between January 2016 and October 2017.

Applicant's summary and conclusion

Interpretation of results:
other: test item is not serious eye damaging (CLP (Cat 1) but the hazardous properties of the test item with regard to corneal irritation cannot be predicted.
Conclusions:
The test substance is not serious eye damaging according to the Regulation (EC) No 1272/2008 and subsequent adaptions (Category 1), but no further prediction regarding the eye irritation potential can be made according to the evaluation criteria.