Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-29
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:
2013-07-26
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-09-14

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: solid, white powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

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 transported to the laboratory in Hank’s Buffered Salt Solution (HBSS) supplemented with streptomycin / penicillin.
- 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 % 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 in a specially designed cornea holder according to the description given in OEDC guideline 437, which 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).
- each corneae with a value of the basal opacity > 7 was 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 the incubation time, the test item or control items were rinsed off with saline.
- fresh incubation medium was added into the anterior compartment and opacity was measured (t240).
- permeability of the corneae was determined.

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 the anterior compartment and replaced by 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.
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.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
0.24
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- for the negative control an increase of neither opacity nor permeability of the corneae could be observed (mean IVIS = 1.38).
- the positive control showed clear opacity and distinctive permeability of the corneae (mean IVIS = 127.81) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

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

Proposedin vitroIrritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

0

0.33

0.065

0.070

0.98

1.38

Not categorized

0

0.068

1.02

1

0.077

2.16

Positive Control

133.67*

0.006*

133.76

127.81

Category 1

122.67*

0.011*

122.83

126.67*

0.011*

126.83

Cesium tetrafluoroaluminate

0.67*

0.002*

0.70

0.24

Not categorized

-0.33*

-0.011*

-0.50

0.67*

-0.010*

0.52

*corrected values

** negative values will be set to zero

Table 2: Historical Data

 

Positive Control

Negative Control

Mean IVIS

115.29

1.28

Standard Deviation IVIS

9.57

0.20

Range of IVIS

98 - 131

1 - 2

Mean Opacity t240min

113.93

0.20

Standard Deviation
Opacity t240min

12.24

0.20

Range of Opacity t240min

82 - 135

000 - 000

Mean Permeability

0.09

0.07

Standard Deviation of Permeability

0.12

0.01

Range of Permeability

000 - 001

000 - 000

Values of 41 studies with solid test items sharing 20 sets of controls, performed between January 2017 and October 2017.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Relative to the negative control, the test item Cesium tetrafluoroaluminate did not cause an increase of the corneal opacity. The calculated mean in vitro irritancy score was 0.24. According to OECD 437 and in accordance with Regulation (EC) 1272/2008 and subsequent regulations the test item is not categorized (GHS).