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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-04-13 to 2016-08-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted: 26th July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
lutetium(3+) yttrium(3+) cerium oxidandiide silicate
EC Number:
941-731-3
Molecular formula:
Lu2(1-x)Y2xSiO5:Ce, 0
IUPAC Name:
lutetium(3+) yttrium(3+) cerium oxidandiide silicate
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: C14-193
- Purity: theoretical composition based on starting materials: 81.25% Lu2O3, 13.51% SiO2, 5.2% Y2O3 and 0.039% CeO2

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability in water at room temperature: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The test item was suspended with physiological saline 0.9% NaCl to gain a 20% concentration.

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: A. Moksel AG, Buchloe, Germany
- Number of animals: 3 corneas
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): On the test day, fresh eyes were collected from the slaughterhouse and were transported in the Hank’s balanced salt solution containing Pen/Strep on ice to the laboratories. Immediately after arrival of eyes, cornea preparation was initiated.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.

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): 750 µL
- Concentration (if solution): 20%

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 0.9%
- Lot/batch no. (if required): 1406788
Duration of treatment / exposure:
4 hours ± 5 min at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
The optical density was measured upon 90 minutes of incubation with sodium fluorescein solution after exposure to the test item.
Number of animals or in vitro replicates:
Three corneas each for the test item, negative control (physiological saline 0.9% NaCl) and positive control (imidazole 20% in physiological saline 0.9% NaCl).
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- The eyes were carefully examined for defects and defectives 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. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 +/- 1 °C.

TREATMENT METHOD: closed chamber

Treatment of the Corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I>I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control.
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 and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed.
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 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. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer.
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After 4 hours incubation either the test substance or the control substance was removed and the epithelium washed at least three times with minimum essential medium (MEM) (containing phenol red).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD490 value)
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) are given in Table 1 (see "Any other information on materials and methods").
An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made.
For this purpose further testing with another suitable method is required.

Evaluation of Results:
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.
The mean OD490 for the blank cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490. The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean of triplicates
Value:
39.42
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
no prediction can be made
Other effects / acceptance of results:
The in vitro irritation score obtained with the positive control fell 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.
For detailed results please refer to Tables 2 to 4 in box “Any other information on results incl. tables”.

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

2.77

3.05

0.27

 

2

3.24

4.51

1.27

 

3

2.66

3.89

1.23

 

MV

2.89

3.82

0.93

 

4

 

Positive Control

3.77

75.02

71.26

70.33

5

3.44

87.04

83.59

82.67

6

3.77

74.45

70.69

69.76

MV

3.66

78.84

75.18

74.25

7

 

Test Item

1.60

37.39

35.79

34.86

8

1.38

42.04

40.65

39.73

9

3.32

47.78

44.45

43.53

MV

2.10

42.40

40.30

39.37

MV = mean value

Table 3: Permeability

Cornea No.

Test Item

OD490

Corrected OD490 Value

1

 

Negative Control

0.013

 

2

0.010

 

3

0.020

 

MV

0.014

 

4

 

Positive Control

2.715

2.701

5

2.240

2.226

6

1.444

1.430

MV

2.133

2.119

7

 

Test Item

0.020

0.006

8

0.020

0.006

9

0.013

-0.001

MV

0.018

0.003

Table 4: In Vitro Irritation Score

Cornea No.

Test Item

Corrected Opacity

Corrected OD490 Value

IVIS

1

 

Negative Control

0.27

0.013

 

2

1.27

0.010

 

3

1.23

0.020

 

MV

0.93

0.014

1.14

4

 

Positive Control

70.33

2.701

 

5

82.67

2.226

 

6

69.76

1.430

 

MV

74.25

2.119

106.03

7

 

Test Item

34.86

0.006

 

8

39.73

0.006

 

9

43.53

-0.001

 

MV

39.37

0.003

39.42

MV = mean value

Applicant's summary and conclusion

Interpretation of results:
other: no prediction can be made
Conclusions:
In conclusion, based on the mean in vitro irritation score of 39.42 obtained in the bovine corneal opacity and permeability assay (BCOP, OECD 437) no prediction can be made regarding the classification of the test substance.
Executive summary:

The eye irritation potential of LYSO was investigated in the bovine corneal opacity and permeability assay (BCOP, OECD 437). The test item was suspended with physiological saline 0.9 % NaCl to gain a 20 % concentration. A mean in vitro irritation score of 39.42 was determined. The positive control induced the appropriate responses, indicating the validity of the assay. According to the UN GHS criteria, this mean in vitro irritation score does not allow to make any prediction regarding classification of the test substance. For the purpose of classification, further testing with another suitable method is required.

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