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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:
key study
Study period:
2016-09-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
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
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Version / remarks:
April 1997
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-09-14

Test material

Constituent 1
Chemical structure
Reference substance name:
Aluminium lanthanum trioxide
EC Number:
234-433-3
EC Name:
Aluminium lanthanum trioxide
Cas Number:
12003-65-5
Molecular formula:
AlLaO3
IUPAC Name:
Aluminium lanthanum trioxide
Test material form:
solid: particulate/powder
Details on test material:
Test Item: Aluminium lanthanum trioxide
Molecular formula: AlLaO3
Molecular weight: 213.89 g/mol
Appearance: Whitish powder
Storage conditions: Ambient temperature, keep container tightly closed, dry
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: abattoir (Schlachthof Aschaffenburg, 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 at ambient temperature.
- Time interval prior to initiating testing: corneae were isolated and used on the same day after delivery of the eyes

Test system

Vehicle:
physiological saline
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:
SELECTION AND PREPARATION OF CORNEAS
- all eyes were examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- cornea was removed from the eye.
- each cornea was mounted in a cornea holder according to the description given in OECD 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 (MEM*, equivalent to EMEM).
- 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.

*MEM supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, and 1% fetal calf serum

QUALITY CHECK OF THE ISOLATED CORNEAS
- at the end of the equilibration period, the basal opacity was determined (t0).
- each cornea with a value of the basal opacity > 7 was discarded.

APPLICATION DOSE / EXPOSURE TIME / REMOVAL OF TEST SUBSTANCE
- 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 period, the test item or control items, respectively, were rinsed off from the application side with saline
- fresh incubation medium was added into the anterior compartment and opacity was measured (t240).
- permeability of the cornea 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 spectrophotometer (Versamax® Molecular Devices) (OD490).
- after the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by a 0.5% (w/v) sodium fluorescein solution in HBSS.
- corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C.
- incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
Evaluation permeability:
- the corrected OD490 value of each cornea treated with positive control and 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 was used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula was used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group was calculated from the IVIS values of each individual treatment and positive control cornea.
Depending on the score obtained, the test item was classified into categories according to OECD guideline 437 (table 1 in the field "Any other information on materials and methods incl. tables" below).

ACCEPTANCE CRITERIA:
The test was 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
Remarks:
(mean)
Value:
17.84
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- with the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.48).
- distinct opacity of the corneae leading to a mean IVIS of 110.15 was caused after treatment with the positive control (10% (w/v) Benzalkonium chloride in saline) corresponding to the classification Irreversible effects on the eye / serious eye damag (CLP (Cat 1)).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: opacity and permeability of the negative control are less than the respective established upper limits for background opacity and
permeability.
- Acceptance criteria met for positive control: positive control falls within two standard deviations of the current historical mean.

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 incubation time


Test Group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

 

 

Mean

 

Mean

 

 

Negative Control

0

0.33

0.079

0.077

1.19

1.48

1

0.076

2.14

0

0.075

1.13

Positive Control

117.67*

0.003*

117.72

110.15

104.67*

0.017*

104.93

107.67*

0.009*

107.81

Aluminium lanthanum trioxide

20.67*

0.114*

22.38

17.84

10.67*

0.190*

13.52

14.67*

0.197*

17.63

*corrected values

Table 2: Historical data

 

Positive Control

Negative Control

Mean IVIS

122.01

1.32

Standard Deviation IVIS

15.51

0.33

Range of IVIS

98.49 – 167.85

0.73 – 2.04

Mean Opacity t240min

120.03

0.17

Standard Deviation
Opacity t240min

18.45

0.25

Range of Opacity t240min

88.67 – 183.00

-0.33 – 0.67

Mean Permeability

0.147

0.184

Standard Deviation Permeability

0.257

0.281

Range of Permeability

0.000 – 1.438

0.055 – 1.100

Values of 32 studies with solid test items performed from February 2015 (calendar week 7) until October 2016 (calendar week 40)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, based on a valid OECD 437 test and under the experimental conditions reported, aluminium lanthanum trioxide does not meet classification criteria of irreversible effects on the eye / serious eye damage (CLP (Cat 1).