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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:
November from 11th to 12th, 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
other: Bovine
Details on test animals or tissues and environmental conditions:
- Bovine eyes source: breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic.
- Collection: eyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death.
- Deterget: no detergent was used.
- Animals: only healthy animals (12 to 60 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test.
- Storage: the risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/ml and streptomycin at 100 μg/ml).

The time interval between collection of the eyes and use of corneas in the BCOP was minimized, so the collected eyes were processed on the same day.

Test system

Vehicle:
physiological saline
Controls:
yes
Amount / concentration applied:
Application form preparation: the test substance was tested as suspension prepared from test substance at 20 % concentration in a 0.9 % sodium chloride solution. 2 g of the test substance was suspended in 10 ml of 0.9 % sodium chloride solution.

Open-chamber method was used, because the test substance at 20 % concentration was a viscous paste. The test substance (the test substance in quantity enough to completely cover the cornea) was applied directly to the epithelial surface of the cornea using the micropipette. After dosing, the glass window was replaced on the anterior chamber to recreate a closed system.
Number of animals or in vitro replicates:
Exposed group (test substance) - 3 corneas
Details on study design:
EYE SELECTION AND EXANMINATION
The eyes, once they arrive at the laboratory, were carefully examined for defects including scratched, and neovascularisation. Only corneas from eyes free of such defects were used. The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.
From 20 eyes the 3 eyes were eliminated after inductive incubation, because the baseline opacity values were >7.

EYE PREPARATION
Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium an endothelium. Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM). The device was then equilibrated at 32 ± 1°C for at least one hour in water bath to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.
Each test group (test substance, concurrent negative and positive controls) consisted of the three eyes. The three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups.

POST EXPOSURE
After the exposure period, the negative control and the positive control substance was removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.
The test substance was removed from the anterior chamber with EMEM (containing phenol red). The corneas (applied the test substance) were also repeatedly rinsed with EMEM (without phenol red), because the test substance is coloured . Rinsing was finalized after complete removal of the test substance. The EMEM (without phenol red) was used as a final rinse to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.

CONTROLS
- Positive control group: 20 % Imidazole in 0.9 % NaCl; 3 corneas
- Negative control group: 0.9 % NaCl; 3 corneS

MEASUREMENTS
Opacity: the amount of light transmission through the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.

Permeability: the amount of sodium fluorescein dye that penetrates all corneal cell layers (i.e., the epithelium on the outer cornea surface through the endothelium on the inner cornea surface) measured indirectly using visible light spectrophotometry. 1 ml sodium fluorescein solution (5 mg/ml) was added to the anterior chamber of the corneal holder, which interfaced with the epithelial side of the cornea, while the posterior chamber, which interfaced with the endothelial side of the cornea, is filled with fresh EMEM. The holder was incubated in horizontal position for 1.5 hours at 32 ± 1 °C.

The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry. The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.

STUDY ACCEPTANCE CRITERIA
A test is considered acceptable if the positive control gives IVIS that falls within one standard deviations of the current historical mean, which is to be updated at least every three months, or each time an acceptable test is conducted in laboratories where tests are conducted infrequently. The negative or solvent/vehicle control responses should result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control.

SCORING SYSTEM
The IVIS cut-off value for identifying the test substance as including serious eye damage (UN GHS Category 1) and the test substance not requiring classification for eye irritation or serious damage (UN GHS No Category) will be given hereafter:
≤ 3: no Category
> 3; ≤ 55: no prediction can be made
≥ 55: category 1

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
3.77
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
probability of weak irritation
Other effects / acceptance of results:
IVIS: 3.77 not irritating

No macroscopic damage was observed on corneas before application.
Corneal opacity was observed on the corneas treated by positive control. The corneas treated by negative control were without macroscopic damage. The corneas treated by the test substance were without macroscopic damage.

NEGATIVE CONTROL
The value of opacity for negative control (0.9 % NaCl) obtained during the study was 1.67 and value of permeability was 0.013.
The values obtained during this study not exceeded upper limits, so the study is considered acceptable.

POSITIVE CONTROL
The value of IVIS for positive control (20 % imidazole in 0.9 % NaCl) obtained during the study was 80.01.
This value is within the acceptance limit (one standard deviations of the current historical mean), so the study is considered acceptable.

Any other information on results incl. tables

IVIS values

Group IVIS
Calculation Result
NC
(0.9 % NaCl)
1.67 + 15 x 0.013   1.87
PC
(20 % Imidazole in 0.9 % NaCl)
51.33 + 15 x 1.912   80.01
EXP
(test item)
3.66 + 15 x 0.007 3.77

NC- negative control; PC- positive control; EXP – test substance application form

Appearance of Corneas after the Test Substance Exposure

Group Cornea No. Appearance after exposure 
Negative control 1 Without macroscopic damage
2 Without macroscopic damage
4 Without macroscopic damage
Positive control 16 Corneal opacity 
19 Corneal opacity 
21 Corneal opacity 
Test substance 5 Without macroscopic damage
6 Without macroscopic damage
7 Without macroscopic damage

Opacity

Group Cornea No. Baseline opacity Opacity after treatment Opacity difference Mean opacity difference Mean Opacity(corrected)
NC
(0.9 % NaCl)
1 5 8 2 1.67 -
2 5 7 1
4 6 8 2
PC
(20 % Imidazole in 0.9 % NaCl)
16 3 57 51 53.00 (53.00 – 1.67)
51.33
19 6 59 53
21 5 61 53
EXP
(test item)
5 5 8 4 5.33 (5.33 – 1.67)
3.66
6 6 10 4
7 7 14 8

NC- negative control; PC- positive control; EXP – test substance application form

Permeability

Group Cornea No. Values of Permeability (Optical density at 490nm) Mean Permeability Mean Permeability(corrected)
NC
(0.9 % NaCl)
1 0.009 0.013 -
2 0.009
4 0.021
PC
(20 % Imidazole in 0.9 % NaCl)
16 2.109 1.925 (1.925 – 0.013)
1.912
19 1.744
21 1.921
EXP
(test item)
5 0.021 0.020 (0.020 – 0.013)
0.007
6 0.019
7 0.020

NC- negative control; PC- positive control; EXP – test substance application form

DAVIATION FROM STUDY PLAN

Change of technique application of the test substance – the open-chamber method was used. The test substance suspension (20 % concentration) was applied in quantities enough to completely cover the cornea. This change had no impact on the results of the study.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Conclusions:
Not irritating
Executive summary:

The test substance was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The test was performed according to the OECD Test Guideline No. 437, Adopted 26th July 2013. 

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used. Open-chamber method was used, because the test substance was higly viscous. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

No macroscopic damage was observed on corneas before application. Corneal opacity was observed on the corneas treated by positive control. The corneas treated by negative control were without macroscopic damage. The corneas treated by the test substance were without macroscopic damage.

The In Vitro Irritancy Score (IVIS) for test item was 3.77. This value of IVIS is higher than 3 and simultaneously lower than 55 therefore the classification according to UN GHS criteria for eye irritation or serious eye damage cannot be made.

Conclusion

Not irritating