Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
Adopted 26th July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid: particulate/powder
Details on test material:
Trade name: Saturn Yellow LFF 200Batch No.: 4003/S

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
TEST SYSTEM:Bovine eyes source: Breeding service CHOVSERVIS a.s., division TORO Hlavečník, Hradec Králové, Czech RepublicEyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death. No detergent was used. 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. 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 (typically collected and used on the same day). The results were based on the selection criteria for the eyes, as well as the positive and negative control responses. All eyes used in the assay were from the same group of eyes collected on a specific day.SELECTION CRITERIA FOR EYES USED IN BCOP: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 a baseline opacity >7 opacity units were discarded. From 22 eyes the 5 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study, 5 eyes were superfluous and the remaining 3 eyes were used for the testing of another substance.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
20% solution of the test substance in physiological saline
Duration of treatment / exposure:
4 hours
Number of animals or in vitro replicates:
Exposed group (test substance) – 3 corneas Positive control group (20% Imidazole in 0.9% NaCl) – 3 corneas Negative control group (0.9% NaCl) – 3 corneas
Details on study design:
PROCEDURE SCHEME - Selection of corneas, mounting in holders - Incubation with EMEM 1hour (32 ± 1°C) - Removed EMEM, measurement of baseline opacity - Treatment by positive and negative control substances and test substance (incubation 4 hours) - Washing of epithelium, measurement of opacity after application - Application of sodium fluorescein (5 mg/ml), incubation 1.5 hour (32 ± 1°C) - Measurement of absorbance (490 nm).PREPARATION OF THE EYESCorneas 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. APPLICATION OF THE TEST SUBSTANCETreatment protocol for non-surfactant solids was used. Non-surfactant solids are typically tested as solutions or suspensions at 20% concentration.Application form preparation:The test substance was tested 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 spatula. After dosing, the glass window was replaced on the anterior chamber to recreate a closed system.CONTROL SUBSTANCES Concurrent negative controls and positive controls were included in experiment. The controls were included in the BCOP test method so that nonspecific changes in the test system could be detected and to provide a baseline for the assay endpoints.POST-EXPOSUREAfter 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) and sequentially was removed mechanically using a cotton swab – very gently (the test substance was coloured); also the test substance was removed from the anterior chamber with EMEM (containing phenol red) once more. The corneas (applied the test substance) were also repeatedly rinsed with EMEM (without phenol red). Rinsing was finalized after complete removal of the test substance. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.EXPERIMENTAL MEASUREMENTS - Opacity: the amount of light transmission through the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) 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 (Spectrophotometer GENESYS 10 UV/VIS Scanning). 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. - Mean opacity:Opacity values of treated corneas were corrected by subtracting individual background opacity values and the mean opacity is calculated. - Mean permeability:Mean OD value of treated corneas was corrected by subtracting the mean OD value of negative control and the mean permeability is calculated. - IVIS calculation:Resulting mean opacity and OD490 values for each treatment group was combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:IVIS = mean opacity value + (15 x mean permeability OD490 value)

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
1.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Opacity

Group

Baseline opacity

Opacity after treatment

Opacity difference

Mean

opacity difference

Mean Opacity (corrected)

Negative Control

(0.9% NaCl)

6

8

2

1.67

-

6

7

1

6

8

2

Positive Control

(20% Imidazole in 0.9% NaCl)

7

63

56

53.67

(53.67 – 1.67)

52.00

5

61

56

6

55

49

Test Substance

(Direct Yellow 28)

5

8

3

2.67

(2.67 – 1.67)

1.00

7

9

2

7

10

3

Table 2: Optical density (permeability)

Group

Values of Permeability (OD490)

Mean Permeability

Mean Permeability

(corrected)

Negative Control

(0.9% NaCl)

0.031

0.034

-

0.042

0.030

Positive Control

(20% Imidazole in 0.9% NaCl)

1.605

1.831

(1.831 – 0.034)

1.797

2.133

1.754

Test Substance

(Direct Yellow 28)

0.056

0.047

(0.047 – 0.034)

0.013

0.042

0.042

 

Table 3: IVIS

Group

IVIS

Calculation

Result

Negative Control

(0.9% NaCl)

1.67 + 15 × 0.034 

2.18

Positive Control

(20% Imidazole in 0.9% NaCl)

52.00 + 15 × 1.797 

78.96

Test Substance

(Direct Yellow 28)

1.00 + 15 × 0.013

1.20

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The In Vitro Irritancy Score (IVIS) for Direct Yellow 28 was 1.20. This value of IVIS is ≤ 3 therefore the classification of test substance effect according to UN GHS criteria for eye irritation or serious eye damage is: No category.
Executive summary:

The test substance, Direct Yellow 28, 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, 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, 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 highly 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.

The In Vitro Irritancy Score (IVIS) for Direct Yellow 28 was 1.20.

This value of IVIS is ≤ 3 therefore the classification of test substance effect according to UN GHS criteria for eye irritation or serious eye damage is: No category.