Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
The Bovine Corneal Opacity and Permeability Assay (BCOP)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 03, 2017 to October 03, 2017
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:
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)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Chemical name (IUPAC), synonym or trade name: Phosphoric acid, mono-and di-C8-10-(even numbered)-alkyl esters
CAS number: 98653-76-0
Molecular structure: UVCB
Molecular formula: UVCB

Specific details on test material used for the study:
- Name of test material (as cited in study report): Phosphoric acid, mono-and di-C8-C10-(even numbered)-alkyl esters
- Batch: CH 205624/001
- Composition: UVCB
- Appearance: Clear colourless liquid
- Test item storage: At room temperature
- Stable under storage conditions until: 01 July 2018 (expiry date)

Test animals / tissue source

Species:
other: Bovine
Details on test animals or tissues and environmental conditions:
Test System: Bovine eyes were used as soon as possible after slaughter.
Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.

Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 µL
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
-
Duration of post- treatment incubation (in vitro):
The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C.
Number of animals or in vitro replicates:
3
Details on study design:
Preparation of Corneas:
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

Cornea Selection and Opacity Reading:
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Treatment of Corneas and Opacity Measurements:
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test substance was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test substance over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ±1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
10 minutes exposure
Value:
112
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The individual in vitro irritancy scores for the negative controls ranged from 0.2 to 0.7. The individual in vitro irritancy scores for the positive control ranged from 55 to 59. The corneas treated with the positive control item were turbid after the 10 minutes of treatment. The corneas treated with the test substance showed opacity values ranging from 54 to 63 and permeability values ranging from 3.384 to 3.918. The corneas were turbid after the 10 minutes of treatment with the test substance. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no color change of the medium was observed. Hence, the in vitro irritancy scores ranged from 107 to 115 after 10 minutes of treatment with the test substance.

Any other information on results incl. tables

In Vitro Irritancy Score:

Treatment

Final Opacity2

Final OD4902

In vitroIrritancy Score1

 

Negative control

0.7

0.000

0.7

0.3

0.003

0.4

0.2

0.002

0.2

 

Positive control

21

2.478

59

23

2.098

55

22

2.322

56

 

The test item

63

3.460

115

56

3.384

107

54

3.918

113

1  In vitro irritancy score (IVIS) = opacity value + (15 x OD490value).

2  Positive control and test item are corrected for the negative control.

Summary of Opacity, Permeability and In Vitro Scores:

Treatment

Mean

Opacity1

Mean

Permeability1

MeanIn vitroIrritation Score1, 2

Negative control

0.4

0.002

0.4

Positive control

(Ethanol)

22

2.299

57

The test item

58

3.587

112

1  Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

2  In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490value).

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the study conditions, the substance was found to be causing serious eye damage in bovine corneal opacity and permeability test.
Executive summary:

A study was conducted to determine the in vitro eye damage potential of the substance according to OECD Guideline 437, in compliance with GLP. The eye damage potential of the test substance was tested through topical application for 10 minutes. The test substance was applied as is (750 µL) directly on top of the freshly isolated bovine cornea sample. The negative control responses for opacity and permeability was less than the upper limits of the laboratory historical control range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 57 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test substance induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 112 after 10 minutes of treatment. Under the study conditions, the substance was found to be causing serious eye damage in bovine corneal opacity and permeability test (Groot, 2017).