Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 April 2018 - 10 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
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)
Version / remarks:
09 October 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
d.d. 22 January 2018

Test material

Reference
Name:
Unnamed
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Pink powder
- Storage conditions: At room temperature

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: young cattle, obtained from the slaughterhouse Vitelco, 's-Hertogenbosch, The Netherlands.
- Storage, temperature, and transport conditions of ocular tissue: eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Subsequently, eyes were collected an transported in physiological saline in a suitable container under cooled conditions.
- The eyes were checked for unacceptable defects and those exhibiting defects were discarded.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 305.6 to 375.0 mg
Duration of treatment / exposure:
240 ± 10 minutes
Duration of post- treatment incubation (in vitro):
90 ± 5 minutes in sodium-fluorescein for permeability determinations
Number of animals or in vitro replicates:
3 replicates for the negative, positive, and treatment group each.
Details on study design:
TREATMENT METHOD: The medium from the anterior compartment was removed and 750 µL of either the negative control or positive control was introduced onto the epithelium of the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32.0 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and test item were removed and the epithelium was washed three times with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) before put in cMEM (MEM containing 1% (v/v) L-glutamine and 1% (v/v) Fetal Bovine Serum) for opacity measurements. Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns.
- POST-EXPOSURE INCUBATION: 90 ±5 minutes in sodium-fluorescein for permeability determinations.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader, OD490). OD490 values of less than 1.500 were used in the permeability calculation.
- Other: possible pH effects of the test substance on the corneas were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).
Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
a) The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
b) The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

DECISION CRITERIA (see table 1):
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces IVIS >55 is defined as a corrosive or severe irritant (UN GHS: category 1);
For a test substance that induces an IVIS >3 and ≤55, no prediction on irritant potency can be made.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of 3 replicates
Value:
1.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Mean IVIS: 1.2
Positive controls validity:
valid
Remarks:
Mean IVIS: 107
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- The corneas treated with the test item showed opacity values of 0.1, 0.6 and 3.6.
- Permeability values were ranging from -0.006 to 0.029.
- Individual IVIS scores of the corneas treated with the test item were 0.5, 0.6 and 3.7 (n=3).
The IVIS of 3.7 is above the cutoff for no classification on eye irritation. However historical control data of IVIS scores for negative controls ranges from -5.3 to 5.3. The IVIS score of 3.7 can therefore be regarded as an exception that may occur and does not indicate injury to the cornea.

OTHER EFFECTS:
- No pH effect of the test item was observed on the rinsing medium.
- The corneas were clear or slightly translucent after 240 minutes of treatment with C.I. Pigment Red 81:2.

ACCEPTANCE OF RESULTS (see table 2) :
- Acceptance criteria met for negative control: yes, results were within historical range (IVIS ranging from 0.3 to 1.9).
- Acceptance criteria met for positive control: yes, results were within historical range (IVIS ranging from 101 to 113). The corneas treated with the positive control were turbid after the 240 minutes of treatment.

Any other information on results incl. tables

Table 2          In Vitro Irritancy Score

Treatment

Final Opacity(b) 

Final OD490(b) 

In vitro Irritancy

Score(a)

 

 

 

 

Negative control

0.9

0.031

1.3

0.1

0.012

0.3

1.5

0.028

1.9

 

 

 

 

Positive control

101

0.796

113

85

1.076

101

91

1.022

107

 

 

 

 

C.I. Pigment Red 81:2

3.6

0.007

3.7

0.1

0.029

0.6

0.6

-0.006

0.5

a) In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

b) Positive control and test item are corrected for the negative control.

            

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
A Bovine Corneal Opacity and Permeability test (BCOP) was performed according to OECD guideline 437 and GLP principles. C.I. Pigment Red 81:2 induced an average IVIS ≤ 3, therefore no classification is required for eye irritation or serious eye damage.