Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 July 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:
adopted on 9 Oct 2017
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Vitelco, 's Hertogenbosch, The Netherlands
- Indication of any existing defects or lesions in ocular tissue samples: No; Eyes with visible defects such as opacity, scratches, pigmentation and neovascularization were discarted.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL

NEGATIV CONTROL
- Amount applied: 750 µL

POSITIV CONTROL
- Amount applied: 750 µL
Duration of treatment / exposure:
10 ± 1 min at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
120 ± 10 min
Number of animals or in vitro replicates:
triplicates for each treatment and control group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). Each isolated cornea was mounted in a corneal holder 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.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the equilibration period, the initial opacity was determined. Corneas with a initial opacity >7 were discarded.

TREATMENT METHOD: closed chamber. The medium from the anterior compartment was removed and either the negative control, positive control or test item 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 item over the entire cornea. Corneas were incubated in a horizontal position.

POST-INCUBATION PERIOD: yes, for 120 ± 10 minutes at 32 ± 1°C

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea; the light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter (OP-KIT).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a microplate reader (OD490) (TECAN Infinite M200 Pro Plate Reader) at 490 nm.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) , IVIS = mean opacity value + (15x mean OD490 value)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as serious eye damage and labelled Category 1 according to CLP/EPS/GHS.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55 no prediction can be made.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
mean value of 3 cormeas
Run / experiment:
10 min
Value:
ca. 0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The mean in vitro irritancy score for the test substance was -0.4.
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes; The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range.
- Acceptance criteria met for positive control: yes; Mean IVIS for the positive control was 59 and was within two standard deviations of the current historical positive control mean.



Any other information on results incl. tables

Table 2. Results after 10 min incubation time

Treatment

Mean Opacity1

Mean Permeability1

Mean In vitro Irritation Score1, 2

Negative control

1.8

0.009

1.9

Positive control (Ethanol)

24

2.311

59

Test item

-0.4

0.000

-0.4

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:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
The irritation potential of the test substance was assessed in the BCOP assay. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of -0.4 (threshold for irritancy ≥ 3) and therefore, no classification is required for eye irritation or serious eye damage.