Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Feb 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 Jul 2013
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

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
- Storage, temperature and transport conditions of ocular tissue: Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: The eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Indication of any existing defects or lesions in ocular tissue samples: 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.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 µL
Duration of treatment / exposure:
10 ± 1 min
Duration of post- treatment incubation (in vitro):
120 ± 10 min
Number of animals or in vitro replicates:
Three corneas were used for each treatment 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)). 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 h at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). 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.

TREATMENT METHOD
After treatment, 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 10 ± 1 min at 32 ± 1 °C. Possible pH effects of the test item on the corneas were recorded.

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 medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 min at 32 ± 1 °C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490) .

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as Category 1.
Test substance with an IVIS ≤ 3 was regarded as 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
Run / experiment:
mean value of 3 corneas
Value:
104
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control (physiological saline) the calculated IVIS ranged from 0.8 to 1.2, and therefore within the standard deviations of the current historical mean of the negative control (IVIS: -2.8 to 3.0) (please refer to Table 1 and 2 under "any other information on results incl. tables").
- Acceptance criteria met for positive control: After treatment with the positive control (ethanol) the calculated IVIS ranged from 56 to 63, and therefore within the standard deviations of the current historical mean of the positive control (IVIS: 28.0 - 110.9) (please refer to Table 1 and 2 under "any other information on results incl. tables").

Any other information on results incl. tables

Table 1: Results

Treatment Permeability score
Final OD*
Final Opacity* In vitro Irritancy Score
Negative control -0.001 1.2 1.2
0.013 0.7 0.9
0.002 0.8 0.8
Positive control 2.615 16 56
5.439 21 103
3.195 15 63
Test item 4.453 44 111
4.343 23 89
4.153 50 112

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

Table 2 Historical control data

  Negative control Positive control
  Opacity Permeability In vitro Irritancy Score In vitro Irritancy Score
Range -2.9 - 3.0 -0.034 - 0.100 -2.8 - 3.0 28.0 - 110.9
Mean 0.18 0.00 0.23 55.28
SD 1.10 0.01 1.13 15.14
n 113 113 113 88

Applicant's summary and conclusion

Interpretation of results:
other: corrosive according to Regulation(EC) No. 1272/2008
Conclusions:
Under the conditions of the BCOP assay, the test substance showed corrosive properties towards eyes.
CLP: Eye Damage 1, H318 according to Regulation(EC) No. 1272/2008