Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 June 2018 - 07 August 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
aqueous solution
Details on test material:
Sodium hydrogen substituted amino acid, FC-C 13403
Apppearance: Clear Colorless solution

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: 750 µL
Duration of treatment / exposure:
Experiment 1: 8 minutes
Experiment 2: 12 minutes
Duration of post- treatment incubation (in vitro):
120 ±10 minutes in cMEM for opacity measurements and subsequently 90 ± 5 minutes in sodium-fluorescein for permeability determinations
Number of animals or in vitro replicates:
2 experiments with 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, positive control or the 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 for 8 minutes in the first experiment and 12 minutes in the second experiment at 32.0 ± 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 (MEM containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum).
- POST-EXPOSURE INCUBATION: 120 ±10 minutes in cMEM for opacity measurements and subsequently 90 ±5 minutes in sodium-fluorescein for permeability determinations

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT) and calculated
- 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).

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

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
Experiment 1: mean of 3 replicates
Value:
-0.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Mean IVIS: 30
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Experiment 2: mean of 3 replicates
Value:
-1.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Mean IVIS: 71
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Experiment 1
- The corneas treated with the test item showed opacity values between -1.5 and 0.4
- Permeability values were ranging from 0.002 to 0.040
- Individual IVIS scores were 0.4, -1.4, 0.7 (n=3)

Experiment 2
- The corneas treated with the test item showed opacity values between -2.5 and -0.2
- Permeability values were ranging from -0.005 to 0.008
- Individual IVIS scores were -2.4, -0.7, -0.3 (n=3)

OTHER EFFECTS:
- No pH effect of the test item was observed on the rinsing medium

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, results were in both experiments within historical range (IVIS ranging from 2.0 to 2.6 and 1.9 to 3.5 in experiment 1 and 2, respectively).
- Acceptance criteria met for positive control: yes, results were in both experiments within historical range (IVIS ranging from 29 to 31 and 66 to 78 in experiment 1 and 2, respectively).

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 test guideline 437 and GLP principles. Sodium hydrogen substituted amino acid solution induced an IVIS ≤ 3, therefore no classification is required for eye irritation or serious eye damage.