Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 May 2016 - 03 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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:
26 July 2016
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
solid
Details on test material:
Identification: Nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide
Appearance: Light yellow lumps
Test item storage: At room temperature; Store in closed container

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 750 μL per cornea


Duration of treatment / exposure:
240 minutes
Details on study design:
SELECTION AND 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.

QUALITY CHECK OF THE ISOLATED CORNEAS
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.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
Physiological saline

POSITIVE CONTROL USED
20% (w/v) Imidazole solution

APPLICATION DOSE AND EXPOSURE TIME
750 μL for 10 minutes.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3

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.
- Corneal permeability: Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader).
- Others: Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean OD490 value).
DECISION CRITERIA:
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces an IVIS > 55 is defined as a corrosive or severe irritan (UN GHS: catgeory 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
Remarks:
mean
Run / experiment:
Single run
Value:
86
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
- Acceptance criteria met for positive control: The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 134 and 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.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD 437 guidiline and GLP principles, it is concluded that the substance induces serious eye damage.

Executive summary:

A Bovine Corneal Opacity and Permeability test (BCOP) was performed with the substance according to OECD guideline 437 and GLP principles. The substance was applied as a 20% dilution in physiological saline (750 µL/ cornea, n=3). The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 134, and the mean in vitro irritancy score of the negative control (physiological saline) was 1.2. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The substance did induce ocular irritation through opacity or permeability, resulting in a mean in vitro irritancy score of 86.3 after 240 minutes of treatment. Since the substance induced an IVIS ≥ 55, the substance should be classified as serious eye damage category 1 and labelled with H318: Causes serious eye damage according to Regulation (EC) No 1272/2008.