Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 6 - November 30, 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
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)
GLP compliance:
no
Remarks:
The methods and procedures used in this assay were consistent with OECD Test Guideline 437.

Test material

Constituent 1
Chemical structure
Reference substance name:
12-[(3R,4S)-4-ethylpyrrolidin-3-yl]-1,5,7,10-tetraazatricyclo[7.3.0.0²,⁶]dodeca-2(6),3,7,9,11-pentaene dihydrochloride
EC Number:
845-622-0
Cas Number:
2050038-84-9
Molecular formula:
C14H19Cl2N5
IUPAC Name:
12-[(3R,4S)-4-ethylpyrrolidin-3-yl]-1,5,7,10-tetraazatricyclo[7.3.0.0²,⁶]dodeca-2(6),3,7,9,11-pentaene dihydrochloride
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Bovine corneas, obtained as a byproduct from freshly slaughtered animals, were mounted in special holders and exposed to the test articles.
Four corneas were incubated in the presence of the test article at 32 ± 1ºC. Three corneas were incubated in the presence of each control at 32 ± 1ºC.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The solid test article was administered to the test system as 20% (w/v) (200 mg/mL) dilutions in sterile, deionized water.
Duration of treatment / exposure:
The solid test article and the concurrent assay controls (20% (w/v) dilution of imidazole and sterile, deionized water) were exposed to the corneas for 4 hours. After removal of the test or control article from the corneas, a final opacity was determined (i.e., the corneas did not receive a post-exposure incubation).
Number of animals or in vitro replicates:
Four corneas were incubated in the presence of the test article. Three corneas were incubated in the presence of each control.
Details on study design:
The Bovine Corneal Opacity and Permeability Assay (BCOP) was used to assess the potential ocular irritancy of the test article to isolated bovine corneas. Bovine corneas, obtained as a byproduct from freshly slaughtered animals, were mounted in special holders and exposed to the test article. An In Vitro Score was determined for the test article based on the induction of opacity and permeability (to fluorescein) in the isolated bovine corneas.

The test article was administered to the test system as 20% (w/v) (200 mg/mL) dilutions in sterile, deionized water. The test article dilution was prepared by weighing the test article into a conical tube, adding sterile, deionized water, until a 20% (w/v) dilution was achieved, and then vortexing the dilution for approximately 1 minute prior to application. The positive control (a 20% (w/v) dilution of imidazole prepared in Complete Minimal Essential Medium (without phenol red)) and the negative control (sterile, deionized water) were tested concurrently.

Four corneas were incubated in the presence of the test article and three in the presence of the control at 32 ± 1ºC .for four hours. After removal of the test or control article from
the corneas, the corneas were returned to the incubator at 32 ± 1ºC for a post-exposure incubation period of 2 hours, after which a final opacity value was determined.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
13 September 2018
Value:
112.1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The positive control in vitro irritancy score was 97.8.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The in vitro score was calculated as 112.1 and as it is > 55, the test material is classified as Category 1, according to OECD TG 437.