Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11.01.2016 to 16.03.2016
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 Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

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

Test animals / tissue source

Species:
other: Freshly isolated bovine cornea (at least 9 month old donor cattle)
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: abattoir: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Characteristics of donor animals (e.g. age, sex, weight): 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The isolated eyes were
transported to the laboratory in HBSS at ambient temperature.
- Time interval prior to initiating testing: The corneae were directly used in the BCOP test on
the same day.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL


Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
two hours
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such
as vascularization, pigmentation, opacity and scratches were discarded. The cornea was
carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of
tissue (sclera) was left for stability and handling of the isolated cornea.


NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Saline (0.9% NaCl in deionised water)

POSITIVE CONTROL USED
2-Ethoxyethanol (purity: 99%)

APPLICATION DOSE AND EXPOSURE TIME
The test item were applied undiluted for ten minutes.

TREATMENT METHOD:
Each isolated cornea was mounted in a specially designed cornea holder according to the
description given in OECD guideline 437, that consists of anterior and posterior
compartments, which interface with the epithelial and endothelial sides of the cornea,
respectively. The endothelial side of the cornea was positioned against the sealing ring (Oring)
of the posterior part of the holder. The cornea was gently flattened over the O-ring but
stretching was avoided. The anterior part of the holder was positioned on top of the cornea
and fixed in place with screws. Both compartments of the holder were filled with incubation
medium. The posterior compartment was filled first to return the cornea to its natural convex
position.

POST-INCUBATION PERIOD: no


REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: one
- POST-EXPOSURE INCUBATION: two hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacitometer determines changes in the light transmission passing through the corneae,
and displays a numerical opacity value. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the
manual and the opacity of each of the corneae was determined by reading each holder placed
in the photoreceptor compartment for treated cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)


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

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the
current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less
than the established upper limits for background opacity and permeability values for
bovine corneae treated with the respective negative control.

Results and discussion

In vitro

Results
Irritation parameter:
cornea opacity score
Value:
ca. 0
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Relative to the negative control, the test item DINCD (Diisononyl 1,4-
cyclohexanedicarboxylate) did not cause any increase of the corneal opacity or permeability.
The calculated mean in vitro irritancy score was 0.00.
In conclusion, according to the current study and under the experimental conditions reported,
DINCD (Diisononyl 1,4-cyclohexanedicarboxylate) is not categorized (GHS).