Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
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)
GLP compliance:
yes (incl. certificate)

Test material

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

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
Bovine eyes (greater than 35 weeks of age) were obtained from an abattoir and transported to the MB Research in a refrigerated container containing
Hanks’ Balanced Salt Solution (HBSS) with penicillin-streptomycin. The bovine eyes were transported to MB Research on 08 Feb 2018, within
24 hours of harvest.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 ml of test article, ethanol, or MEM solution was applied to the epithelium of each of the three test article corneas
Duration of treatment / exposure:
10 (±1) minutes
Duration of post- treatment incubation (in vitro):
All corneas were incubated at 32 (±1)°C for an additional two hours at which time the MEM solution in the
anterior and posterior chambers was removed and the holders refilled with fresh MEM solution
Number of animals or in vitro replicates:
3
Details on study design:
Following the pretest observations, the MEM solution was removed from the anterior chamber. A volume of
0.75 ml of test article, ethanol, or MEM solution was applied to the epithelium of each of the three test
article corneas, three positive control corneas and three negative control corneas in a manner which
ensured that the entire cornea was covered. Test article corneas were dosed via the open-chamber
method. The negative and positive controls were dosed via the closed-chamber method.
All holders and corneas were placed in a horizontal position (anterior chamber facing upward) in the
32 (±1)°C water-jacketed incubator (VWR, model: 1815 TC). After 10 (±1) minutes), the test article,
ethanol, or MEM was removed from the epithelium of the cornea and anterior chamber of the holder by
washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red.
The anterior and posterior chambers of the holders were then refilled with fresh MEM solution. Opacity
measurements were made following the 10-minute exposure and MEM solution refill.
All corneas were incubated at 32 (±1)°C for an additional two hours at which time the MEM solution in the
anterior and posterior chambers was removed and the holders refilled with fresh MEM solution. A
measurement of opacity was taken with each treated cornea compared to the blank supplied wit h the
OP-KIT. This reading was used in the final IVIS calculations.
Immediately following the two-hour opacity measurement, the MEM solution was removed from the
anterior chamber and replaced with 1.0 ml of 0.4% sodium fluorescein solution in Dulbecco's phosphatebuffered
saline (PBS). Each holder was then returned to the 32 (±1)°C incubator in a horizontal position
(anterior chamber facing upward) ensuring contact of the fluorescein with the cornea.
The Spectronic 20-D Colorimeter Spectrophotometer (Milton/Roy, model: 333175) was calibrated prior to
use on the same day of dosing. Calibration entailed first adjusting the wavelength to 490nm and the
transmittance to 0.0%. The transmittance for a sample of fresh distilled water (in a cuvette, inserted into
the sample compartment) was adjusted to 100.0% on the spectrophotometer. The mode was then
switched to absborance before collecting optical density study data. After 90 (±5) minutes, the fluid from
the posterior chamber of each corneal holder was removed and the amount of dye that passed through
the cornea (permeability) was measured as the optical density at 490nm (i.e., the OD490nm) by a
spectrophotometry.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Remarks:
10 minutes exposure
Run / experiment:
1
Value:
8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Remarks:
10 minutes
Run / experiment:
2
Value:
10
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Remarks:
10 minutes
Run / experiment:
3
Value:
7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Remarks:
2 hours
Run / experiment:
1
Value:
25
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Remarks:
2 hours
Run / experiment:
2
Value:
21
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Remarks:
2 hours
Run / experiment:
3
Value:
33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
31.73
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 3-aminopropyldiethylamine and 2-piperazin-1-ylethylamine was determined to moderate eye irritant in BCOP study according OECD 437.
Executive summary:

4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 3-aminopropyldiethylamine and 2-piperazin-1-ylethylamine was determined to moderate eye irritant in BCOP study according OECD 437.