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Toxicological information

Eye irritation

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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the O.E.C.D. test guideline 437 with GLP compliance.

Data source

Reference Type:
study report
Report date:

Materials and methods

Principles of method if other than guideline:
Based on O.E.C.D. test guideline 437, Bovine Corneal Opacity and Permeability Test Method.
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-Isopropylidenedicyclohexanol, oligomeric reaction products with 1-chloro-2,3-epoxypropane
EC Number:
EC Name:
4,4'-Isopropylidenedicyclohexanol, oligomeric reaction products with 1-chloro-2,3-epoxypropane
Cas Number:
Molecular formula:
4,4'-Isopropylidenedicyclohexanol, oligomeric reaction products with 1-chloro-2,3-epoxypropane
Constituent 2
Reference substance name:
Cyclohexanol, 4,4'-(1-methylethylidene)bis-, polymer with 2-(chloromethyl)oxirane
Cyclohexanol, 4,4'-(1-methylethylidene)bis-, polymer with 2-(chloromethyl)oxirane
Details on test material:
As per IUCLID Sections 1.1. 1.2. and 4.1.

Test animals / tissue source

other: bovine
Details on test animals or tissues and environmental conditions:
Study was in vitro.

Test system

unchanged (no vehicle)
Amount / concentration applied:
0.750 ml
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
Two hours
Number of animals or in vitro replicates:
no animals, study was in vitro.
Details on study design:
Bovine eyes were obtained from a local abattoir as a by-product from fleshly slaughtered animals (J.W. TREUTH & SONS, Inc., Baltimore, MD). The eyes were excised and then placed in Hanks’ Balanced Salt Solution, containing Penicillin/Streptornycin (HBSS), and transported to the laboratory on ice packs. Immediately upon receipt of the eyes into the laboratory, preparation of the corneas was initiated. The isolated corneas were then stored in a petri dish containing HBSS until they were mounted in a corneal holder. The corneas were mounted in the holders with the endothelial side against the 0-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and the screws were tightened. Starting with the posterior chamber, the two chambers were then filled with Minimum Essential Medium (EMEM) without phenol red, containing 1% fetal bovine serum and 2mM L-glutamine (Complete MEM). The corneal holders were incubated at 32 ± 1°C for a minimum of 1 hour.

After a minimum of 1 hour of incubation, the corneas were removed from the incubator. The medium was removed from both chambers and replaced with fresh Complete MEM. The initial opacity was determined for each cornea using a Electro Designs OP-KIT opacitometer. Three corneas, whose initial opacity readings were close to the median opacity for all the corneas, were selected as the negative control corneas. The medium was then removed from the anterior chamber and replaced with the test article, positive control (ethanol), or negative control (sterile water)

Due to its viscous nature, the test substance was administered directly onto the exposed cornea using a positive displacement pipet. Each treated cornea was completely covered with the test article. The comeas were incubated in the presence of the test article at 32 +/- 1°C for 10 minutes. After the 10-minute exposure time, the control or test article treatments were removed. The epithelial side of the corneas was washed at four times with Complete MEM to ensure total removal of the control or test substance. The test substance observed to form oil droplets in the media during the rinsing process. Therefore a cotton tipped applicator was used to remove the test article from the chamber. The anterior chambers were refilled with fresh Complete MEM (without phenol red) and an opacity measurement was performed. The corneas were returned to the incubator for approximately 2 hours after which a final measure of opacity was obtained.

After the final two hour opacity nieasurement was performed, the medium was removed from both chambers of the holder, The posterior chamber was filled with flesh Complete MEM and 1 mL of a 4 mg/mL fluorescein solution was added to the anterior chamber. The corneas were then incubated in a horizontal position (anterior side up) for approximately 90 minutes at 32 ± 1°C. At the end of the 90-minute incubation period, the medium was removed from the posterior chamber and placed into tubes Alquots of 360 pL from the numbered tubes were placed into their designated wells on a 96-well plate. The optical density at 490 nm (0D490) was determined using a Molecular Devices Vmax kinetic microplate reader. The change in opacity for each cornea (including the negative control corneas) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting from each the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of each cornea for that treatment condition. The following formula was used to determine the in vitro score: In Vitro Score = Mean Opacity Value + (15 x Mean 0D490 Value).

Results and discussion

In vivo

Irritation parameter:
other: premeability.
Time point:
other: two hours
ca. 0.3
not specified
Remarks on result:
other: The mean In Vitro Score = 0.3
Irritant / corrosive response data:
The mean In Vitro Score for the test substance was 0.3.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Migrated information Criteria used for interpretation of results: other: Based on the modification of the Sina prediction model proposed by Vanparys, et al. (1993), where a substance that induces an in vitro score < 3 would be predicted to be a non-eye irritant.
The test substance is predicited to be a non eye irritant.
Executive summary:

The test substance, 4,4'-Isopropylidenedicyclohexanol, oligomeric reaction products with 1 -chloro-2,3 -epoxypropane, was tested for potential eye irritation effects in the in vitro O.E.C.D. test guideline 437 BCOP test with GLP compliance. Based on an In Vitro score of 0.3 the test substance is not an eye irritant.