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Diss Factsheets

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted on 12 June 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and in compliance with GLP.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
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 2013
according to guideline
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
EC No. 440/2008
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Cas Number:
Molecular formula:
C26 H24 N2 O2
Constituent 2
Chemical structure
Reference substance name:
1,3,5-triazine-2,4,6-triyltris(oxybenzene-4,1-diylpropane-2,2-diylbenzene-3,1-diylpropane-2,2-diylbenzene-4,1-diyl) tricyanate
Molecular formula:
1,3,5-triazine-2,4,6-triyltris(oxybenzene-4,1-diylpropane-2,2-diylbenzene-3,1-diylpropane-2,2-diylbenzene-4,1-diyl) tricyanate
Constituent 3
Chemical structure
Reference substance name:
4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl cyanate
Molecular formula:
4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl cyanate
Constituent 4
Chemical structure
Reference substance name:
4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenyl N-{(E)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate
Molecular formula:
4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenyl N-{(E)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate
Constituent 5
Chemical structure
Reference substance name:
4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl N-{(Z)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate
Molecular formula:
4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl N-{(Z)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate
Constituent 6
Chemical structure
Reference substance name:
4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl N-[(E)-(4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenoxy)methylidene]carbamimidate
Molecular formula:
4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenyl N-[(E)-(4-{2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl}phenoxy)methylidene]carbamimidate
Constituent 7
Chemical structure
Reference substance name:
Molecular formula:
Constituent 8
Chemical structure
Reference substance name:
4-[2-(3-{2-[4-({4,6-bis[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]-1,3,5-triazin-2-yl}oxy)phenyl]propan-2-yl}phenyl)propan-2-yl]phenyl N-{(E)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate
Molecular formula:
4-[2-(3-{2-[4-({4,6-bis[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]-1,3,5-triazin-2-yl}oxy)phenyl]propan-2-yl}phenyl)propan-2-yl]phenyl N-{(E)-[4-(2-{3-[2-(4-cyanatophenyl)propan-2-yl]phenyl}propan-2-yl)phenoxy]methylidene}carbamimidate
Constituent 9
Reference substance name:
Polymeric impurites: reaction product of Bishenol M with cyanogen bromide
Molecular formula:
not available
Polymeric impurites: reaction product of Bishenol M with cyanogen bromide
Details on test material:
Identification: Sodium benzoate
Purity: >99.5%
Physical state/Appearance: white granular solid
Expiry Date: 09 May 2022
Storage Conditions: room temperature over silica gel
Specific details on test material used for the study:
The test item was supplied by or on behalf of the Sponsor including the following information:
Identification: 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate
CAS No.: 127667-44-1
Batch: FAR366085A
Purity: Not indicated by the Sponsor
Appearance: Highly viscous yellowish liquid
Expiry Date: 29 September 2019
Storage Conditions: In the refrigerator
Stability in Solvent: Not indicated by the Sponsor
Purpose of Use: Industrial chemical

Test animals / tissue source

other: Eyes from >9 month old cattle
other: Not applicable
Details on test animals or tissues and environmental conditions:
Test System: Freshly isolated bovine cornea (at least 9 month old donor cattle)
Rationale: OECD 437
Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany

Collection of Bovine Eyes
Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item or control items were applied to the cornea.
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
3 corneas per treatment
Details on study design:
Preparation of Corneae
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. The corneae were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) 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. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).
The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and for the negative and positive controls, respectively.

Exposure of the Corneae to the Test Groups
The corneae were distributed as follows:
Groups Number of Corneae
1 Negative Control 3
2 Positive Control 3
3 Test Item 3
The anterior compartment received the test item suspension or the negative or positive controls at a volume of 0.75 mL ensuring sufficient coverage of the epithelial surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted 240 minutes.
Afterwards, the test item or the control items, respectively, were each rinsed off from the according application sides with saline, and fresh incubation medium was added into the anterior compartment and opacity was measured (t240). The opacity measurement is described in chapter 3.5.
In the second step of the assay, permeability of the cornea was determined. The permeability measurement is described in chapter 3.6.

Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. 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.
After exposure of the corneae to the different test groups, and after rinsing the opacity value was determined again (t240).

Permeability Determination
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Data Recording
The data generated were recorded in the raw data file. The results are presented in tabular form, including experimental groups with the test item as well as negative and positive controls.

Data Evaluation
The change of the opacity value of each treated cornea or of the positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
The corrected OD490 value of each cornea treated with positive control or test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.
IVIS Calculation
The following formula is used to determine the IVIS of the negative control:

IVIS = opacity value + (15 x OD490 value)

The following formula is used to determine the IVIS of the positive control and the test item:

IVIS = (opacity value – corrected opacity value mean negative control) + (15 x corrected OD490 value)

The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the IVIS score obtained, the test item is classified into the following Category according to OECD guideline 437:

≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1

Criteria for Determination of a Valid Test
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

Irritation parameter:
in vitro irritation score
> 0.08 - < 1.56
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation

In vivo

Irritant / corrosive response data:
Corneal epithelium condition
The corneas treated with the test item were cloudy post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation

In vitro irritancy scores:
The in vitro irritancy score for the test item was 0.94
The in vitro irritancy score for the negative control was 1.36
The in vitro irritancy score for the positive control was 126.28
Other effects:
No other effects were observed

Any other information on results incl. tables

Results after 240 Minutes Treatment Time

Test Group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)



Proposedin vitroIrritancy Score









Negative Control







Not categorized







Positive Control





Category 1







Test Item





Not categorized







*corrected values

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
In conclusion, according to the current study and under the experimental conditions reported, 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate is not categorized (GHS).
Executive summary:

This in vitro study was performed to assess thecorneal damage potentialof4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspensionin saline (0.9% (w/v) NaCl in deionised water) of the test item 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate as well as the positive and the negative controls were each applied to different corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae andopacity was measured again (t240).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (CLP/EPA/GHS (Cat 1)).


Relative to the negative control, the test item 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate did not cause an increase of the corneal opacity and permeability. The calculated meanin vitroirritancy score was 0.94.According to OECD 437 the test item is not categorized (GHS).