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Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted on 21 November 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:
2017

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)
Version / remarks:
July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1'-[isopropylidenebis(p-phenyleneoxy)]bis[3-[[2-[(2-aminoethyl)amino]ethyl]amino]-2-propanol
Cas Number:
108248-15-3
Molecular formula:
C29H50N6O4
IUPAC Name:
1,1'-[isopropylidenebis(p-phenyleneoxy)]bis[3-[[2-[(2-aminoethyl)amino]ethyl]amino]-2-propanol
Constituent 2
Chemical structure
Reference substance name:
3,3'-[iminobis(ethane-2,1-diylimino)]bis{1-[4-(2-{4-[3-({2-[(2-aminoethyl)amino]ethyl}amino)-2-hydroxypropoxy]phenyl}propan-2-yl)phenoxy]propan-2-ol}
Molecular formula:
C54H87N9O8
IUPAC Name:
3,3'-[iminobis(ethane-2,1-diylimino)]bis{1-[4-(2-{4-[3-({2-[(2-aminoethyl)amino]ethyl}amino)-2-hydroxypropoxy]phenyl}propan-2-yl)phenoxy]propan-2-ol}
Constituent 3
Chemical structure
Reference substance name:
3-[4-(2-{4-[3-({2-[(2-aminoethyl)amino]ethyl}amino)-2-hydroxypropoxy]phenyl}propan-2-yl)phenoxy]propane-1,2-diol
Molecular formula:
C25H39N3O5
IUPAC Name:
3-[4-(2-{4-[3-({2-[(2-aminoethyl)amino]ethyl}amino)-2-hydroxypropoxy]phenyl}propan-2-yl)phenoxy]propane-1,2-diol
Constituent 4
Reference substance name:
Polymeric Adducts of 2,​2'-​[(1-​methylethylidene)​bis(4,​1-​phenyleneoxymethylen​e)​]​bis-oxirane and N1-​(2-​aminoethyl)​-1,​2-ethanediamine
IUPAC Name:
Polymeric Adducts of 2,​2'-​[(1-​methylethylidene)​bis(4,​1-​phenyleneoxymethylen​e)​]​bis-oxirane and N1-​(2-​aminoethyl)​-1,​2-ethanediamine
Constituent 5
Reference substance name:
Unknown components
IUPAC Name:
Unknown components
Specific details on test material used for the study:
- Oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine
- CAS No. 31326-29-1
- Analytical purity: 94%
- Lot/batch No.: Ei 3041
- Expiration date of the lot/batch: 01 May 2021
- Isomers composition: Not applicable
- Other: Storage conditions: Room tremperature
- Appearance: Colorless solidified liquid as determined by Envigo CRS GmbH laboratory staff

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Number of animals: not specified - multiple
- 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 supplemented with streptomycin / penicillin at ambient temperature.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes.
- indication of any existing defects or lesions in ocular tissue samples: None
- Indication of any antibiotics used: Not reported

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL per cornea - 20% solution (w/v) in saline
Duration of treatment / exposure:
240 Minutes
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
3 replicates
Details on study design:
Experimental Design and Study Conduct

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 transported to the laboratory in HBSS supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes.

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 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 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 the negative and positive controls.

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 solution or negative or positive control at a volume of 0.75 mL each on the 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 control items, respectively, were rinsed off from the application side with saline, and fresh incubation medium was added into the anterior compartment and opacity was measured (t240). The opacity measurement is described below.
In the second step of the assay, permeability of the cornea was determined. The permeability measurement is described below.

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 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 ± 10 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 and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
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).

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Value:
> 41.17 - < 47.99
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Not categorized
Positive controls validity:
valid
Remarks:
Category 1
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Value:
> 36.67 - < 40.67
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Not categorized
Positive controls validity:
valid
Remarks:
Category 1
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
This in vitro study was performed to assess the corneal damage potential of 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine 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) solution in saline of the test item 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity 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 (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.17).
The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 105.73) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).
Relative to the negative control, the test item caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 43.76 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 no prediction for the damage hazard of the test item to the eye can be made.

In vivo

Other effects:
None

Any other information on results incl. tables

Results after 240 minutes incubation time:

 Test Group  Opacity value = Difference (t240-t0) of Opacity  Permeability at 490 nm (OD490)  IVIS  Proposed in vitro Irritancy Score
         
 Negative Control  0  0.055  0.83  Not categorized
   0  0.057  0.86  Not categorized
   1  0.055  1.83  NOt categorized
 Positive Control  104.67  0.106  106.26  Category 1
   95.67  0.168  98.19  Category 1
   109.67  0.204  112.73  Category 1
 Test Item  36.67  0.300  41.17  No hazard prediction can be made
   40.67  0.488  47.99   No hazard prediction can be made
   37.67  0.297  42.13   No hazard prediction can be made

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine is not serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for the damage hazard cannot be made (GHS).
Executive summary:

This in vitro study was performed to assess the corneal damage potential of 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine 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) solution in saline (0.9% (w/v) NaCl in deionised water) of the test item 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine, the positive, and the negative controls were applied to 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 and opacity 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’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with diethylenetriamine caused an increase of the corneal opacity and permeability compared with the values caused by the negative control. The calculated mean in vitro irritancy score was 43.76. According to OECD 437 (see table in chapter 3.8.3) the test item the test item is not classified as serious eye damaging (CLP/EPA/GHS (Cat 1) but the test item’s hazard for eye damaging cannot be predicted.

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