Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 January 2018 - 4 October 2018
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
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
9th October 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction products of 2,3-epoxypropyl phenyl ether and 2,2'-iminodi(ethylamine)
EC Number:
948-518-4
Molecular formula:
(C13H23N3O2 . C31H43N3O6)x
IUPAC Name:
Reaction products of 2,3-epoxypropyl phenyl ether and 2,2'-iminodi(ethylamine)
Test material form:
liquid
Details on test material:
PGE-DETA adduct
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Olin Corporation Batch:17-09-502-04 QM 14K17-54
- Expiration date of the lot/batch: 30 October 2019
- Purity: Approx. 97% (including the oligomeric isomers)
Storage Conditions: At room temperature
Stability in Solvent: Not stable in water


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dosed neat.

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
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. The corneae were isolated on the same day after delivery of the eyes.

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 minute exposure at 32 ± 1 °C in the water-bath.
Duration of post- treatment incubation (in vitro):
Two hour incubatoin at 32 ± 1 °C
Number of animals or in vitro replicates:
3 replicates each for controls and Test Item
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Preparation of Corneae:
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.
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).

QUALITY CHECK OF THE ISOLATED CORNEAS
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.

NUMBER OF REPLICATES
Sets of three corneae were used for treatment with the test item and for the negative and positive controls.

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

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

APPLICATION DOSE AND EXPOSURE TIME
0.75ml, 10 minutes

TREATMENT METHOD: [closed chamber / open chamber]
not specified

POST-INCUBATION PERIOD: yes, 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
12 times with EMEM containing phenol red and 3 times with cMEM, fresh cMEM was added into the anterior compartment.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France)

- Corneal permeability: Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was 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).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
Opacity
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0).
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.
Permeability
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.

In vitro Score (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.

DECISION CRITERIA: The decision criteria used was that indicated in the TG.

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.
If the result in the first testing run is considered borderline (predictions from the 3 corneae are non-concordant), such that:
• 2 of the 3 corneae give discordant predictions from the mean of all corneae, or,
• 1 of the 3 corneae gives a discordant prediction from the mean of all 3 corneae, and the discordant result is >10 IVIS units from the cut-off threshold of 55.
A second testing run will be considered, as well as a third one in case of discordant mean IVIS results between the first two testing runs.






Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Test Item
Value:
89.32
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Any other information on results incl. tables

 Test Group Opacity value = Difference (t130-t0) of Opacity        Permeability at 490 nm (OD490)  IVIS  Mean IVIS  Proposed in vitro Irritancy Score
     Mean   Mean     
 Negative Control  1  0.67  0.0191  0.100 0.55#    3.87   2.17 1.33#  No Category
  Negative Control  1    0.060        
  Negative Control  0   0.050         
 Positive Control  79.33*    1.459*#    101.22#  99.63 100.31#  Category 1
 Positive Control  77.33*    1.300*#    96.83#    
 Positive Control  79.33*   1.569*#     102.87#    
 Test item  57.33*    2.095*#    88.76#  89.32 90.00# Category 1 
 Test item  62.33*    2.099*#    93.82#    
 Test item  56.33*    2.072*#    87.41#    

* corrected values

# The mean IVIS of the negative control is slightly above the Historical Control Data. If the first cornea of the negative control is determined as outlier especially due to the high permeability value of 0.191 and will not be considered in the evaluation (shown as struck through values) and then set to zero. Since the values of the positive control and the test item depend on a correction factor produced with the negative control, the new calculated values designated with # affect only the permeability values and result then in the same categorization as before. Therefore and in accordance with the sponsor a repeat experiment was not considered.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl Ph ether is serious eye damaging (CLP/EPA/GHS (Cat 1).
Executive summary:

This in vitro study was performed to assess the corneal damage potential of 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl Ph ether by means of the BCOP assay using fresh bovine corneae.

The study was performed twice. The first study was declared as invalid due to technical problems. This report reflects only the data of the repeat experiment.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 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. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t130).

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 (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae was observed.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl Ph ether caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 90.00. According to OECD 437 (see table in chapter 3.9.3) the test item is classified as serious eye damaging (CLP/EPA/GHS (Cat 1).