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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From August 31, 2017 to August 31, 2017
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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
updated 26 July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Rape oil, reaction products with diethylenetriamine, di-Me sulfate-quaternized
EC Number:
308-732-5
EC Name:
Rape oil, reaction products with diethylenetriamine, di-Me sulfate-quaternized
Cas Number:
98219-63-7
Molecular formula:
C50H97N3O5S1 (Fatty amides C44:2- representative)
IUPAC Name:
Rape oil, reaction products with diethylenetriamine, di-Me sulfate-quaternized
Test material form:
solid

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Test system

Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
2 h
Number of animals or in vitro replicates:
Triplicate
Details on study design:
Preparation of Corneas: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading: The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test substance and three corneas to the positive control substances.

Treatment of Corneas: The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test substance or control substance were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes. At the end of the exposure period the test substance and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein: Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations: After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
10 minutes
Value:
4.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Inconclusive

Any other information on results incl. tables

Corneal epithelium condition: The corneas treated with the test substance were clear post treatment and partly cloudy post incubation. The corneas treated with the negative control substance were clear post treatment and post incubation. The corneas treated with the positive control substance were cloudy post treatment and post incubation.

Individual and mean corneal opacity and permeability measurements:

Treatment Cornea Number Opacity Permeability (OD) In Vitro Irritancy Score
Pre-Treatment Post-Treatment Post Incubation Post-Incubation - Pre-Treatment Corrected Value   Corrected Value
Negative Control 1 3 3 4 1   0.007    
2 3 2 2 0   0.011    
3 4 2 2 0   0.007    
        0.3*   0.008♦   0.5
Positive Control 4 6 40 37 31 30.7 0.794 0.786  
5 3 31 33 30 29.7 1.96 1.952  
6 2 24 26 24 23.7 1.885 1.877  
          28•   1.538• 51.1
Test substance 10 3 6 6 3 2.7 0.099 0.091  
11 3 4 9 6 5.7 0.073 0.065  
12 1 3 4 3 2.7 0.014 0.006  
          3.7•   0.054• 4.5

OD = Optical density * = Mean of the post-incubation − pre-treatment values ♦ = Mean permeability • = Mean corrected value

Corneal epithelium condition post treatment and post incubation

Treatment Cornea number Observation
Post treatment Post incubation
Negative Control 1 Clear Clear 
2 Clear Clear 
3 Clear Clear 
Positive Control 4 Cloudy Cloudy
5 Cloudy Cloudy
6 Cloudy Cloudy
Test substance 10 Clear Partly Cloudy
11 Clear Partly Cloudy
12 Clear Partly Cloudy

Results:

Treatment In Vitro Irritancy Score
Test substance 4.5
Negative control 0.5
Positive control 51.1

Applicant's summary and conclusion

Interpretation of results:
other: Inconclusive for classification
Conclusions:
Under the study conditions, eye irritation potential of the test substance was determined to be inconclusive based on bovine corneal opacity and permeability test (IVIS score – 4.5).
Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the test substance, ‘di-C16-18-satd. and C18-24-unsatd. AAEMIM-MS' (active: 100%), using Bovine Corneal Opacity Test (BCOP), according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. Preparation, selection and opacity reading of the corneas were performed as per guideline. Prepared corneas in triplicates were treated with each, test substance (750 µL), negative control (Sodium chloride 0.9% w/v) and positive control (Ethanol) substances at 32 ± 1ºC for 120 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete Eagle’s Minimum Essential Medium (EMEM) containing phenol red before a final rinse with complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1ºC for 90 minutes. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In vitro Irritancy Score (IVIS). The positive control IVIS was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied. The negative control resulted in opacity of <3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied. The test substance IVIS score obtained was 4.5, which is well below the corrosive limit of 55 and is slightly above the non-corrosive limit of 3; therefore no predictions could be made. Under the study conditions, no prediction of eye irritation could be made for the test substance (Envigo, 2018).