Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21-27 April 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:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
published in the Official Journal of the European Union L324, dated December 09, 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Yellow to brown, aromatic liquid
- Storage: At +10°C to +25°C under inert gas, kept in a tightly closed container and stored in a dry place, protected from heat and direct sunlight.

Test animals / tissue source

Species:
other: Bovine eyes from cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Source of eyes: Eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse. To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing 1% Penicillin/Streptomycin.

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 minutes
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
Not applicable
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: Only corneas from eyes free of defects were used. The corneas were dissected with a 2-3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with prewarmed Eagle’s Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.

QUALITY CHECK OF THE ISOLATED CORNEAS: After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer. A minimum of three corneaswith opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment, solvent and positive control groups (three corneas/group).

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% sodium chloride solution

POSITIVE CONTROL USED: 1% NaOH solution in aqua ad iniectabilia

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes: 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the exposure period the epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. No discolouration of phenol red was visible after exposure of the test item. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.
- POST-EXPOSURE INCUBATION: After rinsing, the corneas were incubated at 32±1°C for two hours. After this post-exposure incubation period, the corneas were examined.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32±1°C for 90±5 minutes. The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader. Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer using a standard 1 cm path length.
- Others (e.g, pertinent visual observations, histopathology): Corneal injury was assessed by evaluating the opacity and permeability of the cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: As specified in the guideline

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
-0.492
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: see Table 1

Any other information on results incl. tables

The corneas treated with the negative control item 0.9% sodium chloride solution revealed a mean opacity value of 0.359 ± 0.638 and a mean permeability value of 0.015 ± 0.009. The calculated IVIS value of 0.579 ± 0.521 was well below the cut-off value of 3 (UN GHS no category).

The corneas treated with the positive control item 1% NaOH in aqua ad iniectabilia revealed a mean opacity value of 72.589 ± 21.979 and a mean permeability value of 1.654 ± 0.124 compared to the solvent control. The calculated IVIS value of 97.399 ± 21.031 was within two standard deviations of the current historical mean and well above the cut-off value of 55. Hence, the acceptance criteria for the test were fulfilled.

Following treatment with “Karstedt concentrate” a mean opacity of -0.412 ± 0.023 and a mean permeability value of -0.005 ± 0.001 compared to the negative control were determined. The calculated IVIS of -0.492 ± 0.005 is below the cut-off value of 3 (UN GHS no category). Hence, the test item did not show severely irritant or corrosive properties and consequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification.

The IVIS values are given in the table below:

         Cornea No.

 Opacity (opacity units)

 Permeability (OD)

 IVIS      

 Per Cornea   

 Per group   

 Mean

 SD

 0.9% NaCl      

 1  -0.279  0.025  0.096  0.579        0.521      
 2  0.359  0.010  0.509
 3  0.996  0.009  1.131
 1% NaOH        4  96.215  1.626  120.605  97.399        21.031      
 5  68.804  1.546  91.994
 6  52.749  1.790  79.599
 Karstedt concentrate        7  -0.438  -0.004  -0.498  -0.492        0.005      
 8  -0.399  -0.006  -0.489
 9  -0.399  -0.006  -0.489

SD: standard deviation

OD: optical density

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro bovine corneal opacity and permeability assay conducted in accordance with OECD Test Guideline 437, and to GLP, "Karstedt concentrate" did not induce a response indicative of a corrosive or severe irritant. The calculated IVIS of -0.492 is below the cut-off value of 3 (UN GHS no category).
Executive summary:

In an in vitro bovine corneal opacity and permeability (BCOP) assay conducted in accordance with OECD Test Guideline 437 and to GLP, “Karstedt concentrate” was applied to isolated bovine corneas for 10 minutes, followed by an incubation period of 2 hours. The In Vitro Irritancy Score (IVIS) calculated from individual scores for induced opacity (decreased light transmission through the cornea) and permeability (passage of sodium fluorescein dye through the cornea) was -0.492 ± 0.005. The IVIS value is less than the cut-off value of 3 (no category) and consequently the test material is not classified as a severe irritant. The positive and negative controls were considered valid.

Under the conditions of this assay, “Karstedt concentrate” would not be classified as irritating nor corrosive to the eye according to UN GHS classification.