Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed in a GLP lab in accordance with OECD test guidelines with one minor deviation that was considered not to affect the integrity of the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
Control groups data and documentation were shared with another study. This deviation was considered not to affect the integrity of the study.
GLP compliance:
yes (incl. certificate)
Remarks:
OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Off white solid
Details on test material:
- Name of test material (as cited in study report): Glycyl
- Expiration date of the lot/batch: 04 August 2015
- Storage condition of test material: Room temperature in the dark

Test animals / tissue source

Species:
other: Cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Local abattoir
- Age at study initiation: 12 to 60 months old

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

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
Amount / concentration applied:
0.75 mL of the test substance present at 20% w/v in a 0.9% w/v sodium chloride solution.
Duration of treatment / exposure:
240 minutes at 32 ± 1°C followied by three rinces in fresh minimum essential medium (MEM).
Observation period (in vivo):
Opacity readings were taken and coreneas were observed visually following MEM rince. Permeability to sodium fluoresein was also evaluated.
Number of animals or in vitro replicates:
Nine eyes were used during the study, corneas with opacity values close to the median value were used.
Three corneas were issued to each group, test group, positive and negative control group.
Details on study design:
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 (epithelial side uppermost) 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 (MEM) 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.

A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

REMOVAL OF TEST SUBSTANCE
- Washing: Corenas were rinced three times with fresh MEM.
- Time after start of exposure: 240 minutes
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM without phenol red. The anterior chamber was refilled with fresh complete MEM 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 ± 1 ºC for 90 minutes

SCORING SYSTEM:

Opacity Measurement:
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permeability Measurement:
After incubation the medium in the posterior chamber of each holder was decanted and retained.

360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

In Vitro Irritancy Score:
The following formula was used to determine the In Vitro Irritancy Score: In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value).
In Vitro Irritancy Score: ≤ 3 - No category. Not requiring classification to UN GHS or EU CLP
In Vitro Irritancy Score: > 3; ≤55 - No prediction of eye irritation can be made
In Vitro Irritancy Score > 55 -Category 1. UN GHS or EU CLP Causes serious eye damage

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.


TOOL USED TO ASSESS SCORE: fluorescein permeability

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Remarks:
Negative Control
Basis:
mean
Time point:
other: 240 minutes
Score:
2
Reversibility:
not specified
Remarks on result:
other: Mean of the post-treatment − pre-treatment values
Irritation parameter:
cornea opacity score
Remarks:
Positive Control
Basis:
mean
Time point:
other: 240 minutes
Score:
75.3
Reversibility:
not specified
Remarks on result:
other: Mean corrected value
Irritation parameter:
cornea opacity score
Remarks:
Test substance
Basis:
mean
Time point:
other: 240 minutes
Score:
1
Reversibility:
not specified
Remarks on result:
other: Mean corrected value
Irritation parameter:
other: Permeability: Negative Control
Basis:
mean
Time point:
other: 240 minutes
Score:
0.012
Irritation parameter:
other: Permeability: Positive Control
Basis:
mean
Time point:
other: 240 minutes
Score:
1.488
Remarks on result:
other: Mean corrected value
Irritation parameter:
other: Permeability: Test substance
Basis:
mean
Time point:
other: 240 minutes
Score:
0.037
Remarks on result:
other: Mean corrected value
Irritant / corrosive response data:
The In Vitro irritancy scores are summarized as follows:
Test Item: 1.6
Negative Control: 2.2
Positive Control: 97.7
Other effects:
The corneas treated with the test item were cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment

Any other information on results incl. tables

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment Cornea
Number
Opacity Permeability (OD) InVitroIrritancyScore
Pre-Treatment Post-Treatment Post-Treatment−
Pre-Treatment
Corrected
Value
  Corrected
Value
Negative
Control⊕
12 3 6 3   0.009    
18 3 5 2   0.012    
19 3 4 1   0.014    
      2.0*   0.012♦   2.2
Positive
Control⊕
1 6 85 79 77 1.505 1.493  
3 2 77 75 73 1.393 1.381  
5 4 82 78 76 1.601 1.589  
        75.3•   1.488• 97.7
Test Item 22 3 8 5 3 0.061 0.049  
23 1 3 2 0.0 0.064 0.052  
24 2 3 1 0.0 0.022 0.010  
        1.0•   0.037• 1.6

OD = Optical density

* = Mean of the post-treatment − pre-treatment values

♦ = Mean permeability

• = Mean corrected value

⊕ = Control group shared with Harlan study number 41500023

Table 2: Corneal Epithelium Condition Post Treatment

Treatment Cornea Number Observation
Post Treatment
NegativeControl⊕ 12 Clear
18 Clear
19 Clear
PositiveControl⊕ 1 Cloudy
3 Cloudy
5 Cloudy
Test Item 22 Clear
23 Clear
24 Clear

⊕ = Control group shared with Harlan study number 41500023

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
No category. Not requiring classification to UN GHS or EU CLP.
Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine corneain vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

 

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/ EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. 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 test item is classified according to the prediction model below:

IVIS

CLASSIFICATION

≤ 3

No category. Not requiring classification to UN GHS or EU CLP

>3; ≤55

No prediction of eye irritation can be made

>55

Category 1.UN GHS or EU CLP Causes serious eye damage


 

The In-Vitro irritancy scores are summarized as follows:

Treatment

InVitroIrritancy Score

Test Item

1.6

Negative Control

2.2

Positive Control

97.7

 

 

No category. Not requiring classification to UN GHS or EU CLP.