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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:
key study
Study period:
The study was conducted on 19 July 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 guidelineopen allclose all
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of Inspection: 18/07/2017 - 20/07/2017 Date of Issue: 28/11/2017

Test material

Constituent 1
Chemical structure
Reference substance name:
trans-cinnamic acid
EC Number:
205-398-1
EC Name:
trans-cinnamic acid
Cas Number:
140-10-3
Molecular formula:
C9H8O2
IUPAC Name:
3-phenylacrylic acid
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 20171106
- Expiration date of the lot/batch: 10 April 2020
- Purity test date: 99.0% w/w

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark, over silica gel

Test animals / tissue source

Species:
other: excised bovine corneas
Strain:
other: excised bovine corneas
Details on test animals or tissues and environmental conditions:
Source of Bovine Eyes
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

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): 0.1970 g
- Concentration (if solution): N/A

Duration of treatment / exposure:
240 minutes
Observation period (in vivo):
N/A
Duration of post- treatment incubation (in vitro):
N/A
Number of animals or in vitro replicates:
Three
Details on study design:
SELECTION AND 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 65 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

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. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.


NUMBER OF REPLICATES
Three

NEGATIVE CONTROL USED
Identification: Sodium chloride 0.9% w/v
Lot: 3012487
Purity: 0.9%
Supplier: Aguettant Ltd
Expiry Date: 01 November 2018
Storage Conditions: Room temperature

POSITIVE CONTROL USED
Identification: Imidazole
Batch: 162411
Supplier: Fisher Scientific
Purity: >99%
Expiry Date: 10 April 2019
Storage Conditions: Room temperature in the dark

APPLICATION DOSE AND EXPOSURE TIME
0.1970g; 240 minutes

TREATMENT METHOD:
The EMEM was removed from the anterior chamber of the BCOP holder and the test item or control items were applied to the cornea. Approximately 0.1970 g of the solid test item was found to adequately cover the corneal surface. 0.75ml of each control item was 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 240 minutes.


POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the
anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red
- POST-EXPOSURE INCUBATION: N/A. 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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Using a calibrated opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)
- Others: pertinent visual observations

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
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

DECISION CRITERIA: In accordance with OECD TG

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
36.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None noted

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes - the test is considered acceptable if if the negative control produced an In Vitro Irritancy Score which is less than or equal to theupper limit for background opacity and permeability values during 2017 for bovine corneas treated with the respective negative control which in this case was ≤2.3 for opacity and ≤0.044 for permeability. These criteria were satisifed
- Acceptance criteria met for positive control: Yes - the test is considered acceptable if the positive control produced an in vitro irritancy score which fell within two standard deviations of the historical mean during 2017 for this testing facility which in this case was 71.2 to 132.9. The in vitro irritancy score was 81.7 therefore this is satisfied.

Any other information on results incl. tables

Corneal Opacity and Permeability Measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in the table below.

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea

Opacity

Permeability (OD492)

In VitroIrritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment – Pre-Treatment

Corrected Value

 

Corrected Value

 

Negative Control

1

7

12

5

 

0.004

 

 

2

5

6

1

 

0.005

 

 

3

6

6

0

 

0.003

 

 

 

 

 

2.0*

 

0.004*

 

2.1

 

Positive Control

4

5

67

62

60.0

1.815

1.811

 

5

5

74

69

67.0

1.116

1.112

 

6

3

64

61

59.0

1.030

1.026

 

 

 

 

 

62.0

 

1.316*

81.7

 

Test Item**

7

5

55

50

48.0

0.037

0.033

 

10

6

43

37

35.0

0.012

0.008

 

11

5

32

27

25.0

0.005

0.001

 

 

 

 

 

36.0*

 

0.014*

36.2

OD = Optical Density; * = Mean ** = Test item could not be formulated to a concentration of 20% w/v solution in sodium chloride 0.9% w/v.

Corneal Epithelium Condition

The condition of each cornea is given in the table below.

Treatment

Cornea Number

Observation

Post-Treatment

 

Negative Control

1

Clear

2

Clear

3

Clear

 

Positive Control

4

Cloudy

5

Cloudy

6

Cloudy

 

Test Item*

7

Cloudy

10

Cloudy

11

Cloudy

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.

In Vitro Irritancy Score

The in vitro irritancy scores are summarised as follows.

Treatment

In VitroIrritancy Score

Test Item

36.2

Negative Control

2.1

Positive Control

81.7

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
No prediction of eye irritation can be made.
Executive summary:

Introduction

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 cornea in 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.

Method

The test item was applied neat 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).

Data Interpretation

IVIS

UN GHS

3

No category

>3;55

No prediction can be made

> 55

Category 1

Results

The In Vitro irritancy scores are summarized as follows:

Treatment

In VitroIrritancy Score

Test Item

36.2

Negative Control

2.1

Positive Control

81.7

Conclusion

No prediction of eye irritation can be made.