Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 20 September 2018 and Experimental completion date: 02 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

Materials and methods

Test guideline
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)
Version / remarks:
09 October 2017
Deviations:
yes
Remarks:
these deviations were considered to have not affected the integrity or validity of the study (see below)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: crystal powder
Details on test material:
- Synonym (Trade Name): ADDITIVE® 9735
- CAS Number: 102-40-9
- Molecular Formula: C10H14O4
- Molecular Weight: 198 g/mol
- Appearance: White crystal powder
- Expiration date: 30 August 2019
- Purity: 100%

Test animals / tissue source

Species:
other: Eyes from adult cattle (typically 12 to 60 months old)
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Obtained from a local abattoir as a by-product from freshky slaughtered animals.
- Number of animals: not applicable
- Characteristics of donor animals (e.g. age, sex, weight): Eyes from adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 µg/mL

Test system

Vehicle:
other: Sodium Chloride 0.9% w/v.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
For the purpose of this study the test item was prepared as a 20% w/v solution in sodium chloride 0.9% w/v.
Duration of treatment / exposure:
240 minutes at 32°C.
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
90 minutes at 32°C.
Number of animals or in vitro replicates:
- Test item: triplicate
- Negative control: triplicate
-
Positive control: 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.
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 : negative control, positive control and test item were all preformed in triplicate

NEGATIVE CONTROL USED : The negative control item, sodium chloride 0.9% w/v, was used as supplied.

SOLVENT CONTROL USED (if applicable) : negative control and solvent control (vehicle) are the same

POSITIVE CONTROL USED: The positive control item, Imidazole, was used as a 20% w/v solution in sodium chloride 0.9% w/v.

APPLICATION DOSE AND EXPOSURE TIME : 0.75 Ml of test item preparation was applied (test item was prepared as a 20% w/v solution in sodium chloride 0.9% w/v).

TREATMENT METHOD: [closed chamber / open chamber]

POST-INCUBATION PERIOD: yes, 90 minutes at 32°C (application of Sodium Fluorescein)

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Cornea were rinsed 3 times with fresh complete EMEM contaning 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.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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.

- Corneal permeability: 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.
The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

- Others : 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)

Visual Observations: The condition of the cornea was visually assessed post treatment.

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.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS UN GHS
≤ 3: No Category
>3; ≤ 55: No prediction can be made
> 55: Category 1


DECISION CRITERIA: Criteria for an Acceptable test:

For an acceptable test the following positive control criterion should be achieved:
- 20% w/v Imidazole was used for positive control purposes. The test was 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. Therefore the In Vitro Irritancy Score should fall within the range of 71.2 to 132.9.

For an acceptable test the following negative control criteria should be achieved:
- Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2017 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤2.3 and for permeability ≤0.44


Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
test item
Value:
1.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No category. Nor requiring classification to UN GHS or EU CLP.
Irritation parameter:
in vitro irritation score
Run / experiment:
negative control
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
positive control
Value:
88.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Corneal Epithelium Condition
The corneas treated with the test item were clear 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤2.3 and permeability ≤0.44. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the range of 71.2 to 132.9. The positive control acceptance criterion was therefore satisfied.





Any other information on results incl. tables

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD492)

In VitroIrritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

5

4

0

 

0.000

 

 

2

4

3

0

 

0.000

 

 

3

4

4

0

 

0.000

 

 

 

 

 

0.0*

 

0.000¨

 

0.0

Positive
Control

4

4

74

70

70.0

1.007

1.007

 

5

4

68

64

64.0

1.495

1.495

 

6

2

79

77

77.0

1.191

1.191

 

 

 

 

 

70.3·

 

1.231·

88.8

Test Item

7

5

6

1

1.0

0.043

0.043

 

9

4

5

1

1.0

0.016

0.016

 

10

4

6

2

2.0

0.027

0.027

 

 

 

 

 

1.3·

 

0.029·

1.8


OD= Optical density         * = Mean of the post-treatment -pre‑treatment values          ¨= Mean permeability                           ·= Mean corrected value

Corneal Epithelium Condition Post Treatment

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

Clear

9

Clear

10

Clear

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
No category. Not requiring classification to UN GHS or EU CLP.
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 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.

Method

The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v 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 anIn VitroIrritancy Score (IVIS). 

Data Interpretation

The test item is classified according to the prediction model as follows:

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 Vitro Irritancy Score (IVIS)

Test Item

1.8

Negative Control

0.0

Positive Control

88.8

 

Conclusion

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