Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 December 2015 (experimental phase)
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
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
2013
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)
Version / remarks:
Method B.47 of Commission Regulation (EC) No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom

Test material

Constituent 1
Reference substance name:
Octadecanoic acid, reaction products with tetraethylenepentamine
EC Number:
276-033-1
EC Name:
Octadecanoic acid, reaction products with tetraethylenepentamine
Cas Number:
71799-54-7
Molecular formula:
not applicable (UVCB substance)
IUPAC Name:
Octadecanoic acid, reaction products with tetraethylenepentamine
Test material form:
solid: pellets
Details on test material:
- Storage condition of test material: Room temperature in the dark

Test animals / tissue source

Species:
other: bovine corneas
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

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL of the test item preparation
- Concentration (if solution): the test item was ground to a fine powder and prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution.

VEHICLE
- Concentration (if solution): 0.9% w/v sodium chloride solution
Duration of treatment / exposure:
240 min
Observation period (in vivo):
90 min fluorescein incubation
Number of animals or in vitro replicates:
each 3 corneas for test item, negative and positive control
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): The cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.
- Time after start of exposure: 240 min

SCORING SYSTEM: In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value) according to OECD guideline 437

TOOL USED TO ASSESS SCORE: opacity reading / fluorescein

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean after 240 min
Value:
1.5
Vehicle controls validity:
valid
Remarks:
= negative control
Negative controls validity:
valid
Remarks:
IVIS = 0.5 after 240 min
Positive controls validity:
valid
Remarks:
IVIS = 81.6 after 240 min
Other effects / acceptance of results:
Test item is not irritating to the eye.

Any other information on results incl. tables

RESULTS

 

Corneal Opacity and Permeability Measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1.

 

Table 1 Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD)

In VitroIrritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre-Treatment

Corrected Value

 

Corrected Value

Negative Control

5

3

3

0

 

0.016

 

 

18

3

3

0

 

0.014

 

 

22

3

4

1

 

0.008

 

 

 

 

 

0.3*

 

0.013

 

0.5

Positive Control

19

2

65

63

62.7

1.657

1.644

 

23

4

57

53

52.7

1.444

1.431

 

24

3

61

58

57.7

1.718

1.705

 

 

 

 

 

57.7

 

1.594

81.6

Test Item

26

2

3

1

0.7

0.124

0.111

 

27

2

2

0

0.0

0.071

0.058

 

28

2

3

1

0.7

0.058

0.045

 

 

 

 

 

0.4

 

0.072

1.5

OD =Optical density

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

♦ = Mean permeability

• = Mean corrected value

 

Corneal Epithelium Condition

 

The condition of each cornea is given in Table 2.

 

Table2 Corneal Epithelium Condition Post Treatment

Treatment

Cornea Number

Observation Post Treatment

Negative Control

5

Clear

18

Clear

22

Clear

Positive Control

19

Cloudy

23

Cloudy

24

Cloudy

Test Item

26

Clear

27

Clear

28

Clear

 

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.

 

In Vitro Irritancy Score

 

The In Vitro irritancy scores are summarized as follows:

 

Treatment

In Vitro Irritancy Score

Test Item

1.5

Negative Control

0.5

Positive Control

81.6

 

 

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity of <4.1 and permeability <0.105. The negative control acceptance criteria were therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
not irritating
Conclusions:
The study was conducted under GLP according to OECD guideline 437 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritating potential of Octadecanoic acid, reaction products with tetraethylenepentamine to the eye in vitro.
The mean IVIS of the test item is ≤ 3. Hence, the test item was considered to be non-irritating to the eye.
The study is suitable as a standalone study as the present study clearly reveals that Octadecanoic acid, reaction products with tetraethylenepentamine does not need to be classified as irritating to the eye, no further testing is required.
Executive summary:

The purpose of this OECD 437 GLP study 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.

 

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 In Vitro irritancy scores are summarized as follows:

IVIS (Test Item) = 1.5

IVIS (Negative Control) = 0.5

IVIS (Positive Control) = 81.6

 

The test item does not require classification to UN GHS or EU CLP.