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:
Experimental start date: 10th December 2014 Experimental completion date: 12the December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 437 "Bovine corneal Opacity and Permeability Assay"
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
Identification: PP-R-001 (also known as Trigonox 501-CS40)
Chemical name: 1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris (Ethyl and Propyl) derivatives
IUPAC Name: Reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9-n-propyl- 1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3, 6,9-tri-n-propyl-1,2,4,5,7,8-hexoxonane
CAS Number: 1613243-54-1
Batch Number: BYK004856-98
Purity: not applicable
Physical state / Appearance: clear, colorless liquid
Expiry Date: 03 April 2017
Storage Conditions: approximately 4 °C in the dark

Test animals / tissue source

Species:
other: Eyes from adult cattle
Strain:
other: Not applicable
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 refrigerated on arrival and used within 24 hours of receipt.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: The negative control item, 0.9% w/v sodium chloride solution, was used as supplied. The positive control item, Ethanol, was used as supplied.
Amount / concentration applied:
Volume: 0.75 mL of test item

Concentration: not stated in study report
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
Immediately after exposure
Number of animals or in vitro replicates:
Three corneas to each of the following: test material group, negative control group, positive control group
Details on study design:
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 (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) 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 MEM.

A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer (Appendix 1). 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.

Treatment of Corneas
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were 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 10 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. The anterior chamber was refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea was visually observed.

The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.

After incubation the holders were removed from the incubator and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
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 (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
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.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.


Results and discussion

In vitro

Results
Irritation parameter:
cornea opacity score
Run / experiment:
3 corneas
Value:
0.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: reversibility not determined
Other effects / acceptance of results:
The corneas treated with the test item were clear post treatment and clear post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

Any other information on results incl. tables

 In VitroIrritancy Score

TheIn Vitroirritancy scores are summarized as follows:

 

Treatment

In VitroIrritancy Score

Test Item

0.1

Negative Control

0.7

Positive Control

52.4

 

 

Criteria for an Acceptable Test

TheIn VitroIrritancy Score of the positive control group narrowly exceeded the upper acceptable range. The upper acceptable range in the Study Plan was 51.0. In this study the positive control result was 52.4. As the positive control value was greater than the acceptance criterion, demonstrating the sensitivity of the test system, and the test itemIn VitroIrritancy Score was clearly ≤3, it was considered that the results of the test are acceptable and a repeat test would be unnecessary.

 

The negative control gave opacity of ≤4.7 and permeability ≤0.080. The negative control acceptance criteria were therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
The test item, PP-R-001 (CAS#1613243-54-1), was considered to be no category according to the prediction model.
 
Therefore the test item does not require 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 undiluted test item, PP-R-001 (CAS#1613243-54-1), was applied for 10 minutes followed by an incubation period of 120 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).

 

Interpretation

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

 

 Results

TheIn Vitroirritancy scores are summarized as follows:

 

Treatment

In VitroIrritancy Score

Test Item

0.1

Negative Control

0.7

Positive Control

52.4

 

 Conclusion

The test item, PP-R-001 (CAS#1613243-54-1), was considered to be no category according to the prediction model.

 

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