[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted on 03 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

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)

Version / remarks:

26 July 20013

Deviations:

no

Test guideline 2

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:

EC NO. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

EC Number:

289-348-4

EC Name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

Cas Number:

87788-32-7

Molecular formula:

C10H22B2F6N2

IUPAC Name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

impurity 1

Reference substance name:

{3-[(amino-N)methyl]-3,5,5-trimethylcyclohexanamine}(trifluoro)boron

Molecular formula:

C10H22BF3N2

IUPAC Name:

{3-[(amino-N)methyl]-3,5,5-trimethylcyclohexanamine}(trifluoro)boron

additive 1

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

methanol

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir as a by-product from freshly slaughtered animals
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): Adult cattle - 12 to 60 monthe old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes 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
- Time interval prior to initiating testing: The corneas were prepared immediately on arrival at the laboratory.
- indication of any existing defects or lesions in ocular tissue samples: none
- Indication of any antibiotics used: none reported.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of the test item as supplied or control items were applied to the appropriate corneas

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

3

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 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.

Selection of Corneas and Opacity Reading
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. 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 EMEM 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 EMEM containing phenol red before a final rinse with complete EMEM without phenol red. 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.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the final 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:

in vitro irritation score

Value:

ca. 45.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

DEVIATIONS FROM STUDY PLAN
The following deviation from the Study Plan occurred:

The positive control group had an overall IVIS of 53.8. This was marginally higher than the criteria range set for an acceptable test. However, as the score was only marginally exceeded, it was decided that this result was acceptable as the positive control group was still providing its intended function which is to show the sensitivity of the test system to a known ocular irritant.
This deviation was considered to have not affected the integrity or validity of the study.

Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was above the range of 29.6 to 52.0. The positive control acceptance criterion was therefore not satisfied. This is reported as a deviation.
The negative control gave opacity of ≤2.9 and permeability ≤0.103. The negative control acceptance criteria were therefore 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 below.

Corneal Epithelium Condition

The corneas treated with the test item were cloudy post treatment and 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.

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized below:

Treatment	Cornea Number	Opacity					Permeability (OD)		In Vitro Irritancy Score
		Pre-treatment	Post-treatment	Post incubation	Post incubation - pre-treatment	Corrected Value		Corrected Value
Negative Control	1	3	3	3	0		0.017
	2	3	3	3	0		0.012
	3	3	3	4	1		0.025
					0.3*		0.018**		0.6
Positive Control	4	3	37	38	35	34.7	1.150	1.132
	5	2	35	35	33	32.7	1.302	1.284
	6	2	34	35	33	32.7	1.699	1.681
						33.3 ***		1.366***	53.8
Test Item	7	1	50	23	22	21.7	0.349	0.331
	8	5	73	54	49	48.7	0.695	0.677
	9	0	69	43	43	42.7	0.620	0.602
						37.7***		0.537***	45.7

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No prediction of eye irritation can be made

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 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 undiluted test item 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 an In Vitro Irritancy Score (IVIS).  

Data Interpretation

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

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

The In Vitro irritancy scores are summarized as follows:

Treatment	In vitro Irritancy Score
Test Item	45.7
Negative Control	0.6
Positive Control	53.8

Conclusion

No prediction of eye irritation can be made.