1,3,4,6,7,8a-hexahydro-1,1,5,5-tetramethyl-2H-2,4a-methanonaphthalen-8(5H)-one

Eye irritation

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 08 May 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,3,4,6,7,8a-hexahydro-1,1,5,5-tetramethyl-2H-2,4a-methanonaphthalen-8(5H)-one

EC Number:

245-890-3

EC Name:

1,3,4,6,7,8a-hexahydro-1,1,5,5-tetramethyl-2H-2,4a-methanonaphthalen-8(5H)-one

Cas Number:

23787-90-8

Molecular formula:

C15H24O

IUPAC Name:

1,1,5,5-tetramethylhexahydro-2H-2,4a-methanonaphthalen-8(5H)-one

Test material form:

other: Liquid

Details on test material:

Identification: Isolongifolanone
Batch: SC00010626
CAS number: 23787-90-8
EINECS: 245-890-3
Chemical name: 2,2,7,7-TETRAMETHYLTRICYCLO[6.2.1.0^{1,6}]UNDECAN-5-ONE
Purity: 69.9%
Physical state/Appearance: Extremely pale yellow liquid
Expiry Date: 17 Febraury 2016
Storage Conditions: Room temperature in the dark

Test animals / tissue source

Species:

other: 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 prepared immediately on arrival.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

0.75 mL of undiluted test item.

Duration of treatment / exposure:

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes.

Number of animals or in vitro replicates:

Three corneas were used.

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 (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. 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.


Evaluation of Results
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity Measurement
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.

Permeability Measurement
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.

In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:

In Vitro Irritancy Score = mean opacity value + (15 x mean 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.

Visual Observation
The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

3.8

In vivo

Irritant / corrosive response data:

The in vitro irritancy score for the test item was 3.8.
The in vitro irritancy score for the negative control was 1.2.
The in vitro irritancy score for the positive control was 41.1.

Other effects:

Corneal Epithelium Condition
The corneas treated with the test item were clear 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.

Any other information on results incl. tables

Criteria for an Acceptable Test

The positive controlIn VitroIrritancy Score was within the range of 27.8 to 51.0. The positive control acceptance criterion was therefore satisfied.

 

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

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number		Opacity				Permeability (OD)		In VitroIrritancy Score
		Pre-Treatment	Post-Treatment	Post Incubation	Post-Incubation - Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	7	2	2	3	1		0.045
	8	2	3	2	0		0.098
	9	2	2	2	0		0.026
					0.3*		0.056¨		1.2
Positive Control	1	3	24	22	19	18.7	1.347	1.291
	2	2	25	26	24	23.7	1.106	1.050
	3	3	26	25	22	21.7	1.670	1.614
						21.3·		1.318·	41.1
Test Item	4	1	3	4	3	2.7	0.073	0.017
	5	2	5	6	4	3.7	0.058	0.000
	6	3	6	8	5	4.7	0.068	0.012
						3.7·		0.009·	3.8

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

Corneal Epithelium Condition Post Treatment and Post Incubation 

Treatment	Cornea Number	Observation
		Post Treatment	Post Incubation
Negative Control	7	clear	clear
	8	clear	clear
	9	clear	clear
Positive Control	1	cloudy	cloudy
	2	cloudy	cloudy
	3	cloudy	cloudy
Test Item	4	clear	clear
	5	clear	clear
	6	clear	clear

Opacitometer Calibration

The opacitometer was calibrated on the day of the test. 

Calibration of Opacitometer
Balance dial = 0	Target	Reading displayed	Acceptable
Calibrator number 1	75	75	Yes
Calibrator number 2	150 ±2%	150	Yes
Calibrator number 3	225 ±2%	224	Yes

Calibration of the opacitometer was considered to be acceptable.

Applicant's summary and conclusion

Interpretation of results:

other: No prediction of eye irritation can be made.

Remarks:

Criteria used for interpretation of results: EU

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 bioitem 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 it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS 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 Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS 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 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	3.8
Negative Control	1.2
Positive Control	41.1

 

Conclusion

No prediction of eye irritation can be made. However, as the observations post treatment and post incubation are completely clear, it is felt that this 0.8 increase over an IVIS score of 3 is not biologically relevant.