Bicyclo[3.1.1]hept-2-ene-2-ethanol, 6,6-dimethyl-, 2-acetate, (1R,5S)-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline 437 without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 437

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(1R,5S)-2-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl) ethyl acetate

EC Number:

800-940-9

Cas Number:

35836-72-7

Molecular formula:

C13H20O2

IUPAC Name:

(1R,5S)-2-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl) ethyl acetate

impurity 1

Reference substance name:

Non identified impurities

Molecular formula:

Not applicable

IUPAC Name:

Non identified impurities

Test material form:

liquid

Details on test material:

Batch No.: 117042
Purity: 99.3%
Name of test material (as cited in study report): NOPYL ACETATE
Physical state: colourless - slightly amber liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 10 April 2013

Test animals / tissue source

Species:

other: bovine eye

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
Bovine eyes (from cattle less than 12 months old) collected at the slaughterhouses of La Talaudière - France (42350) and carried in a stable medium (Hanks medium buffered with sodium bicarbonate), at room temperature, and used within 4 h after killing the animals.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: yes, 2 groups (positive and negative controls) containing 3 corneas each.

Amount / concentration applied:

TEST MATERIAL
Amount(s) applied (volume or weight with unit): 750 ± 8 µL

Concentrations:
- test material: undiluted
- negative control: 0.9 % (w/v) sodium chloride
- positive control: 10 % (w/v) sodium hydroxide

Duration of treatment / exposure:

10 ± 1 min

Observation period (in vivo):

Corneal opacity was measured after 2 h ± 10 min of incubation.
Corneal permeability was measured after 90 ± 5 min of incubation.

Number of animals or in vitro replicates:

Total: 9 corneas (3 corneas/group)

Details on study design:

Details of test procedure: bovine eyes recovered from a slaughterhouse were inspected and undamaged corneas were dissected and mounted in specially constructed BCOP corneal holders. After 1 h incubation in media, the basal opacity of each cornea was recorded using an opacitometer. 750 ± 8 µL of the test item and of each control were applied directly on the cornea using a positive-displacement micropipette and incubated in a water-bath at 32 ± 1 °C, in a vertical position (screw upward) for 10 ± 1 min. Before reading the final opacity, the corneas were rinsed and incubated for 2 h ± 10 min in fresh media to equilibrate. Then the permeability of each cornea was determined with a fluorescein solution after an incubation of 90 ± 5 min. An aliquot (360 µL) of the media from the posterior compartment was read in a plate reader at 490 nm to determine the permeability of the cornea to the fluorescein solution. The opacity and permeability values were combined to obtain an in vitro irritancy score (IVIS).

Incubation temperature: 32 ± 1 °C

Calibration information for devices used for measuring opacity and permeability (e.g., opacitometer and spectrophotometer): opacitometer (OPKIT) was calibrated with specific calibrators before each measurement of the corneal opacity. Values of opacity after three calibrations were 73-77, 147-153 and 220-240, respectively.

Controls: 0.9 % (w/v) sodium chloride and 10 % (w/v) sodium hydroxide were used as negative and positive controls, respectively.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

ca. -1.6

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

ca. -3.5

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

ca. -4.3

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 7.3.2/1: Opacity and optical density

Treatment	Opacity				Optical density		IVIS
	Before treatment (OPT0)	After 2 h (OPT2)	Difference (OPT2-OPT0)	Corrected	OD	Corrected	Score
Negative control	1	8	7		0.037
	0	3	3		0.018
	1	5	4		0.034
Mean				4.7	0.03
Standard deviation				2.1	0.01
10 % (w/v) NaOH	3	126	123	118.3	7.848	7.818	235.6
	4	130	126	121.3	7.888	7.858	239.2
	1	96	95	90.3	7.864	7.834	207.8
Mean				110.0	7.837		227.6
Standard deviation				17.1	0.020		17.2
Test item	0	3	3	-1.7	0.034	0.004	-1.6
	1	2	1	-3.7	0.038	0.008	-3.5
	3	3	0	-4.7	0.051	0.021	-4.3
Mean				-3.3	0.011		-3.2
Standard deviation				1.5	0.009		1.4

In vitro irritancy score (IVIS) = (OPT2-OPT0) + (15 x OD)

O.D. of the fluorescein solution (8 µg/mL): 1.490

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information (IVIS<55.1) Criteria used for interpretation of results: EU

Conclusions:

IVIS of nopyl acetate was 0.0. Therefore it is not classified as corrosive or severe irritant for the isolated bovine cornea, after a 10 min contact period.

Executive summary:

In an in vitro eye irritation study performed according to OECD Guideline No 437 and in compliance with GLP, 750 ± 8 µL of nopyl acetate were applied directly to isolated bovine corneas (3 corneas/group), mounted in specially constructed BCOP corneal holders, and incubated in a water-bath at 32 ± 1°C for 10 ± 1 min. Before reading the final opacity, the corneas were rinsed and incubated for 2 h in fresh media to equilibrate. Then the permeability of each cornea was determined with a fluorescein solution after an incubation of 90 min. An aliquot (360 µL) of the media from posterior compartment was read in a plate reader at 490 nm to determine the permeability of the cornea to the fluorescein solution. The opacity and permeability values were combined to obtain an in vitro irritancy score (IVIS). 0.9% (w/v) sodium chloride and 10% (w/v) sodium hydroxide were used as negative and positive controls, respectively.

The IVIS of the test item and positive control were 0.0 and 227.6 ± 17.2 respectively. Optical density values for the negative control corneas (nutritive medium) and fluorescein solution (8 µg/mL) were within the desired range and thus confirmed the validity of the test.

IVIS of nopyl acetate is 0.0. Therefore it is not classified as corrosive or severe irritant for the isolated bovine cornea, after 10 min of contact.