Reaction mass of ethyl (1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate and ethyl (1R,2R,4R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate

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 between 10 June 2014 and 11 June 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 437 using the Bovine Corneal Opacity & Permeability Assay method and in compliance with GLP.

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:

ethyl (1R,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1R,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate

Molecular formula:

C10H14O2

IUPAC Name:

ethyl (1R,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1R,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate

Test material form:

other: Liquid

Details on test material:

Identity: IFF TM 09-218
Alternative Identity: TM 09-218
Chemical name: BICYCLO[2.2.1]HEPT-5-ENE-2-CARBOXYLIC ACID, ETHYL ESTER
CAS number: 10138-32-6
Intended use: Fragrance ingredient in household products
Appearance: Clear liquid
Storage conditions: Room temperature (ca. 20°C, in the dark)

Test animals / tissue source

Species:

other: Eyes from adult cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Bovine eyes
The bovine eyes, supplied by a reputable supplier, were excised by an abattoir employee and collected as soon after slaughter as possible (excised at 12:00 hours, 10 June 2014). Eyes were obtained from cattle aged approximately 25 months. Instructions were given to avoid damaging the corneas
during excision. Excised eyes were maintained and transported to the laboratory, at ambient temperature, in sufficient HBSS, containing 1% (v/v)
penicillin/streptomycin solution, to cover all the eyes in the receptacle. The eyes were used within 4 hours of excision (incubation of mounted corneas commenced at 14:48 hours, 10 June 2014).

Test system

Vehicle:

other: 0.9% Sodium chloride solution

Controls:

yes

Amount / concentration applied:

750 µL of the test substance.

Duration of treatment / exposure:

10 minutes (± 30 seconds)

Observation period (in vivo):

Two hours ± 10 minutes

Number of animals or in vitro replicates:

Not applicable

Details on study design:

Preparation of corneas
All eyes were carefully examined, macroscopically, for defects (opacity, scratches, pigmentation, cuts, etc.) and those exhibiting defects were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea dissected leaving approximately 2 to 3 mm of sclera present around the cornea. The isolated corneas were stored in a petri dish containing HBSS plus 1% penicillin/streptomycin solution until all the corneas had been dissected.
Once all the corneas had been dissected, they were rinsed in fresh HBSS plus 1% penicillin/streptomycin solution prior to mounting.
The corneas were mounted in the cornea holders with the endothelial side against the O-ring of the posterior half of the holder. Each cornea was gently
flattened over the O-ring and holder surface with a wetted, gloved finger to expel any air. The anterior half of the holder was then positioned on top of the cornea and secured with screws. Both compartments of the holder were filled with HBSS plus 1% penicillin/streptomycin, using a syringe. The posterior
compartment was always filled first to return the cornea to its natural shape. Care was taken to ensure no air bubbles were present within the holders. The holders were then plugged and incubated, in an upright position, overnight at room temperature (approximately 20°C).
After overnight incubation at room temperature (approximately 20°C) the HBSS plus 1% penicillin/streptomycin was removed from both chambers. Both chambers were filled with cMEM (posterior chamber first), taking care to exclude air bubbles and plugged. The corneas were then incubated in the upright position for 60 minutes ± 5 minutes at 32°C ± 1°C in a waterbath. The waterbath temperature remained within the limits of 32°C ± 1°C throughout
the experiment.
At the end of the 60 minute incubation period, the medium was removed from both the anterior and posterior compartments using a pipette tip attached to a vacuum pump. The compartments were refilled with fresh cMEM (posterior chamber first). Again care was taken to ensure no air bubbles were present within the holders. The posterior compartment was then plugged and the basal opacity measurements performed.

Opacity measurement
The opacitometer measured the light transmission through the centre of each mounted cornea, displaying a numerical opacity value (arbitrary unit). The opacity of each cornea was measured by reading each holder in the right-hand chamber of the calibrated opacitometer. Corneas with an opacity value greater than 7 units were discarded. The mean basal opacity value of the corneas was then calculated and three corneas with opacity values close to the mean value were chosen for use as negative control corneas.

Test substance pH
An estimate of the pH of the test substance, diluted 1 in 10 with 0.9% saline, was determined using pH test sticks and recorded.

Treatment groups
Corneas were treated in triplicate with either the test substance, positive control (ethanol) or negative control (0.9% sodium chloride solution).

Treatment of corneas
The test substance and the positive control were tested undiluted. Immediately prior to treatment, the medium was removed from the anterior compartment of the holder using a pipette tip attached to a vacuum pump, taking extra care to ensure all excess liquid had been removed. Seven hundred and fifty µL (750 µL) of the test substance, positive control or negative control was introduced into the anterior part of each holder. Following application, the anterior compartment was plugged and the holder turned to a horizontal position and slightly rotated to ensure uniform distribution of the test substance over the surface of the cornea. The test substance or controls were in contact with the cornea for a total of 10 minutes (± 30 seconds). Each holder was incubated in a horizontal position at 32°C ± 1°C in a waterbath.
Following incubation, the test substance, positive and negative controls were removed from the epithelial surface of the cornea by washing, at least three times or until the wash medium (EMEM with phenol red) was clear and there was no discolouration. The wash medium was added via the holes on
the top of the holder. After each wash, the wash medium was removed using a pipette tip attached to a vacuum pump. The test substance required
three washes.
The anterior compartment was then filled with cMEM taking care to ensure no air bubbles were present in the compartment. Once all the air bubbles
had been removed, the anterior compartment was re-plugged and the holders returned to the waterbath and incubated, in an upright position, for two hours ± 10 minutes at 32°C ± 1°C.
Following completion of the two hour incubation period, the medium was removed from both compartments and replaced with fresh cMEM. The posterior compartment was re-plugged and the opacity of each cornea measured and recorded. The opacity values obtained at this stage were used in calculating the final In Vitro Irritancy Score. Throughout the assay the corneas were examined for opaque spots or other irregularities.

Permeability determination
The sodium fluorescein stock solution (4 mg/mL) was thawed prior to commencing the permeability incubation. Following the final opacity measurement, the medium was removed from the anterior compartment of the holder. One mL (1 mL) of the sodium fluorescein solution was added to the anterior compartment using a micropipette. Following addition of the sodium fluorescein solution to the anterior side of the holder, the compartment was
plugged and the corneas incubated in a horizontal position at 32°C ± 1°C for 90 ± 5 minutes in a waterbath.
Following incubation, the medium in the posterior compartment was mixed by drawing approximately 2.5 mL gently up and down a 5 mL syringe, with a needle attached, three times. An aliquot of the mixed medium from the posterior compartment was removed and transferred to a 1 cm path length cuvette. A spectrophotometer was adjusted to read at 490 nm (OD490) and a sample of cMEM read (OD = 0.064). The spectrophotometer was blanked using this solution prior to reading the permeability samples. Any solution giving an OD490 value above 1.8 was diluted 1 in 5 with cMEM.

Results and discussion

In vivo

Results

Irritation parameter:

other: In vitro irritancy score

Basis:

mean

Time point:

other: 2 hours

Score:

2.8

Reversibility:

not specified

Irritant / corrosive response data:

Throughout the assay the corneas were examined for opaque spots or other irregularities. Following treatment with test substance, IFF TM 09-218, the corneas were noted as clear. The corneas treated with the positive control, ethanol, were opaque and the corneas treated with the negative control, 0.9% sodium chloride solution, were clear.

Any other information on results incl. tables

Sample: IFF TM 09-218

0.9 % sodium chloride solution (negative control)

Cornea	Opacity pre-	Opacity post-	Change	Permeability OD 490
1	6	8	2	0.019
2	5	6	1	0.014
5	4	5	1	0.016
Mean change			1.333	Mean 0.016
SD			0.577	SD 0.003

 

Sample: IFF TM 09-218

Cornea	Opacity pre-	Opacity post-	Change	Corrected change	Permeability OD 490	Corrected
9	6	11	5	3.667	0.040	0024
10	4	7	3	1.667	0.020	0.004
11	6	10	4	2.667	0.015	-0.001
Mean change				2.667	Mean corrected OD490	0.009
SD				1.000	SD	0.013

 

In vitro irritancy score of IFF TM 09-218

(Opacity + (15 x corrected OD490))

Cornea	Opacity	15 x OD490	In vitro irritancy score
9	3.667	0.355	4.022
10	1.667	0.055	1.722
11	2.667	-0.020	2.647
Mean in vitro irritancy score			2.8
SD			1.2

SD = Standard deviation

Note. Rounded values only are displayed, unrounded numbers are used for calculations by excel spreadsheet.

 

Sample: Ethanol (positive control)

0.9 % sodium chloride solution (negative control)

Cornea	Opacity pre-	Opacity post-	Change	Permeability OD 490
1	6	8	2	0.019
2	5	6	1	0.014
5	4	5	1	0.016
Mean change			1.333	0.016
SD			0.577	0.003

 

Sample: Ethanol (positive control)

 

Cornea	Opacity pre-	Opacity post-	Change	Corrected change	Permeability OD 490	Corrected
3	6	36	30	28.667	0.930	0.914
6	4	28	24	22.667	0.995	0.979
8	6	36	30	28.667	1.180	1.164
Mean corrected change				26.667	Mean corrected OD 490	1.019
SD				3.464	SD	0.130

 

In vitro irritancy score of ethanol (positive control)

(Opacity + (15 x corrected OD490))

Cornea	Opacity	15 x OD490	In vitro irritancy score
3	28.667	13.705	42.372
6	22.667	14.680	37.347
8	28.667	17.455	46.122
Mean in vitro irritancy score			41.9
SD			4.4

 

SD = Standard deviation

Note. Rounded values only are displayed, unrounded numbers are used for calculations by Excel spreadsheet.

Assay validity

The positive control, ethanol, elicited an In Vitro Irritancy Score of 41.9. This value was within the historical range (mean ± 2 x SD = 25.0 – 49.8) for the assays performed to date. The negative control, 0.9% sodium chloride solution, opacity mean change value was 1.333 which was below the maximum acceptance value of 2.0. The permeability mean of the negative control was 0.016, which was below the maximum acceptance value of 0.1.

pH determination

The pH of the test substance, IFF TM 09-218, diluted 1 in 10 in 0.9% saline, measured using pH sticks, was approximately 7.0.

The results of the BCOP assay are summarised in the table below.

Sample	Opacity±SD	Permeability±SD	In vitro irritancy score±SD	In vitro classification
Test substance	2.667 ± 1.000	0.009 ± 0.013	2.8 ± 1.2	No category
Ethanol	26.667 ± 3.464	1.019 ± 0.130	41.9 ± 4.4	No prediction can be made
0.9 % sodium chloride solution	1.333 ± 0.577	0.016 ± 0.003	Not applicable	Not applicable

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

It was concluded that IFF TM 09-218 was predicted to have a classification of No Category for ocular irritation according to the UN GHS.

Executive summary:

The eye irritation potential of the test substance, TM 09-218, was assessed as not irritating according to OECD Test Guideline 437 using the Bovine Corneal Opacity & Permeability Assay method.