4-(2-methylpropyl)benzyl propyl ether

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 31 October 2017 and 1 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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-(2-methylpropyl)-4-(propoxymethyl)benzene

EC Number:

826-704-5

Cas Number:

1631962-93-0

Molecular formula:

C14H22O

IUPAC Name:

1-(2-methylpropyl)-4-(propoxymethyl)benzene

Test material form:

liquid

Specific details on test material used for the study:

Test item: FRET 11-0539
Identity: Benzene, 1-(2-methylpropyl)-4-(propoxymethyl)-; 4-(2-methylpropyl) benzyl propyl ether
Storage conditions: Room temperature (15 - 25˚C)
Appearance: Clear, colorless liquid
Expiry: September 2019

Test animals / tissue source

Species:

other: Eyes were obtained from cattle aged less than 30 months

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

The bovine eyes, supplied by Joseph Morris Abattoir, were excised by an abattoir employee and collected as soon after slaughter as possible (last excised at 12:45 hours, 31 October 2017). Eyes were obtained from cattle aged less than 30 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 slaughter (incubation of mounted corneas commenced at 15:32 hours, 31 October 2017).

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 or control items were applied to the cornea.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

triplicate

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% (v/v) 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% (v/v) 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% (v/v) 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% (v/v) penicillin/streptomycin was removed from both chambers using a pipette tip attached to a vacuum pump. 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 ± 1°C in a waterbath. The waterbath temperature remained within the limits of 32 ± 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 used was a STAG BIO OP-KIT CA-1. It 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 item pH
An estimate of the pH of the test item FRET 11-0539, 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 item- FRET 11-0539, positive control (100% ethanol) or negative control (0.9% sodium chloride solution).
The controls and their results were shared with several studies performed in the same assay.

Treatment of corneas
The test item 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 each of the test item, 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 item over the surface of the cornea. The test item 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 item, 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 item required three washes. The anterior chamber 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 = 0.063). 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.

ASSESSMENT OF RESULTS
Opacity
The change in the opacity of each cornea was calculated by subtracting the initial basal opacity from the post-treatment opacity measurement.
The mean change in opacity for the negative control corneas was calculated and was subtracted from the change in opacity of each treated cornea to obtain the corrected opacity value.
The mean corrected opacity change value of each treatment group (of three corneas) was calculated from the individual corrected opacity values of the treated corneas.

Permeability (OD490)
The corrected permeability value (OD490) of each treated cornea was calculated by subtracting the mean negative control cornea value from the permeability value of each cornea.
The mean corrected permeability value of each treatment group was calculated from the individual corrected permeability values of the treated corneas.

In Vitro Irritancy score calculation
The In Vitro Irritancy Score (IVIS) was calculated using the following formula:
In Vitro Irritancy Score = Corrected Opacity Value + (15 x Corrected OD490 Value)
The IVIS was calculated for each individual treatment and positive control cornea. The mean IVIS value for each treatment group was calculated from the IVIS of each individual cornea in the treatment group.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

-1.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

 Assay validity

The positive control, ethanol,elicited an In Vitro Irritancy Score of 40.2. This value was within the historical range (mean ± 2 x SD=24.8- 49.8) for the assays performed to date.

 

The negative control, 0.9% sodium chloride solution, opacity mean change value was 1.667 which was below the maximum acceptance value of 3.0. The permeability mean of the negative control was 0.013, which was below the maximum acceptance value of 0.1.

pH determination

The pH of the test item, FRET 11-0539, diluted 1 in 10 in 0.9% saline, measured using pH sticks, was approximately 7.0. 

BCOP results

Throughout the assay the corneas were examined for opaque spots or other irregularities. Following treatment withtest item, FRET 11-0539, the corneas were noted as clear. The corneas treated with the positive control, ethanol, were very opaqueand the corneas treated with the negative control, 0.9% sodium chloride solution, were clear. The results of the BCOP assay are summarised in the table below.

Test item	Opacity ± SD	Permeability ± SD	In vitro irritancy Score ± SD	In vitro classification
FRET 11-0539	-1.333 ± 0.577	0.002 ±0.005	-1.3 ±0.6	No Category
Ethanol	22.000 ±3.055	1.213 ±0.091	40.2 ±4.3	No prediction can be made
0.9% Sodium Chloride Solution	1.667 ±2.082	0.013 ± 0.002	Not applicable	Not applicable

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item was considered not to be an ocular corrosive or severe irritant.

Executive summary:

The eye irritancy potential of the test substance was assessed according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and is not classified, according to EU CLP criteria.