dipropyl cyclohexane-1,2-dicarboxylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4th August 2015 - 5th August 2015

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,2-dipropyl cyclohexane-1,2-dicarboxylate

EC Number:

805-172-8

Cas Number:

65646-25-5

Molecular formula:

C14 H24 O4

IUPAC Name:

1,2-dipropyl cyclohexane-1,2-dicarboxylate

Test material form:

other: liquid

Details on test material:

Identity: CHP
Chemical name: Cyclohexane-1,2-dicarboxylic n-propyl ester
CAS number: 65646-25-5
Intended use: Chemical process regulator
Lot number: ST111209
Expiry: 31 December 2016
Appearance: Colourless liquid
Storage conditions: Room temperature ca. 20°C, in the dark,
under nitrogen gas
Purity/Assay: 98.6%

Test animals / tissue source

Species:

other: Bovine eyes

Strain:

not specified

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 13:15 hours, 4 August 2015).

Test system

Vehicle:

physiological saline

Controls:

not specified

Amount / concentration applied:

The test substance, CHP, was diluted to 10% with 0.9% saline.

Duration of treatment / exposure:

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.

Duration of post- treatment incubation (in vitro):

two hours +/- 10 minutes at 32°C +/- 1°C.

Number of animals or in vitro replicates:

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

Details on study design:

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 four 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. 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 = 0.062).


The positive control should elicit an In Vitro Irritancy Score that falls within two standard deviations of the historical mean for the laboratory. The negative control mean opacity change value should be ≤3.0 and the permeability mean value ≤0.1.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

0.3

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance, CHP, elicited an In Vitro Irritancy Score of 0.3 ± 0.6 and was predicted to have a classification of No Category according to the UN GHS.

Executive summary:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was performed to assess the

potential of the test substance, CHP, to cause serious eye damage. Ethanol was tested in

parallel as a positive control.

The BCOP assay is an organotypic model that uses isolated bovine corneas from freshly

slaughtered cattle as a means of assessing the potential of a test substance to cause serious

eye damage. Two endpoints, corneal opacity and permeability, were measured and combined

to give an In Vitro Irritancy Score which was used to assign an in vitro irritancy hazard

classification category for prediction of the ocular irritation potential of the test substance.

The negative and positive controls met the acceptance criteria for this assay.

The test substance, CHP, elicited an In Vitro Irritancy Score of 0.3 ± 0.6 and was predicted to

have a classification of No Category according to the UN GHS