Methyl 2-hexyl-3-oxocyclopentanecarboxylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted 9 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 2-hexyl-3-oxocyclopentanecarboxylate

EC Number:

253-379-1

EC Name:

Methyl 2-hexyl-3-oxocyclopentanecarboxylate

Cas Number:

37172-53-5

Molecular formula:

C13H22O3

IUPAC Name:

methyl 2-hexyl-3-oxocyclopentane-1-carboxylate

Test material form:

liquid

Specific details on test material used for the study:

Description: Colourless to pale yellow liquid
Storage conditions: Room temperature, protected from light
Expiry date: 18 January 2020

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Abattoir A. Moksel AG, Buchloe, Germany.
- Storage, temperature and transport conditions of ocular tissue: On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
- Time interval prior to initiating testing: The corneas were incubated for one hour at 32 ± 1 °C
- indication of any existing defects or lesions in ocular tissue samples: Any defective cornea had been discarded.
- Indication of any antibiotics used: Pen/Strep

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 μL of the test substance or the control substance was introduced into the anterior chamber.

Duration of treatment / exposure:

10 minutes incubation at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

- Illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C.
- Optical density at 490 nm (OD490) was determined, 90 minutes after the illuminance measurement was performed.

Number of animals or in vitro replicates:

Triplicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were carefully examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay.

NUMBER OF REPLICATES
Triplicates

NEGATIVE CONTROL USED
As negative controls physiological saline (0.9% NaCl) was used.

POSITIVE CONTROL USED
As positive controls ethanol (100%) was used.

APPLICATION DOSE AND EXPOSURE TIME
The medium was removed from the anterior chamber and replaced with the test item or control. 750 μL of the test substance or the control substance was introduced into the anterior chamber. Exposure time was 10 minutes incubation at 32 ± 1 °C.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).
- POST-EXPOSURE INCUBATION: The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C. After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Optical density at 490 nm (OD490) was determined, 90 minutes after the illuminance measurement was performed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: An initial measurement was performed on each of the corneas using the opacitometer. The illuminance of each cornea was read and recorded before and after treatment.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: See explanation and Table 1 in ‘Any other information on materials and methods incl. tables’.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean based on #1, #2, #3

Value:

0.06

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Mean #1, #2, #3

Value:

0.15

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

fluorescein leakage

Run / experiment:

Mean #1, #2, #3

Value:

-0.006

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

CORNEAL EPITHELIUM CONDITION
None of the 3 corneas treated with Dihydro Isojasmonate showed opacity of the tissue.

CRITERIA FOR AN ACCEPTABLE TEST
The positive control In Vitro Irritancy Score was 42.99. The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid (see Table 4 in 'Any other information on results incl. tables'.)
The negative control gave opacity of ≤0.77 and permeability ≤0.027. The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Individual and mean corneal opacity, permeability measurements and invitro irritation scores can be found in Table 1-3 in 'Any other information on results incl. tables'.

Any other information on results incl. tables

Table 1. Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1		1.11	1.61	0.50
2	Negative	1.11	1.87	0.75
3	Control	1.11	2.16	1.05
MV		1.11	1.88	0.77
4		3.19	34.99	31.80	31.03
5	Positive	2.16	29.80	27.64	26.87
6	Control	2.27	29.49	27.22	26.45
MV		2.54	31.43	28.89	28.12
7		-0.51	1.04	1.55	0.79
8	Test Item	-0.61	-0.21	0.39	-0.38
9		-0.15	0.66	0.81	0.04
MV		-0.42	0.50	0.92	0.15

MV = mean value

 

Table 2. Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1		0.022
2	Negative	0.045
3	Control	0.015
MV		0.027
4		0.985	0.958
5	Positive	1.166	1.139
6	Control	0.905	0.878
MV		1.019	0.991
7		0.020	-0.007
8	Test Item	0.019	-0.008
9		0.025	-0.002
MV		0.021	-0.006

MV = mean value

 

Table 3. In Vitro Irritation Score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1		0.50	0.022
2	Negative	0.75	0.045
3	Control	1.05	0.015
MV		0.77	0.027	1.18
4		31.03	0.958
5	Positive	26.87	1.139
6	Control	26.45	0.878
MV		28.12	0.991	42.99
7		0.79	-0.007
8	Test Item	-0.38	-0.008
9		0.04	-0.002
MV		0.15	-0.006	0.06

MV = mean value

 

Table 4. Historical Mean In Vitro Irritation Score of the Positive Control

	IVIS Positive Control - Ethanol 100 %
Mean Value (MV) Standard Deviation (SD) MV- 2xSD MV+2xSD	48.57 9.69
	29.20 67.94
Number of Replicates providing Historical Mean: 50

Positive controls are updated after every single experiment or at least every 3 months

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met