1-ethylazepan-2-one

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Feb 2018

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 Jul 2013

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-ethylazepan-2-one

EC Number:

606-384-1

Cas Number:

19797-08-1

Molecular formula:

C8H15NO

IUPAC Name:

1-ethylazepan-2-one

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Vitelco, 's Hertogenbosch, The Netherlands
- Storage, temperature and transport conditions of ocular tissue: Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: The eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

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

Duration of treatment / exposure:

10 ± 1 min

Duration of post- treatment incubation (in vitro):

120 ± 10 min

Number of animals or in vitro replicates:

Three corneas were used for each treatment group.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine
(Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for the minimum of 1 h at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

TREATMENT METHOD
After treatment, the holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 min at 32 ± 1 °C. Possible pH effects of the test item on the corneas were recorded.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 min at 32 ± 1 °C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490) .

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as Category 1.
Test substance with an IVIS ≤ 3 was regarded as No Category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean value of 3 corneas

Value:

104

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control (physiological saline) the calculated IVIS ranged from 0.8 to 1.2, and therefore within the standard deviations of the current historical mean of the negative control (IVIS: -2.8 to 3.0) (please refer to Table 1 and 2 under "any other information on results incl. tables").
- Acceptance criteria met for positive control: After treatment with the positive control (ethanol) the calculated IVIS ranged from 56 to 63, and therefore within the standard deviations of the current historical mean of the positive control (IVIS: 28.0 - 110.9) (please refer to Table 1 and 2 under "any other information on results incl. tables").

Any other information on results incl. tables

Table 1: Results

Treatment	Permeability score Final OD*	Final Opacity*	In vitro Irritancy Score
Negative control	-0.001	1.2	1.2
	0.013	0.7	0.9
	0.002	0.8	0.8
Positive control	2.615	16	56
	5.439	21	103
	3.195	15	63
Test item	4.453	44	111
	4.343	23	89
	4.153	50	112

* Positive control and test item are corrected for the negative control.

Table 2 Historical control data

	Negative control			Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-2.9 - 3.0	-0.034 - 0.100	-2.8 - 3.0	28.0 - 110.9
Mean	0.18	0.00	0.23	55.28
SD	1.10	0.01	1.13	15.14
n	113	113	113	88

Applicant's summary and conclusion

Interpretation of results:

other: corrosive according to Regulation (EC) No. 1272/2008

Conclusions:

Under the conditions of the BCOP assay, the test substance showed corrosive properties towards eyes.
CLP: Eye Damage 1, H318 according to Regulation(EC) No. 1272/2008