Oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Apr 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted Oct 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

EC Number:

248-813-1

EC Name:

Oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

Cas Number:

28065-97-6

Molecular formula:

C34H69NO2 C36H67NO2 C36H69NO2 C36H71NO2 C36H73NO2

IUPAC Name:

oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: the isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 Units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: the corneas were isolated on the same day after delivery of the eyes.
- indication of any existing defects or lesions in ocular tissue samples: all eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: 1% (v/v) Penicillin/Streptomycin (100 Units/mL penicillin and 100 µg/mL streptomycin)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL

NEGATIVE CONTROL
- Amount(s) applied: 0.75 mL

POSITIVE CONTROL
- Amount(s) applied: 0.75 mL
- Purity: 99%

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 h at 32 ± 1 °C

Number of animals or in vitro replicates:

triplicates for each treatment and control groups

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each isolated cornea was mounted in a specially designed cornea holder, which consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively.

QUALITY CHECK OF THE ISOLATED CORNEAS
For equilibration, the corneas in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the basal opacity was determined (t0). The basal opacity of all corneas was recorded. Each cornea with basal opacity value > 7 was discarded.

APPLICATION DOSE AND EXPOSURE TIME
Each isolated cornea was mounted in a specially designed cornea holder, which consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments. The cornea were incubated in a horizontal position at 32 ± 1 °C 10 min in the water-bath.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The test substance was rinsed off from the application side with saline.
- POST-EXPOSURE INCUBATION: at 32 ± 1 °C for further two hours in a vertical position

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as severe irritant/corrosive and labelled Category 1.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in 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 corneae

Value:

0.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

With the negative control (saline) neither an increase of opacity nor permeability of the corneas could be observed (mean IVIS = 0.75).
The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneas (mean IVIS = 76.00) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).
Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 0.90 (threshold for serious eye damage: IVIS > 55).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the results in opacity and permeability values are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for positive control: yes, the IVIS falls within two standard deviations of the current historical mean.

Any other information on results incl. tables

Table 1: Results after 10 min exposure time

Test Group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposed in vitro Irritancy Score
		Mean		Mean
Negative Control	0	0.00	0.050	0.050	0.75	0.75	Not categorized
	0		0.051		0.77
	0		0.048		0.72
Positive Control	67.00*		0.896*		80.45	76.00	Category 1
	56.00*		1.093*		72.40
	65.00*		0.677*		75.16
Test substance	0.00*		0.045*		0.68	0.90	Not categorized
	0.00*		0.090*		1.36
	0.00*		0.043*		0.65

*corrected values

Table 2: Historical control data (Values of 116 studies with liquid test items performed from February 2015 (calendar week 7) to January 2017)

	Positive Control	Negative Control
Mean IVIS	83.61	1.03
Standard Deviation IVIS	11.75	0.31
Range of IVIS	56.25 – 113.06	0.56 – 2.38
Mean Opacity t130min	65.75	0.04
Standard Deviation	14.56	0.20
Opacity t130min
Range of Opacity t130min	26.00 – 112.00	-0,33 – 1.00
Mean Permeability	1.23	0.07
Standard Deviation Permeability	0.39	0.01
Range of Permeability	0.32 – 2.09	0.05 – 0.09

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Conclusions:

Under the conditions of this test, the test substance did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 0.90 in the in vitro Bovine Corneal Opacity and Permeability Test. Therefore, the test substance does not require classification for eye irritation or serious eye damage.
CLP: not classified