9-Octadecenedioic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Dec 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions. The solid test substance was applied undiluted with no justification, the evaluation criteria specified an IVIS < 55 to be labelled in no category.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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)

Version / remarks:

adopted Jul 2013

Deviations:

yes

Remarks:

the solid test substance was applied undiluted without justification

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

802-122-7

EC Number:

802-122-7

IUPAC Name:

802-122-7

Constituent 2

Reference substance name:

Octadec-9-enedioic acid

IUPAC Name:

Octadec-9-enedioic acid

Details on test material:

- Name of test material (as cited in study report): Newdioic 12358, C18:1 dioic acid
- Physical state: loose white powder
- Analytical purity: 95.1%
- Lot/batch No.: B2
- Expiration date of the lot/batch: 4 Apr 2014
- Storage condition of test material: at room temperature

Test animals / tissue source

Species:

other: cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an in-vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: slaughter houses of La Talaudière, France
- Donor animals: cattle less than 12 months old
- Date and time of eye collection: 12 Dec 2013
- Time interval prior to initiating testing: maximum 4 h after killing the animal
- Transport medium and temperature conditions: buffered Hanks´ medium at room temperature with 0.2% penicillin/streptomycin

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, pigmentation, opacity, neovascularisation): yes
- Dissection of the eyes and treatment: Corneas were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were immersed in buffered Hanks medium and mounted in cornea holders.
- Description of the cornea holder: The cornea holders consist of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea. Holders were incubated in a water bath at 32 ± 1 °C.
- Test medium and temperature conditions used in the cornea holder: Eagle Minimum Essential Medium (EMEM) with and without phenol red, supplemented with 1% fetal calf serum and 0.25% penicillin/streptomycin; prior to use: pre-warmed to room temperature.
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: initial opacity of 0 - 7

DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Specification of the device: OPKIT

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: number of eyes for the negative control: 3; number of eyes for the positive control: 3

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied in the test: 750 ± 75 mg

NEGATIVE CONTROL
- Substance: physiological saline
- Concentration (if solution): 0.9% sodium chloride solution in distilled water
- Amount(s) applied in the test: no data

POSITIVE SUBSTANCE
- Substance: imidazole
- Concentration (if solution): 20% (w/w) imidazole solution in physiological saline
- Amount(s) applied in the test: no data

Duration of treatment / exposure:

4 h ± 10 min at 32 ± 1 °C

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

number of eyes for the test item: 3

Details on study design:

TEST CONDITIONS
- Short description of the method used: open-chamber method
Open-Chamber method: The glass window from the anterior chamber was removed prior to treatment. The controls or test substance were applied directly to the epithelial surface of the cornea. After dosing, the glass window was replaced on the anterior chamber to recreate a closed system. Corneas were exposed for 4 h with the test substance or the controls.

POST-EXPOSURE TREATMENT
- Removal of the test substance: The test substance was removed from the anterior chamber and the epithelium washed three times.
- Medium for washing the corneas: EMEM containing phenol red
- Medium for final rinsing: EMEM without phenol red

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Time of determination: Directly after refilling fresh EMEM without phenol red in the anterior chamber.
- Specification of the device: OPKIT

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: fluorescein solution was added to the anterior chamber of the cornea holder while the posterior chamber was filled with fresh medium. The amount of fluorescein that crosses into the posterior chamber was quantitatively measured via UV/VIS spectrophotometry at 490 nm recorded as optical density (OD490).
- Amount and concentration of the dye: 5 mg/mL sodium fluorescein solution (final concentration 0.5% in medium)
- Incubation time: 90 ± 5 min at 32 ± 1 °C
- Treatment for measuring: OD490 of a 360 µL aliquot was determined in a flat-bottomed 96-well plate.
- Dilution of the medium: A 1:5 dilution of the medium was made.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: opacity

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 4 h

Score:

26.7

Reversibility:

other: not applicable

Remarks on result:

other: test substance

Results 2

Irritation parameter:

other: permeability

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 4 h

Score:

0.048

Reversibility:

other: not applicable

Remarks on result:

other: test substance

Results 3

Irritation parameter:

other: IVIS

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 4 h

Score:

27.4

Reversibility:

other: not applicable

Remarks on result:

other: test substance

Results 4

Irritation parameter:

other: opacity

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 4 h

Score:

138.7

Reversibility:

other: not applicable

Remarks on result:

other: positive control

Results 5

Irritation parameter:

other: permeability

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 4 h

Score:

3.808

Reversibility:

other: not applicable

Remarks on result:

other: positive control

Results 6

Irritation parameter:

other: IVIS

Basis:

mean

Remarks:

of all 3 eyes

Time point:

other: 4 h

Score:

195.8

Reversibility:

other: not applicable

Remarks on result:

other: positive control

Any other information on results incl. tables

 Table 1: Opacity values

Parameter	Initial opacity	Final opacity	Opacity change	Mean opacity change of NC	Corrected opacity change	Mean opacity value
Negative control	0	4	4	-		3.3
	5	8	3		3.3
	5	8	3
Test substance	1	31	30	-	26.7	26.1
	5	34	29		25.7
	2	33	31		27.7
Positive control	4	147	143	-	139.7	138.7
	6	146	140		136.7
	4	147	143		139.7

Table 2: Permeability values (optical density (OD) at 490 nm)

Parameter	OD490	Mean Blank OD490	OD490 change	Mean OD490 change of NC	Corrected OD490 change	Mean OD490 value
Negative control	0.025	0.028	-	0.028	-	-
	0.032		-
	0.026		-
Test substance	0.079	-	-	-	0.051	0.048
	0.089		-		0.061
	0.060		-		0.032
Positive control	3.740	-	-	-	3.712	3.808
	3.792		-		3.764
	3.976		-		3.948

Table 3: In vitro irritancy score (IVIS) values

	IVIS	Mean IVIS
Test substance	27.4	27.4 ± 0.8
	26.6
	28.2
Positive control	195.4	195.8 ± 2.9
	193.1
	198.9

 

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

No prediction on the irritation potential can be made based on the results of the test, but the test substance does not have to be classified as serious eye irritant (Eye Dam Cat. 1).