Fatty acids, C18 unsaturated, trimers, hydrogenated

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Oct 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study with acceptable restrictions. The positive control subtance was ethanol (no justification provided). The positive control substance failed to meet the definition of ocular corrosive or severe irritant (IVIS ≥ 55.1) but met the acceptability criterion of the testing facility based on historical background data.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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)

Deviations:

yes

Remarks:

positive control subtance was ethanol (no justification provided). Ethanol failed to meet the definition of ocular corrosive or severe irritant (IVIS ≥ 55.1) but met the acceptability criterion of the testing facility based on historical background data.

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C18-unsaturated, trimers, hydrogenated

IUPAC Name:

Fatty acids, C18-unsaturated, trimers, hydrogenated

Details on test material:

- Name of test material (as cited in study report): Hydrogenated Trimer Fatty Acid
- Physical state: amber coloured viscous liquid
- Substance type: UVCB
- Analytical purity: 100%
- Lot/batch No.: 12-92-29
- Expiration date of the lot/batch: 08 March 2014
- Storage condition of test material: at room temperature in the dark

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 to classify substances as “ocular corrosives and severe irritants”. 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: Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Time interval prior to initiating testing: max. 24 h
- Transport medium and temperature conditions: Hanks´ Balanced Salt Solution (HBSS) supplemented with penicillin/streptomycin, on ice

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
- Test medium and temperature conditions used in the cornea holder: complete minimum essential medium (MEM) at 32 ± 1 °C
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: free of macroscopic defects, initial opacity of 3 ± 1

DETERMINATION OF THE INITIAL OPACITY
- Method: Example: Corneal opacity was determined by the amount of light transmission through the cornea using a calibrated opacitometer.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

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

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied in the test: 750 µL

NEGATIVE CONTROL SUBSTANCE
- Substance: sodium chloride
- Concentration: 0.9% w/v in water
- Amount(s) applied in the test: 750 µL

POSITIVE CONTROL SUBSTANCE
- Substance: ethanol
- Concentration: 100%
- Amount(s) applied in the test: 750 µL

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

number of corneas: 3

Details on study design:

TEST CONDITIONS
- Short description of the method used: closed-chamber method
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 10 min.

POST-EXPOSURE TREATMENT
- Removal of the test substance: The test substance was removed from the anterior chamber and the epithelium washed four times.
- Medium for washing the corneas: complete MEM containing phenol red
- Medium for final rinsing: Example: complete MEM without phenol red
- Post-exposure incubation conditions: The holders were incubated, anterior chamber facing forward, at 32 ± 1 °C for 120 min ± 10 min.

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea using a calibrated opacitometer.
- Time of determination: Final opacity reading was taken at the end of the 120-minute post-exposure incubation period.

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: Sodium fluorescein solution was added to the anterior chamber of the cornea holder while the posterior chamber was filled with fresh medium. The amount of sodium fluorescein that crossed into the posterior chamber was quantitatively measured via UV/VIS spectrophotometry at 492 nm recorded as optical density (OD492).
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (4 mg/mL)
- Incubation time: 90 min at 32 ± 1 °C
- Treatment for measuring: Example: OD490 of a 360 µL aliquot was determined in a 96-well plate.
- Dilution of the medium: If values greater than 1.500 OD492 were obtained a 1 in 5 dilution of the medium in complete MEM was performed and the measurement repeated. The modified value was multiplied by 5 to reflect the 1 in 5 dilution.

HISTOPATHOLOGY
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

EVALUATION OF RESULTS
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

- Opacity Measurement: The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

- Permeability Measurement: The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

- In Vitro Irritancy Score: The following formula was used to determine the In Vitro Irritancy Score:

In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

- Visual Observation: The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.

DATA INTERPRETATION
A test item that induces an In Vitro Irritancy Score ≥ 55.1 is defined as an ocular corrosive or severe irritant.

CRITERION FOR AN ACCEPTABLE TEST
For an acceptable test the following positive control criterion must be achieved:
Ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean for the testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 30.9 to 67.7.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: In Vitro Irritancy Score

Basis:

mean

Remarks:

out of all 3 corneas

Time point:

other: 10 min exposure + 120 min post-exposure incubation

Score:

1.1

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: test item

Results 2

Irritation parameter:

other: In Vitro Irritancy Score

Basis:

mean

Remarks:

out of all 3 corneas

Time point:

other: 10 min exposure + 120 min post-exposure incubation

Score:

1.7

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: negative control

Results 3

Irritation parameter:

other: In Vitro Irritancy Score

Basis:

mean

Remarks:

out of all 3 corneas

Time point:

other: 10 min exposure + 120 min post-exposure incubation

Score:

31.6

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: positive control

Irritant / corrosive response data:

Corneal Opacity and Permeability Measurement: Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1.
Corneal Epithelium Condition: The corneas treated with the test item and the negative control were clear post-treatment and post-incubation. The corneas treated with the positive control were cloudy post-treatment and post-incubation.

Any other information on results incl. tables

Table 1. Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Opacity						Permeability (OD)		In vitro Irritancy Score
	Cornea Number	Pre-Treatment	Post-Treatment	Post-Incubation	Post-Incubation −Pre-Treatment	Corrected Value		Corrected Value
Negative Control	1	2	2	3	1		0.019
	2	2	2	3	1		0.035
	3	4	4	6	2		0.026
					1.3 a		0.027 b		1.7
Positive Control	4	3	31	27	24	22.7	0.602	0.575
	5	3	23	28	25	23.7	0.403	0.376
	6	4	33	24	20	18.7	1.071	1.044
						21.7 c		0.665 c	31.6
Test Item	7	3	3	7	4	2.7	0.055	0.028
	8	2	2	2	0	0.0	0.043	0.016
	9	3	3	4	1	0.0	0.017	0.000
						0.9 c		0.015 c	1.1

  

OD: optical density

a: Mean of the post incubation − pre-treatment values

b: Mean permeability

c: Mean corrected value

Criterion for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 30.9 to 67.7. The positive control acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive or severe irritant

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item was considered not to be an ocular corrosive or severe irritant.
There is regulatory acceptance in the EU that a substance can be considered as severe eye irritant (Serious eye damage Category 1/R41) based on a positive result in the Bovine Corneal Opacity and Permeability (BCOP) test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant (Category 2 according to Regulation (EC) No 1272/2008 or R36 according to Directive 67/548/EEC) and shall therefore be subject to further evaluation.