Fatty acids, C16-18 and C18-unsatd., branched and linear, reaction products with diethanolamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed on 03 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection : 10 July 2012 Date of Signature : 07 February 2013

Test material

Test material information

Constituent 1

Reference substance name:

Isostearamide DEA

IUPAC Name:

Isostearamide DEA

Test material form:

other: Liquid

Details on test material:

Sponsor's identification : Isostearamide DEA
Description : amber coloured liquid
Batch number : OE11124 (6/202593/00)
Purity : 100% - UVCB
Date received : 14 May 2012
Expiry date : not supplied
Storage conditions : room temperature in the dark

Test animals / tissue source

Species:

other: Excised Bovine Cornea

Strain:

other: Not Applicable

Details on test animals or tissues and environmental conditions:

Not applicable

Test system

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST ITEM

-Amounts(s) applied (volume or weight with unit):
0.75 mL of the test item was applied to triplicate corneas.

-Concentration (if solution):
The test item was used as supplied.

VEHICLE
No vehicle used

Duration of treatment / exposure:

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes.

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Not applicable

Details on study design:

TEST SITE
-Area of exposure
0.75 mL of the test item was applied to triplicate corneas.

-% coverage:
The test item was topically applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea.

-Type of wrap used:
None used

REMOVAL OF TEST ITEM
-Washing (if done):
At the end of the exposure period the test item was removed from the anterior chamber and each cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM without phenol red.

-Time after start of exposure:
10 minutes post exposure

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 from each 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 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 greater than or equal to 55.1 is defined as an ocular corrosive or severe irritant.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not ocular corrosive or severe irritant

Any other information on results incl. tables

RESULTS

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 condition of the cornea immediately after rinsing and at the final opacity measurement is given in Table 2.

The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

In Vitro Irritancy Score

The results are summarised as follows:

Treatment	In Vitro Irritancy Score
Test Item	1.0
Negative Control	1.5
Positive Control	33.4

Criteria 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.

Table 1          Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity					Permeability (OD)		In vitroIrritancy Score
		Pre-Treatment	Post-Treatment	Post-Incubation	Post-Incubation-Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	1	4	4	5	1		0.042
	2	3	2	4	1		0.030
	3	4	4	5	1		0.031
					1.0*		0.034+		1.5
Positive Control	4	2	27	28	26	25.0	0.408	0.374
	5	2	28	26	24	23.0	0.556	0.522
	6	4	37	37	33	32.0	0.476	0.442
						26.7·		0.446·	33.4
Test Item	7	2	5	6	4	3.0	0.025	0.0
	8	5	4	5	0	0.0	0.016	0.0
	9	4	3	5	1	0.0	0.013	0.0
						1.0·		0.000·	1.0

OD = Optical density  * = Mean of the post incubation-pre‑treatment values  + = Mean permeability       · = Mean corrected value

Table 2          Corneal Epithelium Condition

Treatment	Cornea Number	Observation
		Post Treatment	Post Incubation
Negative Control	1	clear	clear
	2	clear	clear
	3	clear	clear
Positive Control	4	cloudy	cloudy
	5	cloudy	cloudy
	6	cloudy	cloudy
Test Item	7	clear	clear
	8	clear	clear
	9	clear	clear

Appendix Opacitometer Calibration The opacitometer was calibrated on the day of the test.

Calibration of Opacitometer TX307
Balance dial = 0	Target	Readingdisplayed	Acceptable
Calibrator number 1	75	75	Yes
Calibrator number 2	150±2%	149	Yes
Calibrator number 3	225±2%	224	Yes

Calibration of the opacitometer was considered to be acceptable.

Applicant's summary and conclusion

Interpretation of results:

other: Non-Corrosive

Remarks:

Criteria used for interpretation of results: expert judgment

Conclusions:

The test item was considered not to be an ocular corrosive or severe irritant.

Executive summary:

Introduction. A study was performed to assess the ocular irritancy potential of the test item to the isolated bovine cornea. The method was designed to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No. 437 (2009) “Bovine Corneal Opacity and Permeability Assay”

Method. The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate anIn VitroIrritancy Score (IVIS). 

Results. Thein vitroIrritancy scores are summarised as follows:

Treatment	In VitroIrritancy Score
Test Item	1.0
Negative Control	1.5
Positive Control	33.4

Conclusion. The test item was considered not to be an ocular corrosive or severe irritant.