Amides, coco, N,N-bis(hydroxyethyl); C10-12 and C18-unsatd. DEA

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 05, 2017 to December 05, 2017

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

Test guideline 1

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:

updated 26 July 2013

Deviations:

yes

Remarks:

This deviation was considered to have not affected the integrity or validity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

yes

Remarks:

This deviation was considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Amides, C10-12 (even numbered) and C18-unsatd., N, N-bis(hydroxyethyl)

IUPAC Name:

Amides, C10-12 (even numbered) and C18-unsatd., N, N-bis(hydroxyethyl)

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

Triplicate

Details on study design:

Preparation of Corneas: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. 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 in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading: The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test substance and three corneas to the positive control substances.

Treatment of Corneas: The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test substance or control substance were applied to the appropriate corneas. 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 ºC for 10 minutes. At the end of the exposure period the test substance and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein: Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations: After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

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

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

10 minutes

Value:

62.7

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Any other information on results incl. tables

Individual and mean corneal opacity and permeability measurements:

Treatment	Cornea Number	Opacity					Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post Incubation	Post-Incubation - Pre-Treatment	Corrected Value		Corrected Value
Negative Control	1	3	2	4	1		0.003
	2	3	3	7	4		0.005
	3	4	5	8	4		0.035
					0.3*		0.014♦		3.2
Positive Control	4	4	30	30	26	23.0	0.340	0.326
	5	4	37	33	29	26.0	0.466	0.452
	6	3	33	28	25	22.0	0.448	0.434
						23.7•		0.404•	29.7
Test substance	10	2	29	71	69	66	0.561	0.547
	11	1	43	66	65	65	0.076	0.062
	12	1	48	53	52	52	0.155	0.141
						59.0•		0.250•	62.7

OD = Optical density * = Mean of the post-incubation − pre-treatment values ♦ = Mean permeability • = Mean corrected value

Corneal epithelium condition post treatment and post incubation:

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 substance	10	Cloudy	Cloudy
	11	Cloudy	Cloudy
	12	Cloudy	Cloudy

Results:

Treatment	In Vitro Irritancy Score
Test substance	62.7
Negative control	3.2
Positive control	29.7

Applicant's summary and conclusion

Interpretation of results:

other: Category 1 (irreversible effects on the eye) based on CLP criteria

Conclusions:

Under the study conditions, the test substance was found to be causes serious eye damage on bovine corneal opacity and permeability test (IVIS score – 62.7).

Executive summary:

An in vitro study was conducted to determine the eye damage potential of the test substance, C10-12 and C18-unsatd. DEA, according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. In this study, eye damage of the test substance was tested through topical application for 10 minutes, followed by an incubation period of 120 minutes. The test substance was applied as is (750 µL) directly on top of the freshly isolated bovine cornea sample. 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 an In Vitro Irritancy Score (IVIS). The negative control responses for opacity and permeability was less than the upper limits of the laboratory historical range, indicating that the negative control did not induce irritancy on the corneas. The mean IVIS of the positive control (Ethanol) was 29.7, which was marginally lower than the criteria range set for an acceptable test. As the score was only marginally lower, it was decided that the result was acceptable as the positive control group still provided its intended function, which was to show the sensitivity of the test system to a known ocular irritant. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The IVIS score of the test substance was determined to be 62.7 after 10 minutes of treatment, which is above the IVIS threshold of 55, indicating Category 1 conclusion. Under the study conditions, the test substance was concluded to cause serious eye damage (Envigo, 2018).