Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 April 2018

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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 9 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized

EC Number:

291-707-5

EC Name:

Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized

Cas Number:

90459-62-4

Molecular formula:

C24H55N3O6S

IUPAC Name:

bis(2-aminoethyl)amine octadecanoic acid dimethyl sulfate

Test material form:

solid

Remarks:

paste

Test animals / tissue source

Species:

chicken

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: The eyes of healthy male chickens of approximately 7 to 8 weeks old and weighing around 2.0 to 2.1 kg were obtained from a poultry slaughterhouse where they were killed for human consumption.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The intact heads were transported from the slaughterhouse at ambient temperature (typically between 18°C and 25°C) in plastic boxes humidified with tissues moistened with isotonic saline. The procedure involving the collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was completed within one hour to minimize deterioration and/or bacterial contamination.
- indication of any existing defects or lesions in ocular tissue samples: Corneal integrity was quickly assessed with a drop of 2% (w/v) sodium fluorescein applied to the corneal surface for few seconds, and then rinsed with isotonic saline. Fluorescein retention, if any by damaged epithelial cells was examined with a slit lamp microscope. (i.e., fluorescein retention and corneal opacity scores ≤ 0.5).

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

moistoned with drop of physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg

Duration of treatment / exposure:

10 s

Duration of post- treatment incubation (in vitro):

240 min

Number of animals or in vitro replicates:

3 (test item, positive control), 1 (negative control)

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Upon receipt of the chicken heads to the laboratory, the eyelids were carefully excised, taking care not to damage the cornea. Corneal integrity was quickly assessed with a drop of 2% (w/v) sodium fluorescein applied to the corneal surface for few seconds, and then rinsed with isotonic saline. Fluorescein retention, if any by damaged epithelial cells was examined with a slit lamp microscope. (i.e., fluorescein retention and corneal opacity scores ≤ 0.5).
After confirming that the eyes were undamaged, the eyes were further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles was cut with a bent, blunt-tipped scissor. All necessary precautions were taken to aviod any corneal damage due to excessive pressure (i.e, compression artifacts). The visible portion of the optic nerve was left attached to the eye when it was removed from the orbit. Immediately after removing the eye from the orbit, the eye was placed on an absorbent pad and the nictitaing membrane and other connective tissue were removed.
The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The entire cornea was supplied with the isotonic saline drip (3 to 4 drops per minute or 0.1 to 0.15 mL/min) by positioning the clamps in the superfusion apparatus. The temperature of the chambers of the superfusion apparatus was maintained at 32 ± 1.5°C.
After being placed in the superfusion apparatus, the eyes were again examined with a slitp-lamp microscope to ensure that there were no damage during the dissection procedure. Corneal thickness was measured at this time at the corneal apex using the depth measuring device (Pachymeter). Eyes with (i), a fluorescein retention score of >0.5, (ii) corneal opacity >0.5 or (iii) any additional signs of damage were not selected for the study. Out of the eyes that were not rejected based on any of these criteria, all individual eyes with a corneal thickness not deviating more than 10% from the mean value for all eyes were only selected for the study.

EQUILIBRATION AND BASELINE RECORDINGS
Immediately after examination and approval of all eyes, they were incubated for approximately 50 minutes to equilibrate them to the test system prior to treatment. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as baseline (i.e., time=0). Additionally, the fluorescein retention score was also orecorded at 0 hr as baseline measurment value.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
physiological saline (30 µL)

POSITIVE CONTROL USED
Imidazole (30 mg)

OBSERVATION PERIOD
240 min

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: isotonic saline (approximately 20 mL) at ambient temperature

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was evaluated by using the area of the cornea that was most densely opacified for scoring as shown in below. The mean corneal opacity value for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was provided for test item, negative control and positive control group
- Damage to epithelium based on fluorescein retention: Fluorescein retention was evaluated at the 30 minutes observation time point only as shown in below. The mean fluorescein retention value of all test eyes was calculated for the 30 minute observation time point and used for the overall category score given for test item.
- Swelling: measured with optical pachymeter on a slit-lamp microscope
- Macroscopic morphological damage to the surface: Post treatment, the control and test eyes were evaluated for morphological effects, if any such as “pitting of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea. The morphological effects were evaluated and recorded individually for all the eyes.

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: as indicated in the guideline

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Value:

48.31

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class

Remarks:

IV

Results 2

Irritation parameter:

cornea opacity score

Value:

3.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class

Remarks:

IV

Results 3

Irritation parameter:

fluorescein retention score

Value:

2.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class

Remarks:

III

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

INDIVIDUAL DATA of Corneal swelling/Thickness and isolated chicken eye (ICE) Classification

Groups	Eye Clamp No.	Corneal Thickness in Instrument Units (µm) at t = (mins)							ICE Class
		-50	0	30	75	120	180	240
Negative Control	1 Sw%	289 NA	284 NA	268 -5.63	283 -0.35	284 0.00	285 0.35	286 0.70	I
Positive Control	2 Sw%	291 NA	284 NA	462 62.68	477 67.96	527 85.56	584 105.63	577 103.17	IV
	3 Sw%	286 NA	291 NA	466 60.14	481 65.29	529 81.79	576 97.94	571 96.22
	4 Sw%	287 NA	282 NA	451 59.93	483 71.28	526 86.52	556 97.16	582 106.38
	Mean sw% SD	NA	NA	60.91 1.53	68.18 3.00	84.63 2.50	100.25 4.68	101.92 5.19
Test Item	5 Sw%	288 NA	286 NA	348 21.68	338 18.18	357 24.83	376 31.47	367 28.32	IV
	6 Sw%	283 NA	270 NA	322 19.26	333 23.33	358 32.59	384 42.22	374 38.52
	7 Sw%	311 NA	267 NA	319 19.48	335 25.47	344 28.84	396 48.31	375 40.45
	Mean Sw% SD	NA	NA	20.14 1.34	22.33 3.75	28.75 3.88	40.67 8.53	35.76 6.52

Sw%: Corneal Swelling percentage, SD: Standard deviation, t: time.

 

ICE classification criteria for corneal swelling
Mean Corneal Swelling (%)	ICE Class
0 to 5	I
>5 to 12	II
>12 to 18 (>75min after treatment)	II
>12 to 18 (≤75min after treatment)	III
>18 to 26	III
>26 to 32 (>75 min after treatment)	III
>26 to 32 (≤75 min after treatment)	IV
>32	IV

 

 

INDIVIDUAL DATA of Corneal opacity scores and isolated chicken eye (ICE) Classification

Groups	Eye Clamp No.	Corneal Opacity Scores at t =							ICE Class
		-50	0	30	75	120	180	240
Negative Control	1	0	0	0	0	0	0	0	I
Positive Control	2	0	0	3	4	4	4	4	IV
	3	0	0	4	4	4	4	4
	4	0	0	3	4	4	4	4
	Mean	0	0.0	3.3	4.0	4.0	4.0	4.0
	±SD	0.00	0.00	0.58	0.00	0.00	0.00	0.00
Test Item	5	0	0	2	3	3	3	3	IV
	6	0	0	3	3	3	3	3
	7	0	0	2	4	4	4	4
	Mean	0	0.0	2.3	3.3	3.3	3.3	3.3
	±SD	0.00	0.00	0.58	0.58	0.58	0.58	0.58

SD: Standard deviation, NC: Negative control (physiological saline (0.9 % w/v)), PC: Positive control (Imidazole), TI: Test Item (Leomin KP), t: time.

 

Corneal opacity scores
Scores	Observation
0	No opacity
0.5	Very faint opacity
1	Scattered or diffuse areas, details of the iris are clearly visible
2	Easily discernible translucent area, details of the iris are slightly obscured
3	Severe corneal opacity, no specific details of the iris are visible, size of the pupil is barely discernible
4	Complete corneal opacity, iris invisible

 

Maximum Mean Opacity Score	ICE Class
0.0 – 0.5	I
0.6 – 1.5	II
1.6-2.5	III
2.6-4.0	IV

 

INDIVIDUAL DATA of fluorescein retention, Morphological effects andisolated chicken eye (ICE) Classification

Groups	Eye Clamp No.	Fluorescein retention at t = (Min)			Morphological effects	ICE Class
		-50	0	30
Negative Control	1	0	0	0	No morphological effects observed	I
Positive Control	2	0	0	3	Loosening of epithelium	III
	3	0	0	2	Loosening of epithelium
	4	0	0	3	Loosening of epithelium
	Mean	0	0.0	2.7	NA
	SD	0.00	0.00	0.58
Test Item	5	0	0	2	Roughening of the corneal surface	III
	6	0	0	2	Roughening of the corneal surface
	7	0	0	3	Roughening of the corneal surface
	Mean	0	0.0	2.3	NA
	SD	0.00	0.00	0.58

SD: Standard deviation, t: time.

 

Mean Fluorescein retention score at 30 minutes post treatment	ICE Class
0.0 – 0.5	I
0.6 – 1.5	II
1.6-2.5	III
2.6-3.0	IV

 

Score	Observation
0	No fluorescein retention
0.5	Very minor single cell staining
1	Single cell staining scattered throughout the treated area of the cornea
2	Focal or confluent dense single cell staining
3	Confluent large areas of the cornea retaining fluorescein

 

 

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on the percentage of corneal swelling, corneal opacity score, fluroscein retention score and mophologial effects obtained under the laboratory testing conditions and on the basis of overall combination of ICE categories obtained for all three end pointsOctadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternizedwas identified as UN GHS Category 1 (Chemicals Inducing Serious Eye Damage).

Executive summary:

The test item Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized was evaluated in the “Isolated Chicken eye test method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage” as per OECD Guideline 438 (adopted on 9 October 2017).

Heads of chickensapproximately7 to 8 weeks old and weighing around 1.5 to 2.5 kg were collected from a slaughterhouse. With in 2 hr of killing, enucleated eyes were placed in a susperfusion appratus. The test item was applied onto the cornnea of three eyes in one single dose of 30 mg for 10 sec. Similalry, positive control,Imidazole was applied onto the cornnea of three eyes in one single dose of 30 mg for 10 sec. Additionally, normal saline was used as negative control and was applied onto the cornea of one eye in one single dose of 30 µL for 10 sec. Before dosing, the eyes were incubated for 50 minutes to equilibrate them to the test system prior to treatment. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as baseline (i.e.,time=0). Additionally, the fluorescein retention score was also recorded at 0 hr as baseline measurment value. 

The control and test eyes were examined for corneal thickness and corneal opacity and morphology at 0, 30, 75, 120, 180 and 240 min after treatment, and results were recorded according to a fixed scoring system. Fluorescin retention by damaged epithelial cells was scored at 30 min post-treatment only. Additionally, the control and test item treated eyes were evaluated for morphological effects and recorded individually for all the eyes. All examinations were carried out with a slit-lamp microscope and Pachymeter.

Thein vitroclassification for the test itemwasassessed by reading the UNGHS classification that corresponds to the combination of categories obtained for corneal swelling, corneal opacity, and fluorescein retention. For the test item the combination of ICE categories obtained for corneal swelling, corneal opacity, and fluorescein retention was 2 x IV and 1 x III (ICE class of IV observed in all 3 endpoints).

This test is considered acceptable as the concurrent negative control and the positive control were identified as GHS Non-Classified and UN GHS Category 1 respectively.

 

CONCLUSION

Based on the percentage of corneal swelling, corneal opacity score, fluroscein retention score and mophologial effects obtained under the laboratory testing conditions and on the basis of overall combination of ICE categories obtained for all three end pointsOctadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternizedwas identified as UN GHS Category 1 (Chemicals Inducing Serious Eye Damage).