1-Propanaminium, 2-hydroxy-N-(2-hydroxyethyl)-N-methyl-N-[2-[(1-oxo-9-octadecen-1-yl)amino]ethyl]-3-sulfo-, methyl sulfate, sodium salt (1:1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 April 2018 - 10 May 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:

2019

Materials and methods

Test guideline

Test guideline 1

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:

2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of fatty acids, C16 and C18 (unsaturated) and 2-(2-aminoethylamino)ethanol, subsequently reacted with 1-chloro-2,3-epoxypropane and sodium hydrogensulfite, partially quaternized with dimethyl sulfate, sodium salts

EC Number:

701-473-2

Cas Number:

73791-61-4

Molecular formula:

C26H53N2O6S.CH3O4S.Na

IUPAC Name:

Reaction products of fatty acids, C16 and C18 (unsaturated) and 2-(2-aminoethylamino)ethanol, subsequently reacted with 1-chloro-2,3-epoxypropane and sodium hydrogensulfite, partially quaternized with dimethyl sulfate, sodium salts

Test material form:

other: paste

Test animals / tissue source

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local slaughterhouse
- Storage, temperature and transport conditions of ocular tissue: The intact heads are 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 will be completed within two hours to minimize deterioration and/or bacterial contamination.
- Time interval prior to initiating testing: maximally 2 hours
- indication of any existing defects or lesions in ocular tissue samples: eyes applied in the test were unremarkable
- Indication of any antibiotics used: no

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 μL

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

30, 75, 120, 180 and 240 min

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES :
Upon receipt of the chicken heads to the laboratory, first the eyelids were carefully excised, taking care not to damage the cornea. Corneal intergrity was asssessed with a drop of 2% (w/v) sodium fluorescein applied to the corneal surface for few seconds, and then rinsed with isotonic saline. Fluorescein-treated eyes were then examined with a slit lamp microscope to ensure that the cornea is undamaged (i.e., fluorescein retention and corneal opacity scores ≤ 0.5).
Only undamaged 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 were 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 was 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 appratus. The clamps were positioned in the superfusion appratus in such a way that the entire cornea was supplied with the isotonic saline drip (3 to 4 drops per minute or 0.1 to 0.15 mL/min). The temperature of the chambers of the superfusion appratus was maintained at 32 ± 1.5 °C.
After being placed in the superfusion appratus, the eyes were again examined with a slip-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit –lamp microscope. Eyes with (i), a fluorescein retention score of >0.5, (ii) corneal opacity >0.5 or (iii) any additional signs of damage were replaced. Out of the eyes that were not rejected based on the beforementioned criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes of this batch were rejected. During corneal thickness measurements, slit width of slit-lamp microscope was set at 0.095 mm.

EQUILIBRATION AND BASELINE RECORDINGS : Immediately after examination and approval of all eyes, they were incubated for approximately 45 - 60 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as baseline (i.e., time=0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES 3

NEGATIVE CONTROL USED : physilogical saline, Sodium Chloride injection IP, 0.9 % w/v

POSITIVE CONTROL USED : Imidazole (30 mg)

APPLICATION DOSE AND EXPOSURE TIME : 30 μL for 10 seconds

OBSERVATION PERIOD : approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse (+/- 5 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: evaluated by using the area of the cornea that was most densely opacified for scoring
- Damage to epithelium based on fluorescein retention: Fluorescein retention was evaluated at the 30 minutes observation time point
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: 0.095 mm
- Macroscopic morphological damage to the surface: none

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment
- morphological effects (e.g., pitting or loosening of the epithelium)

DECISION CRITERIA: as indicated in OECD TG 438

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

1st trial - at up to 75 min

Value:

11.26

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

percent corneal swelling

Run / experiment:

1 st trial - at up to 240 min

Value:

15.72

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

2nd trial - at up to 75 min

Value:

11.79

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

percent corneal swelling

Run / experiment:

2nd trial - at up to 240 min

Value:

14.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

1 st trial - at up to 240 min

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation parameter:

cornea opacity score

Run / experiment:

2nd trial - at up to 240 min

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 7

Irritation parameter:

fluorescein retention score

Run / experiment:

1 st trial

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 8

Irritation parameter:

fluorescein retention score

Run / experiment:

2nd trial

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. Based on the overall ICE Class the negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.
- Acceptance criteria met for positive control: yes. Based on the overall ICE Class the positive control Imidazole was classified as corrosive/severely irritating, UN GHS Classification: Category 1.

Any other information on results incl. tables

Table 1: Data of corneal swelling/thickness and isolated chicken eye (ICE) classification

Treatment	Eye No. and Sw%	Corneal Thickness (µm) at t (minutes)							ICE Class
		-50	0	30	75	120	180	240
Negative Control	1	291	264	266	267	263	269	264	I
	Sw%	NA	NA	0.76	1.14	-0.38	1.89	0.00
Positive control	2	291	269	486	533	493	570	546	IV
	Sw%	NA	NA	80.67	98.14	83.27	111.90	102.97
	3	267	261	457	477	459	566	523
	Sw%	NA	NA	75.10	82.76	75.86	116.86	100.38
	4	268	266	474	510	474	559	507
	Sw%	NA	NA	78.20	91.73	78.20	110.15	90.60
	Mean sw% ±SD	NA	NA	77.99 2.79	90.88 7.73	79.11 3.79	112.97 3.48	97.99 6.53
Test Item (Trial 1)	5	271	270	296	302	322	320	329	II
	Sw%	NA	NA	9.63	11.85	19.26	18.52	21.85
	6	273	271	310	298	304	306	310
	Sw%	NA	NA	14.39	9.96	12.18	12.92	14.39
	7	280	284	314	318	314	317	315
	Sw%	NA	NA	10.56	11.97	10.56	12.92	14.39
	Mean Sw% ±SD	NA	NA	11.53  2.52	11.26 1.13	14.00 4.63	14.35 3.67	15.72 5.59
Test Item (Trial 2)	5	258	273	296	309	316	319	309	II
	Sw%	NA	NA	8.42	13.19	15.75	16.85	13.19
	6	266	276	302	309	314	318	317
	Sw%	NA	NA	9.42	11.96	13.77	15.22	14.86
	7	271	274	303	302	307	312	318
	Sw%	NA	NA	10.58	10.22	12.04	13.87	16.06
	Mean Sw% ±SD	NA	NA	9.48 1.08	11.79 1.49	13.85 1.86	15.31 1.49	14.70 1.44

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

Table 2: Data of Corneal opacity scores and isolated chicken eye (ICE) classification

Treatment	Eye No.	Corneal Opacity Scores at t (Minutes)							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	3	4	4	4	IV
	3	0	0	3	3	4	4	4
	4	0	0	2	3	4	4	4
	Mean	0	0.0	2.7	3.0	4.0	4.0	4.0
Test Item (Trial 1)	5	0	0	0	0	0	0	0	I
	6	0	0	0	0	0	0	0
	7	0	0	0	0	0	0	0
	Mean	0	0.0	0	0.0	0	0.0	0
Test Item (Trial 2)	1	0	0	0	0	0	0	0	I
	2	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0
	Mean	0	0.0	0	0.0	0	0.0	0

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

Table 3: Data of fluorescein retention, morphological effects and isolated chicken eye (ICE) classification

Treatment	Eye No.	Fluorescein retention at t = (Minutes)			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	IV
	3	0	0	2	Loosening of epithelium
	4	0	0	3	Loosening of epithelium
	Mean	0	0.0	2.7	NA
Test Item (Trial 1)	5	0	0	0	No morphological effects observed	I
	6	0	0	0	No morphological effects observed
	7	0	0	0	No morphological effects observed
	Mean	0	0.0	0.0	NA
Test Item (Trial 2)	1	0	0	0	No morphological effects observed	I
	2	0	0	0	No morphological effects observed
	3	0	0	0	No morphological effects observed
	Mean	0	0.0	0.0	NA

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:

other: no catagory

Conclusions:

Based on the percentage of corneal swelling, corneal opacity score, fluorescein retention score and morphologial effects obtained under the laboratory testing conditions and on the basis of overall combination of ICE categories obtained for all three end points the test item is characterized as “No Category”.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes according to OECD TG 438. The test compound was applied in a single dose (~30 µL /eye) onto the cornea of isolated chicken eyes. The control and test eyes were examined for corneal thickness and corneal opacity at 0, 30, 75, 120, 180 and 240 min after treatment and results were recorded according to a fixed scoring system. Fluorescein retention by damaged epithelial cells was scored at 30 min post-treatment. Additionally, the eyes treated with negative (0.9% NaCl), positive control (30 mg Imidazole) and test item were evaluated for morphological effects. All examinations were carried out with a slit-lamp microscope and Pachymeter. Three test item treated eyes and three positive control eyes and one negative control eye were used in this study.

For the test item the combination of ICE categories obtained for corneal swelling, corneal opacity, and fluorescein retention was 2 x I, 1 x II ( ICE class of I observed for fluorescein retention and opacity and ICE class II observed for corneal swelling).  Positive and negative controls showed the expected results. The experiments were considered to be valid.

Based on the percentage of corneal swelling, corneal opacity score, fluorescein retention score and morphologial effects obtained under the laboratory testing conditions and on the basis of overall combination of ICE categories obtained for all three end points the test item is characterized as “No Category”.