Ethanol, 2,2'-iminobis-, N-tallow alkyl derivs., N-oxides

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April - 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:

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:

9 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethanol, 2,2'-iminobis-, N-tallow alkyl derivs., N-oxides

EC Number:

263-179-6

EC Name:

Ethanol, 2,2'-iminobis-, N-tallow alkyl derivs., N-oxides

Cas Number:

61791-46-6

Molecular formula:

n.a

IUPAC Name:

Ethanol, 2,2'-iminobis-, N-tallow alkyl derivs., N-oxides

Test material form:

semi-solid (amorphous): gel

Test animals / tissue source

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: poultry slaughterhouse
- Number of animals: 7
- Characteristics of donor animals: 6 to 7 weeks old and weighing around 1.5 to 2.5 kg
- Storage, temperature and transport conditions of ocular tissue. Heads were removed immediately after sedation of the chickens, by electric shock and incision of the neck for bleeding. 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.
- Time interval prior to initiating testing: The procedure involving the collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was completed within two hours to minimize deterioration and/or bacterial contamination.
- indication of any existing defects or lesions in ocular tissue samples: The eyelids of chicken heads were carefully excised, without damaging 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 – 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).
- Indication of any antibiotics used: not specified

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 mg

Duration of treatment / exposure:

10 s

Duration of post- treatment incubation (in vitro):

30, 75, 120, 180 and 240 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids of chicken heads were carefully excised, without damaging the cornea. Corneal integrity was quickly assessed with drop a 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).

EQUILIBRATION AND BASELINE RECORDINGS
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 avoid 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 nictitating 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 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.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL (NC) USED
Yes, sodium chloride (0.9 % w/v)

POSITIVE CONTROL (PC) USED
Yes, imidazole

APPLICATION DOSE AND EXPOSURE TIME
Test item: 30 mg, 10 s
PC: 30 mg, 10 s
NC: 30 µL, 10s

OBSERVATION PERIOD
30, 75, 120, 180 and 240 minutes

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. 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 minute observation time point only. 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; slit-width setting: Corneal swelling was determined from corneal thickness measurements made with a pachymeter and was expressed as a percentage and is calculated from corneal thickness measurements according to the following formula:

[(Corneal thickness at time t – Corneal thickness at time t = 0) / Corneal thickness at time t = 0] ×100

The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was provided for test item, negative and positive control group.

- 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: The decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Value:

<= 5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Results 2

Irritation parameter:

cornea opacity score

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Results 3

Irritation parameter:

fluorescein retention score

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

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

Table 7: Data of corneal sweling/thickness and ICE classification

Treatment	Eye No. and Sw%	Corneal Thickness (µm) at t (minutes)
		Selection	0	30	75	120	180	240	ICE Class
Negative Control	1	306	303	288	301	304	298	280	I
	Sw%	NA	NA	-4.95	-0.66	0.33	-1.65	-7.59
Positive control	2	309	297	383	464	487	481	550	IV
	Sw%	NA	NA	28.96	56.23	63.97	61.95	85.19
	3	306	302	377	461	467	486	546
	Sw%	NA	NA	24.83	52.65	54.64	60.93	80.79
	4	305	301	384	468	488	482	544
	Sw%	NA	NA	27.57	55.48	62.13	60.13	80.73
	Mean sw% ±SD	NA	NA	27.12 2.10	54.79 1.89	60.25 4.94	61.00 0.91	82.24 2.55
Test Item (Trial 1)	5	302	294	288	297	301	292	299	I
	Sw%	NA	NA	-2.04	1.02	2.38	-0.68	1.70
	6	298	300	285	299	290	294	297
	Sw%	NA	NA	-5.00	-0.33	-3.33	-2.00	-1.00
	7	298	307	298	285	286	290	284
	Sw%	NA	NA	-2.93	-7.17	-6.84	-5.54	-7.49
	Mean Sw% ±SD	NA	NA	-3.32 1.52	-2.16 4.39	-2.60 4.65	-2.74 2.51	-2.26 4.72
Test Item (Trial 2)	1	302	281	286	286	282	285	292	I
	Sw%	NA	NA	1.78	1.78	0.36	1.42	3.91
	2	276	286	295	292	295	288	285
	Sw%	NA	NA	3.15	2.10	3.15	0.70	-0.35
	3	279	281	287	287	292	281	286
	Sw%	NA	NA	2.14	2.14	3.91	0.00	1.78
	Mean Sw% ±SD	NA	NA	2.35 0.71	2.00 0.20	2.47 1.87	0.71 0.71	1.78 2.13
Sw%: Corneal Swelling percentage, SD: Standard deviation, t: time.

Table 8: Data of corneal opacity and ICE classification

Treatment	Eye No.	Corneal Opacity Scores at t (Minutes)
		Selection	0	30	75	120	180	240	ICE Class
Negative Control	1	0	0	0	0	0	0	0	I
Positive Control	2	0	0	2	3	4	4	4	IV
	3	0	0	2	3	4	4	4
	4	0	0	2	2	4	4	4
	Mean	0	0.0	2.0	2.7	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

Table 9: Data of fluorescein retention, morphological effects and ICE classification

Groups	Eye No.	Fluorescein retention at t = (Minutes)			Morphological effects	ICE Class
		Selection	0	30
Negative Control	1	0	0	0	No morphological effects observed	I
Positive Control	2	0	0	1	Loosening of epithelium	III
	3	0	0	1	Loosening of epithelium
	4	0	0	1	Loosening of epithelium
	Mean	0.0	0.0	1.0	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 ±SD	0.0	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 ±SD	0.0	0.0	0.0	NA

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an ex vivo chicken eye irritation/corrosion assay according to OECD guideline 438, evaluation of the test item resulted in an ICE category classification of 3x I. Thus, the test item does not show eye damaging properties.

Executive summary:

The test item 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. Heads of chickens approximately 6 to 7 weeks old and weighing around 1.5 to 2.5 kg were collected at poultry slaughterhouse. Within 2 hours after killing, enucleated eyes were placed in a susperfusion apparatus. Before dosing, the eyes were incubated for 60 minutes at 32 ± 1.5 °C 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 hour as baseline measurement value. Eyes with corneal opacity <0.5 and fluorescein retention score <0.5 were selected. 30 mg of test item and 30 mg imidazole was applied onto the cornea of three eyes for 10 seconds. Similarly, 30 µL of Sodium Chloride injection IP, 0.9 % w/v was used as negative control and was applied onto the cornea of each eye for 10 seconds. 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 were evaluated for morphological effects. All examinations were carried out with a slit-lamp microscope and Pachymeter.

The in vitro classification for a test item was assessed by reading the GHS classification that corresponds to the combination of categories obtained for corneal swelling, corneal opacity and fluorescein retention. The combination of ICE categories obtained for corneal swelling, corneal opacity, and fluorescein retention was 3 x I (ICE class of I 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 GHS Category 1, respectively.

Based on the percentage of corneal swelling, corneal opacity score, fluorescein retention score and morphological 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 was identified for classification as UN GHS No Category.