Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-17 and C17 unsatd. alkyl) derivs. and sodium hydroxide and 2-propenoic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

July 26th 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ß-Alanine, N-(2-aminoethyl)-N-(2-hydroxyethyl)-, N-(C12-C18 and C18unsatd. acyl) derivs., monosodium salts

Cas Number:

93820-52-1

Molecular formula:

C12 fatty acid based: C19H37N2NaO4 - C18 fatty acids based: C23H45N2NaO4

IUPAC Name:

ß-Alanine, N-(2-aminoethyl)-N-(2-hydroxyethyl)-, N-(C12-C18 and C18unsatd. acyl) derivs., monosodium salts

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

dihydrogen oxide

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Aqueous solution of beta-Alanine, N-(2-aminoethyl)-N-(2-hydroxyethyl)-, N-cocoacyl derivs., monosodium salts

Test animals / tissue source

Species:

chicken

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Chicken heads (Spring chickens (Gallus Gallus e.g. Ross 308 Broiler)), supplied by Baileys Turkeys Ltd., Cheshire, UK
- weight: 1.5 to 2.5 kg
- age: 7 weeks prior to being humanely killed for human consumption

PREPARATION OF THE EYES
Eyelids were carefully excised while taking care not to damage the cornea. The integrity of the cornea was measured usinf sodium fluorescein. An
acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores were ≤0.5.

Acceptable eyes were dissected from the skull, enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. The temperature of the chambers was at 32 ±1.5 °C.
Eyes were examined again with a slit-lamp microscope. Corneal thickness was measured with an optical pachymeter on the slit lamp microscope at the center of each cornea. Eyes were replaced when:
i. the fluorescein score is >0.5
ii. the corneal opacity score is >0.5
iii. there is any additional signs of damage
iv. the corneal thickness measurements for individual eyes deviate more than 10% from the mean value for all eyes

Eyes were then incubated for 45 minutes for equilibrium purposes.
Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 mL
- Concentration (if solution): 40% (as supplied by the sponsor)

Duration of treatment / exposure:

10 seconds, then rinsed from the eye using 20 mL of isotonic saline

Observation period (in vivo):

30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been rinsed

Number of animals or in vitro replicates:

negative control (0.9% sodium chloride): 2
positive control (BAC 5% (w/v)): 3
test group: 3

Details on study design:

REMOVAL OF TEST SUBSTANCE - Washing (if done): 20 mL saline
- Time after start of exposure: 10 s

SCORING SYSTEM: according to ICE classification criteria OECD TG 438

TOOL USED TO ASSESS SCORE: slit lamp microscope / fluorescein

ENDPOINTS EXAMINED: corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium)

All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test substance exposure) were determined at each of the above time points.
After the final examinations at the 240 minute time point, the eyes were preserved in neutral buffered formalin, sectioned, stained ans histopathologically evaluated.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: corneal swelling [%]

Value:

20.09

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Value:

2.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

fluorescein retention score

Value:

0.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 4

Irritation parameter:

histopathological observations

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Serious eye damage based on corneal epithelial changes (erosion, necrosis and vacuolation of the corneal epithelium).

In vivo

Irritant / corrosive response data:

Corneal Opacity
Easily discernible translucent areas or severe opacity was noted in the test item treated eyes. Very faint opacity was noted in the negative control treated eyes during the study period. Complete corneal opacity was noted in all the positive control eyes. No morphological effects were noted in the test item or negative control eyes. Sloughing (loosening of the epithelium) was noted in all the positive control eyes.

Fluorescein Retention
Very minor single cell staining or single cell staining scattered throughout the treated area of the corneas was noted on the test eyes. Very minor single cell staining was noted in one of the negative control eyes. Confluent large areas of the cornea retaining fluorescein was noted in all the positive control eyes.

Histopathology
Application of test item resulted in changes indicating serious eye damage, based on corneal epithelial changes. Changes comprised erosion, necrosis and vacuolation of the corneal epithelium. The classification was based on vacuolation being graded as ½ in the lower third of the epithelium in two out of three eyes

Any other information on results incl. tables

Test Item

Maximal mean score for corneal opacity : 2.3, ICE Class III

Mean score of Fluorescein Retention : 0.7, ICE Class II

Maximal Corneal swelling : 20.09%, ICE Class III

Positive Control Item

Maximal mean score for corneal opacity : 4.0, ICE Class IV

Mean score of Fluorescein Retention : 3.0, ICE Class IV

Maximal Corneal swelling : 52.36%, ICE Class IV

Negative Control Item

Maximal mean score for corneal opacity : 0.5, ICE Class I

Mean score of Fluorescein Retention : 0.3, ICE Class I

Maximal Corneal swelling : 3.73%, ICE Class I

Individual Scores and Mean Scores for Corneal Effects – Test Item

 

Endpoint	Eye number	Time (after washing) [min]
		0	30	75	120	180	240
Corneal Opacity	3A	0.5	0.5	2	3	3	3
	4A	0.5	0.5	1	2	2	2
	7A	0	0.5	2	2	2	2
Mean		0.3	0.5	1.7	2.3	2.3	2.3
ICE class		III
Fluorescein Retention	3A	---	0.5	---	---	---	---
	4A	---	1	---	---	---	---
	7A	---	0.5	---	---	---	---
Mean		---	0.7	---	---	---	---
ICE class		II
Corneal Thickness	3A	0.74	0.78	0.82	0.82	0.84	0.83
	4A	0.72	0.70	0.78	0.76	0.79	0.77
	7A	0.68	0.71	0.84	0.92	0.94	0.94
Mean		0.71	0.73	0.81	0.83	0.86	0.85
Mean Corneal Swelling (%)		---	2.34	14.02	16.82	20.09	18.69
ICE class		III
ICE classes combined: 2 x III, 1 x II

 

Individual Scores and Mean Scores for Corneal Effects – Positive Control Item

Endpoint	Eye number	Time (after washing) [min]
		0	30	75	120	180	240
Corneal Opacity	2A	0.5	3	4	4	4	4
	6A	0	4	4	4	4	4
	8A	0.5	4	4	4	4	4
Mean		0.3	3.7	4	4	4	4
ICE class		IV
Fluorescein Retention	2A	---	3	---	---	---	---
	6A	---	3	---	---	---	---
	8A	---	3	---	---	---	---
Mean		---	3	---	---	---	---
ICE class		IV
Corneal Thickness	2A	0.72	0.78	0.96	0.88	0.98	0.98
	6A	0.68	0.90	0.88	0.88	0.98	1.10
	8A	0.72	0.92	0.95	1.00	1.09	1.15
Mean		0.71	0.87	0.93	0.92	1.02	1.08
Mean Corneal Swelling (%)		---	22.64	31.60	30.19	43.87	52.36
ICE class		IV
ICE classes combined: 3 x IV (sloughing in all eyes)

 

Individual Scores and Mean Scores for Corneal Effects – Negative Control Item

Endpoint	Eye number	Time (after washing) [min]
		0	30	75	120	180	240
Corneal Opacity	1A	0.5	0.5	0.5	0.5	0.5	0.5
	5A	0	0.5	0.5	0.5	0.5	0.5
Mean		0.3	0.5	0.5	0.5	0.5	0.5
ICE class		I
Fluorescein Retention	1A	---	0	---	---	---	---
	5A	---	0.5	---	---	---	---
Mean		---	0.3	---	---	---	---
ICE class		I
Corneal Thickness	1A	0.67	0.66	0.68	0.70	0.69	0.70
	5A	0.67	0.66	0.65	0.69	0.68	0.66
Mean		0.67	0.66	0.67	0.70	0.69	0.68
Mean Corneal Swelling (%)		---	-1.49	-0.75	3.73	2.24	1.49
ICE class		I
ICE classes combined: 3 x I

 

 

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Based on the results from this Isolated Chicken Eye test, Amphopropionates C12-18 is considered to cause irreversible effects to the eyes under the experimental conditions described in this report.

Executive summary:

The eye irritation potential of Amphopropionates C12-18 (40% a.i.) was examined in an in vitro eye irritation study according to OECD Guideline 438 (adopted on July 26th 2013).

Approximately 7 weeks old chickens (obtained from slaughter animals for human consumption) were used as eye-donors. 0.03 mL of the test item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item (5% (w/v) Benzalkonium Chloride (BAC)). A further two enucleated eyes were treated with saline to serve as the negative control (0.9% NaCl in water).

The isolated chicken eyes were exposed to a single application of the test sample for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured 30, 75, 120, 180 and 240 minutes after rinsing to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells.

 

Corneal Opacity: Easily discernible translucent areas or severe opacity was noted in the test item treated eyes.

 

Fluorescein Retention: Very minor single cell staining or single cell staining scattered throughout the treated area of the corneas was noted on the test eyes.

 

Histopathology: Application of test item resulted in changes indicating serious eye damage, based on corneal epithelial changes. Changes comprised erosion, necrosis and vacuolation of the corneal epithelium. The classification was based on vacuolation being graded as ½ in the lower third of the epithelium in two out of three eyes.

 

Based on the overall in vitro irritancy criteria, no prediction for eye irritation could be made.

However, semi-quantitative microscopic evaluation of the corneas from eyes exposed to Amphopropionates C12-18 (40% a.i.) gave scores which exceeded the threshold for classification of this material as Category 1 (irreversible effects on the eyes).