1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C8-10 (even numbered) acyl derivs., hydroxides, inner salts

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:

2014

Report date:

2014

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:

adopted 26 July 2013

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[(3-decanamidopropyl)dimethylazaniumyl]acetate 2-[dimethyl(3-octanamidopropyl)azaniumyl]acetate

EC Number:

944-170-2

Molecular formula:

not applicable, UVCB substance

IUPAC Name:

2-[(3-decanamidopropyl)dimethylazaniumyl]acetate 2-[dimethyl(3-octanamidopropyl)azaniumyl]acetate

Test material form:

solid

Test animals / tissue source

Species:

chicken

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: ROSS, spring chickens from poultry slaughterhouse v.d. Bor, Nijkerkerveen, the Netherlands
- Age at study initiation: Approximately 7 weeks old
- Weight at study initiation: body weight range approximately 1.5 2.5 kg

Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.

Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes that showed opacity (score higher than 0.5), were unacceptably stained with fluorescein (score higher than 0.5), or showed any other signs of damage were rejected and were replaced.

After an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured once more to determine the zero reference value for corneal swelling calculations.

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

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

Duration of treatment / exposure:

10 s

Observation period (in vivo):

eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment

Number of animals or in vitro replicates:

negative control (physiological saline, 30 µL): 1
positive control (NaOH, 30 mg): 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

At time t=0, i.e. immediately after the zero reference measurement, the following procedure was applied for each test eye:
The clamp holding the test eye was placed on paper tissues outside the chamber with the cornea facing upwards. Next, three corneas were treated with 30 mg test item. After an exposure period of 10 seconds, the corneal surface was rinsed thoroughly with 20 mL of isotonic saline at ambient temperature. After rinsing, each eye in the holder was returned to its chamber. The eyes were examined at 0, 30, 75, 120, 180 and 240 minutes after treatment. All examinations were performed with a slit-lamp microscope. Fluorescein retention was scored only at 30 minutes after treatment.
After the final examination, the test and control eyes were preserved in a neutral aqueous phosphate-buffered solution of 4% formaldehyde.
The tissues selected were embedded in paraffin wax, sectioned at 5 μm and stained with Periodic Acid-Schiff for histopathology examination. Ocular effects were evaluated using the endpoints of corneal thickness (swelling), corneal opacity and fluorescein retention.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Value:

14

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Value:

2.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

fluorescein retention score

Value:

2.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

After administration of the test item, a small lump of the test substance adhered to the cornea of one eye. After ca 5 minutes it was rinsed off by the saline drip.
The test item caused slight corneal swelling (14%), moderate or severe opacity (2.3) and moderate or moderate to severe fluorescein retention (2.2).
The one cornea with the prolonged exposure to the test item showed severe opacity and moderate to severe fluorescein retention.
The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate.
The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.
Microscopic examination of the corneas treated with the test item revealed very slight erosion of the epithelium.
Microscopic examination of the corneas treated with the negative control (saline) did not reveal any abnormalities. The positive control NaOH caused severe erosion of the epithelium, the majority of the nuclei of the stroma (inner region) not visible and necrosis of the endothelium.

Any other information on results incl. tables

Summary of results

	Maximum mean score for:			Irritation categories^1	Irritation Index^2	Classifications (EU-CLP^3/UN-GHS^4)
	Swelling %	Opacity	Fluorescein retention
Test item	14	2.3^5	2.2	II;III;III	104	2/2A
NaOH (positive control)	47	4.0^6	3.0	IV;IV;IV	187	1/1

 

1.    I = no effect; II = slight effect; III = moderate effect; IV = severe effect.

2.    Irritation Index = maximum mean corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20)

3.    EU-CLP: NC = not classified; Category 2 = Irritating to eyes; Category 1 = irreversible effects on the eye/serious damage to the eye. Regulation (EC) No 1272/2808 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2806.

4.    UN-GHS: NC = not classified; Category 2B = mild irritant, causes eye irritation; Category 2A = irritant, causes eye irritation; Category 1 = irreversible effects on the eye/serious damage to the eye. United Nations-Economic Commission for Europe (UN/ECE) (2003). Globally Harmonised System of Classification and Labelling of Chemicals (GHS).UN, New York and Geneva, 2007.

5.    a small lump of the test substance adhered to the cornea of one eye. After ca 5 minutes it was rinsed off by the saline drip

6.    Immediate iris constriction and severe loosening of epithelium

Individual histopathological findings

	Eye no.	Epithelium						Stroma			Endothelium
		Erosion	Necrosis	Vacuolation†			^Notes	Disorder of fibers	Pyknotic nuclei		Necrosis
				top	mid	low			outer region (adjacent to epithelium)	inner region (adjacent to endothelium)
Test item	1	½	-	-	-	-		-	-	-	-
	3	½	-	-	-	-		-	-	-	-
	5	½	-	-	-	-		-	-	-	-
NaOH (positive control)	2	3	-	-	-	-	‡	-	-	-	P
	4	3	-	-	-	-	‡	-	-	-	P
	6	3	-	-	-	-		-	-	-	P
Saline (negative control)	7	-	-	-	-	-		-	-	-	-

- = not observed; P = present; ½ = very slight; 1 = slight; 2 = moderate; 3 = severe; † = scored in the top/mid/low section of the epithelium; ‡ = majority of the nuclei of the stroma (inner region) not visible

 

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on the results from this Isolated Chicken Eye test, C8-10 Alkylamidopropyl betaine is considered to be irritating to the eyes (Category 2) under the experimental conditions described in this report.

Executive summary:

In an Isolated Chicken Eye (ICE) test according to OECD Guideline 438 (adopted 26 July 2013), the eye irritation potential of C8-10

Alkylamidopropyl betaine was assessed. In addition, the test included a negative control (saline) and a positive control (NaOH). Chicken eyes were obtained from slaughter animals used for human consumption.

The isolated chicken eyes were exposed to a single application of 30 mg for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed.

After administration of the test item, a small lump of the test substance adhered to the cornea of one eye. After ca 5 minutes it was rinsed off by the saline drip.

The test item caused slight corneal swelling (14%), moderate or severe opacity (2.3) and moderate or moderate to severe fluorescein retention (2.2).

The one cornea with the prolonged exposure tothe test item showed severe opacity and moderate to severefluorescein retention. 

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate.

The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Microscopic examination of the corneas treated with C8-10 Alkylamidopropyl betaine revealed very slight erosion of the epithelium.

Microscopic examination of the corneas treated with the negative control (saline) did not reveal any abnormalities. The positive control NaOH caused severe erosion of the epithelium, the majority of the nuclei of the stroma (inner region) not visible and necrosis of the endothelium.

Applying the classification criteria of the ICE, C8-10 Alkylamidopropyl betaine is classifed as Category 2 “Irritating to eyes”.

It is recognized that the prediction models adopted in the OECD Test Guideline 438 are for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. However, further support for identification of C8-10

Alkylamidopropyl betaine as a Category 2 irritant is derived from histopathology examination within this ICE test.

The effects observed in one cornea treated withthe test item demonstrates thatprolonged contact (mimicking exposure conditions of the Draize eye irritation test in rabbits) will dramatically increase the corneal effects.