1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with C18-unsatd. fatty acids, Me sulfates (salts)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Remarks:

used to justify read-across for other endpoints

Adequacy of study:

supporting 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 on 7 September 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C16-18 (even numbered) and C18 unsatd., Me sulfates (salts)

EC Number:

941-174-6

IUPAC Name:

1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C16-18 (even numbered) and C18 unsatd., Me sulfates (salts)

Test material form:

other: white solid wax

Details on test material:

- Name of test material: MDIPA-Esterquat C16-18 and C18 unsatd.
- Physical state: waxy solid
- Analytical purity: 100%

Specific details on test material used for the study:

- Name of test material: MDIPA-Esterquat C16-18 and C18 unsatd.
- Physical state: solid
- Analytical purity: 100%

Test animals / tissue source

Species:

other: isolated chicken eye

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: Approximately 7 weeks old - Weight: 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 µL

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): 1
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

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 µL of neat MDIPA-Esterquat C16-18 and C18 unsatd. 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 negative control eyes were treated with physiological saline only and the 3 positive control eyes with 5 % (w/v) BAC. 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:

9

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Value:

2

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

fluorescein retention score

Value:

1.5

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

in vitro irritation score

Value:

79

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test substance caused slight corneal swelling (mean swelling of 9%), moderate corneal opacity (mean score of 2) and slight to moderate fluorescein retention (mean score of 1.5). In addition, the top-layer of epithelium had a vesicular appearance. The calculated Irritation Index was 79.
Microscopic examination of the corneas treated with the test substance revealed very slight erosion of the epithelium only. No abnormalities of the stroma and endothelium were observed.
The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control eyes showed severe corneal effects and demonstrated the ICE test as meeting the acceptance criteria to be considered a valid study.
Microscopic examination of the one cornea treated with the negative control (saline) did not reveal any abnormalities. The positive control BAC 5% (w/v) caused moderate or severe erosion of the epithelium in the three corneas and vacuolation in the epithelium of two corneas.

Any other information on results incl. tables

Summary of results

Test material	Maximum mean score for:			Irritation categories^1	Irritation Index^2	Classifications (EU-CLP^3/UN-GHS^4)
	Swelling %	Opacity	Fluorescein retention
test item	9	2.0^5	1.5	II;III;II	79	2/2B
Saline (negative control)	0	0.0	0.0	Not applicable; one eye tested
BAC 5% (w/v) (positive control)	24	3.0^6	3.0	III;IV;IV	144	Category 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 1272/2008 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 1907/2006.

4 UN-GHS: NC = not classified; Category2B = mild irritant, causes eye irritation;Category2A = 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   Vesicular appearance of epithelium

6   Severe loosening of epithelium

Individual values test substance

Eye no. 1	Corneal thickness (instrument units)									Corneal opacity								Fluorescein Retention
T (min)= ->	-45	0	30	75	120	180	240			0	30	75	120	180	240			0	30
	60	58	62	63	63	64	64			0	1.5	1.5	2	2	2			0	1.5
swelling %			6.9	8.6	8.6	10.3	10.3
Eye no. 3	Corneal thickness (instrument units)									Corneal opacity								Fluorescein Retention
T (min)= ->	-45	0	30	75	120	180	240			0	30	75	120	180	240			0	30
	59	57	60	61	62	63	63			0	1.5	1.5	2	2	2			0	1.5
swelling %			5.3	7.0	8.8	10.5	10.5
Eye no. 5	Corneal thickness (instrument units)									Corneal opacity								Fluorescein Retention
T (min)= ->	-45	0	30	75	120	180	240			0	30	75	120	180	240			0	30
	63	62	65	66	66	66	66			0	1.5	1.5	2	2	2			0	1.5
swelling %			4.8	6.5	6.5	6.5	6.5
mean			6	7	8	9	9			mean	1.5	1.5	2.0	2.0	2.0			mean	1.5

In all corneas the top-layer of epithelium had a vesicular appearance

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, MDIPA-Esterquat C16-18 and C18 unsatd. is considered to be irritating to the eyes under the experimental conditions described in this report.

Executive summary:

For the assessment of the eye irritation potential of MDIPA-Esterquat C16-18 and C18 unsatd. Data from an Isolated Chicken Eye (ICE) test are available This in vitro study was performed in accordance with OECD Test 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) adopted on 7 September 2009.

In details the testing has already been performed according to the most recent version of the guideline adopted on July 26^th2013.

 

Approximately 7 weeks old chickens (obtained from slaughter animals for human consumption) were used as eye-donors. A total of 7 eyes were selected for testing: 3 for the test substance, 3 for the positive control (5 % (w/v) Benzalkonium Chloride (BAC) and 1 for the negative control (physiological saline).

 

The isolated chicken eyes were exposed to a single application of 30 µL of the test sample 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.

 

MDIPA-Esterquat C16-18 and C18 unsatd. tested neat causedcaused slight corneal swelling (mean swelling of 9%), moderate corneal opacity (mean score of 2) and slight to moderate fluorescein retention (mean score of 1.5). In addition, the top-layer of epithelium had a vesicular appearance. The calculated Irritation Index was 79.

Microscopic examination of the corneas treated with the test substance revealed very slight erosion of the epithelium only. No abnormalities of the stroma and endothelium were observed

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control eyes showed moderate swelling (24%), severe corneal opacity (mean score of 3) and severe fluorescein retention (mean score of 3) and demonstrated the ICE test as meeting the acceptance criteria to be considered a valid study. The calculated Irritation Index of the positive control (5% (w/v) BAC) was 144. Microscopic examination of the one cornea treated with the negative control (saline) did not reveal abnormalities. The positive control 5% (w/v) BAC caused moderate or severe erosion of the epithelium in the three corneas and vacuolation in the epithelium of two corneas. 

According to the classification schemes identified within the ICE test protocol, MDIPA-Esterquat C16-18 and C18 unsatd. is classified as Category 2, Irritating to eyes. It is recognized that the prediction models adopted in the OECD Test Guideline 438 are foridentifying 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 MDIPA-Esterquat C16-18 and C18 unsatd. tested neat in this ICE study as a Category 2 irritant is derived from histopathology examination within the ICE test.