2-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl]benzamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental conduct date: 24 April 2017, Histopathology completion date: 29 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

adopted 26 July 2013

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl]benzamide

EC Number:

855-895-8

Cas Number:

212908-67-3

Molecular formula:

C27H47NO5

IUPAC Name:

2-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl]benzamide

Test material form:

solid: particulate/powder

Details on test material:

N-Acyl Phytosphingosine 95.2%, of which N-Salicyloyl 99.8%
Batch: E817200799

Specific details on test material used for the study:

The test item was applied in its original form

Test animals / tissue source

Species:

chicken

Details on test animals or tissues and environmental conditions:

Strain of chicken: ROSS, spring chickens


Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to Charles River Laboratories Hungary Kft. at ambient temperature at the earliest convenience.

After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at Charles River Laboratories Hungary Kft. and processed within 2 hours of collection.

After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit.

The eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied in its original form.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
test item: 30 mg, 10 seconds, rinsing: 20 ml saline, number of eyes: 3

Duration of treatment / exposure:

10 s

Observation period (in vivo):

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

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control treated eyes and one negative control eye were examined during the study.

Details on study design:

Experimental design

Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v (Minims, Chauvin, England) was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short.
The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus (Triskelion, Zeist, the Netherlands; see Figure 1). The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10-0.15 mL/min (peristaltic pump set at speed 5.00, Watson-Marlow 205CA, Rotterdam, the Netherlands). The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32oC (water pump set at 36.4oC; Lauda 103, Germany).
After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. Corneal thickness was measured using the Depth Measuring Attachment No. I for the Haag-Streit slit-lamp microscope, set at 0.095 mm. Corneal thickness was expressed in instrument units. An accurate measurement was taken at the corneal apex of each eye.
Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluores-cein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.
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, the eyes (corneas) were treated with the study substances according to the following scheme:

Negative control: Physiological saline, 30 μL, 10 seconds, rinsing with 20 mL saline, 1 eye
Positive control: NaOH, 30 mg, 10 seconds, rinsing with 20 mL saline, 3 eyes
Test item: 30 mg N-Acyl Phytosphingosine 95.2%, of which N-Salicyloyl 99.8%, 10 seconds, rinsing with 20 mL saline, 3 eyes

After rinsing, each eye in the holder was returned to its chamber.
The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment, using the criteria and scoring system given in Annex 1. Fluorescein retention was only scored at approximately 30 minutes after treatment. All examinations were carried out with the slit-lamp microscope.
After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

Maximum mean score

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Maximum mean score

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

Maximum mean score

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Microscopic examination of the corneas treated with the test item did not reveal any abnormalities.
Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control NaOH revealed severe erosion (3/3 corneas), severe necrosis (1/3 corneas) of the epithelium, stromal necrosis (3/3 corneas) and endothelial necrosis (3/3 corneas).

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination of the cornea did not reveal any abnormalities.
The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas revealed severe erosion (3/3 corneas), severe necrosis (1/3 corneas) of the epithelium, stromal necrosis (3/3 corneas) and endothelial necrosis (3/3 corneas).

Applying the classification criteria of the ICE, the following irritation classifications can be assigned:
Test item:
NC:“Not Classified.” (UN-GHS and EU-CLP classifications).

Any other information on results incl. tables

Summary results of the slit-lamp examination

Test material	Maximum mean score for:			Irritation categories1	Irritation Index2	Classifications (EU-CLP3/UN-GHS4)
	Swelling %	Opacity	Fluorescein retention
Test item	1	0.8	0.0	I;II;I	17	NC/NC
NaOH	42	4.0	3.0	IV;IV;IV	182	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 Immediate iris constriction and severe loosening of epithelium Test material Maximum mean score for: Irritation categories1 Irritation Index2 Classifications (EU-CLP3/UN-GHS4)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Applying the classification criteria of the ICE, the following irritation classifications can be assigned for the test item:
NC:“Not Classified.” (UN-GHS and EU-CLP classifications).

Executive summary:

The test item was evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. 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 of the test substance 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.

The test item caused corneal effects leading to a Not Classified classification, consisting of no or very slight corneal swelling (mean of 1%), very slight or slight opacity (mean score of 0.8) and no fluorescein retention (mean score of 0.0). Microscopic examination of the corneas did not reveal any abnormalities.

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination of the cornea did not reveal any abnormalities.

The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas revealed severe erosion (3/3 corneas), severe necrosis (1/3 corneas) of the epithelium, stromal necrosis (3/3 corneas) and endothelial necrosis (3/3 corneas).

Applying the classification criteria of the ICE, the following irritation classifications can be assigned for the tesi item:

NC: “Not Classified.” (UN-GHS and EU-CLP classifications).