(2S,3S,4R)-2-aminooctadecane-1,3,4-triol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 February 2002- 06 February 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

OECD TG 405

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1987

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

439-210-6

EC Name:

-

Cas Number:

554-62-1

Molecular formula:

C18H39NO3

IUPAC Name:

(2S,3S,4R)-2-aminooctadecane-1,3,4-triol

Test material form:

solid: particulate/powder

Details on test material:

SOURCE OF TEST MATERIAL
- Purity: 94.0 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark.

Specific details on test material used for the study:

Batch: 62630012
Expiry date: 01 November 2002

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Remarks:

Albino Rabbit(SPF-Quality)

Details on test animals or tissues and environmental conditions:

Test animals
- Source: Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: Animals used within the study were at least 6 weeks old
- Weight at study initiation: weights were between 1.0 and 3.5 kg
- Housing: Individually in labelled cages with perforated floors (Scanbur, Denmark, dimensions53.5x63x38.5 cm). Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.
- Diet: Standard laboratory rabbit diet (Charles River Breeding and Maintenance Diet for Rabbits,Altromin, Germany) approx. 100 g. per day
- Water: Free access to tap-water

Environmental conditions
- A controlled environment was maintained in the room with optimal conditions considered as being approximately 15 air changes per hour, a temperature of 21±3°C, a relative humidity of 30-70% and 12 hours artificial fluorescent light and 12 hours dark per day.

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

Single samples of approximately 36 mg of PHYTOSPHINGOSINE (a volume of approximately 0.1 ml) were instilled into one eye of each of three rabbits.

Duration of treatment / exposure:

Immediately after starting the experiment

Observation period (in vivo):

Observations were made 1, 24 and 48 hours after instillation.

Duration of post- treatment incubation (in vitro):

None, based on the severity of the ocular lesions observed dunng the study, the animals were sacrificed for ethical reasons immediately after the 48 hours observation.

Number of animals or in vitro replicates:

3

Details on study design:

Treatment
A health inspection was performed prior to application of treatment to ensure that the animals were in a good state of health. Special attention was paid to the eyes, which were free from any abnormality. Each animal was treated by application of 35.8 (35.6 - 36.0) mg of the test substance (a volume of approximately 0.1 ml), in the confilactival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control.
Immediately after the 24 hr observation, a solution of 2% fluorescein in water (adjusted to pH 7.0) was instilled into both yes of each animal to quantitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area. To remove residual test substance, the treated eye of each animal was rinsed with approximately 50 ml tepid tap-water, using a velocity of flow which did not affect the eye, immediately after fluorescein examination on day 2. For reference control the other eye was also rinsed.

Observation
- Mortality/Viability: twice daily
- Toxicity: at least once daily
- Body Weight: Day of treatment (prior to application)
- Irritation: The eyes of each animal were examined approximately 1, 24 and 48 hours after instillation of the test substance. The irritation scores and a description of all other (local) effects were recorded.

The irritation was assessed according to the following numerical scoring system (according to guideline).

Tool used to assess score
Fluor area(%): green staining (Percentage of total corneal area) after fluorescein treatment.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.5

Max. score:

3

Reversibility:

not reversible

Remarks on result:

probability of severe irritation

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.5

Max. score:

3

Reversibility:

not reversible

Remarks on result:

probability of severe irritation

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.5

Max. score:

3

Reversibility:

not reversible

Remarks on result:

probability of severe irritation

Results 4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

not reversible

Remarks on result:

probability of severe irritation

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

not reversible

Remarks on result:

probability of severe irritation

Results 6

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

not reversible

Remarks on result:

probability of severe irritation

Results 7

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

not reversible

Remarks on result:

probability of severe irritation

Results 8

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3.5

Max. score:

4

Reversibility:

not reversible

Remarks on result:

probability of severe irritation

Results 9

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

not reversible

Remarks on result:

probability of severe irritation

Results 10

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not reversible

Remarks on result:

probability of severe irritation

Results 11

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not reversible

Remarks on result:

probability of severe irritation

Results 12

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not reversible

Remarks on result:

probability of severe irritation

Irritant / corrosive response data:

Irritation and Corrosion
Application of approximately 36 mg of the test substance (a volume of approximately 0.1 ml) into one eye of each of three rabbits resulted in effects on the cornea, iris and conjunctivae. Corneal injury was seen as opacity (maximum grade 3) and epithelial damage (maximum 75% of the corneal area). The corneal injury persisted until termination (at 48 hours after instillation). Iridial irritation (grade 1) was observed in all animals and persisted until termination. Irritation of the conjunctivae was seen as redness, chemosis and discharge, which persisted until termination in all animals.
At the 48 hours observation, the outside of the eyelids was red and swollen and grey/white discolouration of the eyelids and/or nictitating membrane (sign of necrosis) was noted.
Based on the severity of the ocular lesions observed during the study, the animals were sacrificed for ethical reasons immediately after the 48 hours observation.

Other effects:

Colouration
No staining of (peri) ocular tissues by the test substance was observed.
Remnants of the test substance were present in the eyes of all animals during the observation period.

Toxicity / Mortality
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Any other information on results incl. tables

Table1: INDIVIDUAL EYE IRRITATION SCORES

Time after dosing	Cornea			Iris	Conjunctivae			Comments
	Opacity	Area	Fluor area(%)		Redness	Chemosis	Discharge
No 116#
1 hour	1	1	75	1	2	2	2	b
24 hours	2	3		1	3	4	3	b
48 hours	3	1		1	3	4	2	b c k
No 118#
1 hour	1	2	75	1	2	3	2	b
24 hours	2	3		1	3	4	2	b
48 hours	3	1		1	3	3	3	b c k l
No 119#
1 hour	1	1	75	1	2	3	2	b
24 hours	2	3		1	3	4	2	b
48 hours	3	1		1	3	4	3	b c k l

Fluor area (%): green staining (percentage of total corneal area) after fluorescein treatment.

Comments

b: Remnants of the test substance in the eye

c: Outside of the eyelids were red and swollen

k: Grey/white discolouration of the nictitating membrane (sign of necrosis).

l: Grey/white discolouration of the eyelid (sign of necrosis)

Table2. MEAN VALUE EYE IRRITATION SCORES

Animal#		Mean 24-48hrs
		corneal opacity			Iris			Conjunctivae
								Redness	Chemosis
116		2.5			1.0			3.0	4.0
118		2.5			1.0			3.0	3.5
119		2.5			1.0			3.0	4.0

# Animal specifications (at start of study):

Animal no		Sex			Age(weeks)			Body weight(grams)
116		male			10-12			2110
118		male			10-12			2335
119		male			10-12			1915

Applicant's summary and conclusion

Interpretation of results:

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

Remarks:

acc. to CLP Regulation

Conclusions:

The test item phytosphingosine causes serious and irreversible damage to eye (corrosive).

Executive summary:

Acute eye irritation/corrosion of PHYTOSPHINGOSINE was investigated in the rabbit according to OECD 405 and in compliance with GLP.

Single samples of approximately 36 mg of PHYTOSPHINGOSINE (a volume of approximately 0.1 ml) were instilled into one eye of each of three rabbits. Observations were made 1, 24 and 48 hours after instillation. Application of the test substance resulted in severe adverse effects on the cornea, iris and conjunctivae. Corneal injury was seen as opacity (maximum grade 3) and epithelial damage (maximum 75% of the corneal area). The corneal injury persisted until termination (at 48 hours after instillation). Iridial irritation (grade 1) was observed in all animals and persisted until termination. Irritation of the conjunctivae was seen as redness, chemosis and discharge, which persisted until termination in all animals. Based on the severity of the ocular lesions observed during the study, the animals were sacrificed for ethical reasons immediately after the 48 hours observation. In conclusion, phytosphingosine causes serious and irreversible damage to eye (corrosive).

The study was considered reliable and adequate for hazard assessment of human health.

Based on these results and according to the classification criteria set out in the CLP Regulation, phytosphingosine is classified for Serious eye damage, Category 1, H318 "Causes serious eye damage".