ethyl N2-dodecanoyl-.sc.l.sc.-argininate hydrochloride

Endpoint summary

Description of key information

Skin irritation

Test method according to OECD 404 . GLP study. The substance object of study, LAE, is not irritating to the skin.

Eye irritation:

Tests assessed with LAE showed that this substance causes severe ocular damage. Tests assessed with different concentrations showed that there is no risk of damage to eyes in the range of concentrations tested which corresponds to 0.02%-0.82% LAE.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 8, 2000 to December 15, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study with GLP.

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Substance: L.A.E.
Batch number: 2625
Expiry date: March 14, 2000.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia, S.A.A.
- Age at study initiation: 8-10 weeks
- Weight at study initiation: approximately 2 Kg
- Housing: Individually housed in stainless steel cages measuring 69x45x51 cm and equipped with grid floors
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days prior to test

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-21ºC
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark by fluorescent tubes

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Remarks:

Administered as supplied.

Controls:

no

Amount / concentration applied:

500 mg

Duration of treatment / exposure:

4 h

Observation period:

14 days
Examinations after dosing: 1, 24, 48 and 72 hours, 7 and 14 days.

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 2.5x2.5 cm of animal's dorsum
- Type of wrap if used: Strip of aluminium foil assemblied by encircling the trunk of the animal with an elastic adhesive bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, by gentle swabbing of the skin with cotton wool soaked in water at body temperature
- Time after start of exposure: 4 hours

SCORING SYSTEM:

ERYTHEMA AND ESCHAR FORMATION
No erythema: 0
Very slight erythema (barely perceptible): 1
Well defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema: 4

OEDEMA FORMATION
No oedema: 0
Very slight oedema (barely perceptible): 1
Slight oedema (adges of area defined by definite raising): 2
Moderate oedema (raised approximately 1mm): 3
Severe oedema (raised more than 1 mm and extending beyond area of exposure): 4

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

(mean score 1)

Time point:

24/48/72 h

Score:

0.67

Max. score:

1

Reversibility:

fully reversible within: 72 h

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

(mean score 2)

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 14 d

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

(mean score 3)

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 14 d

Remarks on result:

other: Day 14: erythema score = 1

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

(mean score 1)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

(mean score 2)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

(mean score 3)

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 14 d

Irritant / corrosive response data:

Reversibility of any observed effect: Changes fully reversible within 15 days.

Other effects:

A slight erythema was observed in all the three treated animals ca. 1 hour after the end of the exposure period. A slight oedema was also observed in one of the three animals at this examination. The same response was maintained in two of the three animals at 24 and 48 hour examinations, while a well defined erythema and slight oedema were observed in the remaining animals at these examinations. Recovery was observed in one of the three animals ca. 72 hours after the end of the exposure, while the other two animals maintained the same erythema scores. A very slight erythema and oedema were still present in two of the three animals at the 7 Day examination. In addition, desquamation of the treated skin was noted in the three animals. Recovery from irritation occurred on Day 15 in two of the three animals, while the desquamation was still evident in two and a slight erythema in one of the treated animals. There was no indication of a systemic effect of treatment. No changes in bodyweight occurred during the course of the study.

Table 1: Individual data and means

Time after the end of exposure	Erythema/Eschar			Oedema
	Animal Nº			Animal Nº
	1	2	3	1	2	3
1 hour	1	1	1	0	0	1
24 hours	1	1	2	0	0	1
48 hours	1	1	2	0	0	0
72 hours	0	1	2	0	0	0
7 day	0	1	1	0	0	1
14 day	0	0	1	0	0	0

Interpretation of results:

GHS criteria not met

Remarks:

CLP Implementation.

Conclusions:

To investigate the degree of irritation of Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE), the presented study was assessed on the intact skin of 3 rabbits. The results of the study showed that LAE had some irritant effect on the skin of the rabbit. The incidence and severity of this reaction were however not sufficient for classification. There was no indication of a systemic effect of treatment.

Executive summary:

The study was performed according to OECD 404 and in agreement with those of B4 detailed in the Council Directive 67/548/EEC EU method.

The purpose of this study was to investigate the degree of irritation produced on the intact skin of the rabbit following 4 hours contact with N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE). A 0.5 g aliquot of the test item was applied to the prepared dorsal skin of 3 animals for a period of 4 hours. The resulting reaction to treatment was assessed approximately 1, 24, 48 and 72 hours, 7 and 14 days after the end of the exposure period.

A slight well defined erythema (scores of 1 and 2) was observed in the 3 treated animals following a 4 hour period of exposure to the test item. A slight erythema was still present 7 days after the end of the exposure period in 2 animals, with a slight oedema also present in one of these 2 animals. One of the 3 animals still exhibited a slight erythema at the Day 15 examination. Desquamation of the treated skin was also noted 7 and 14 days after the end of the exposure.

These results indicate that LAE has an irritant effect on the skin at the rabbit. However, this reaction was not sufficient to require classiffication according to CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 11, 1996 to February 18, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: B&K Universal GJ, S. L.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 2.4 Kg
- Housing: Individually in steel cages (48x59x37 cm)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22 ºC
- Humidity (%): 45-80 %
- Photoperiod (hrs dark / hrs light): 12 hours each 24 hours.

IN-LIFE DATES: From: November 11, 1996; To: December 2, 1996

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.1 mL (84 mg)

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM:

Cornea Opacity: Degree of density (area most dense taken for reading)
- No ulceration or opacity: 0
- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre),
details of iris clearly visible: 1
- Easily discernible translucent area, details of iris slightly obscured: 2
- Nacreous area, no details of iris visible, size of pupil barely discernible: 3
- Opaque cornea, iris not discernible through the opacity: 4

Iris:
- Normal: 0
- Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia,
or injection, any of these or combination of any thereof, iris still reacting to light
(sluggish reaction is positive): 1
- No reaction to light, haemorrhage, gross destruction (any or all of these): 2

Conjunctivae: Redness (refers to palpebral and bulbar conjunctivae)
- Blood vessels normal: 0
- Some blood vessels definitely hyperaemic (injected): 1
- Diffuse, crimson colour, individual vessels not easily discernible: 2
- Diffuse beefy red: 3

Edema: Lids and/or nictitating membranes
- No swelling: 0
- Slight swelling (includes nictitating membranes): 1
- Obvious swelling with partial eversion of lids: 2
- Swelling with lids about half-closed: 3
- Swelling with lids more than half-closed: 4

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

4

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

4

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

4

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

7 d

Score:

3

Remarks on result:

other: At 24h: not possible (white corneal opacity and suppuration)

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

4

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

4

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

4

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Remarks on result:

not determinable

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Remarks on result:

not determinable

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Remarks on result:

not determinable

Irritant / corrosive response data:

Reversibility of any observed effect: Changes not fully reversible within 21 days

Other effects:

It was not possible to assess the effects of the test substance on the iris due to the opacity of the cornea.

Table 1: Acute eye irritation: Results.

Animal Nº.	REGION OF EYE		1 h	24 h	48 h	72 h	7d	14 d	21 d
1	Cornea	Density	3	4	4	4	4	3	4
	Iris		0	-	-	-	-	-	-
	Conjunctiva	Hyperaemia	2	*	*	*	3	2	2
		Edema	4	4	4	4	4	3	3
2	Cornea	Density	3	4	4	4	4	4	4
	Iris		0	-	-	-	-	-	-
	Conjunctiva	Hyperaemia	2	*	3	3	3	2	2
		Edema	4	4	4	4	4	4	3
3	Cornea	Density	3	4	4	4	4	4	4
	Iris		0	-	-	-	-	-	-
	Conjunctiva	Hyperaemia	2	*	*	3	3	2	2
		Edema	4	4	4	3	3	2	2

-: Not possible (corneal opacity)

*: Not possible (white corneal opacity and suppuration)

Interpretation of results:

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

Remarks:

CLP Implementation.

Conclusions:

The scope of this experimental study was to evaluate the degree of eye irritation or corrosion caused by Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) after a single application to the surface of 3 rabbit's eye. Observations to obtain current results were recorded until 21 days after administration. Results showed that the substance is causing severe ocular damage and is classified as eye irritating.

Executive summary:

The aim of this Study was to determine the degree of eye irritation or corrosion caused by N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) after a single application to the surface of the rabbit's eye.

A total of 3 albino rabbits were tested by a single application of 0.1 ml (90% purity of the test substance) to one of the eye, the untreated eye serves as the control. The degree of eye irritation/corrosion was evaluated by scoring lesions of conjunctiva, cornea and iris at specific intervals.

The animal behavior was observed immediately after instillation of the product and then were returned to their cages and checked at regular intervals.The degree of ocular irritation was evaluated in each rabbit at 1, 24, 48 and 72 hours after treatment, following the criteria set out below. Additional observations were made at 7, 14 and 21 days after administration to assess the reversibility or irreversibility of the same.

After assessment and evaluation of the alterations according to the OECD GUIDELINE FOR TESTING OF CHEMICALS, protocol no. 405, of 24^thFebruary 1987 and the EEC Directive of the 31^stMy 1992 (92/69/EEC) Annex, PartB, Method B.5, it is considered to cause irreversible effects on the eye (Category I) according to CLP Regulation.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 11, 1996 to June 18, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: B&K Universal GJ, S. L.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 2.1-2.2 Kg
- Housing: Individually in steel cages (48x59x37 cm)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22 ºC
- Humidity (%): 42-70 %
- Photoperiod (hrs dark / hrs light): 12 hours each 24 hours.

IN-LIFE DATES: From: January 28, 1997; To: February 18, 1997

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

100 mg

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM:

Cornea Opacity: Degree of density (area most dense taken for reading)
- No ulceration or opacity: 0
- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre),
details of iris clearly visible: 1
- Easily discernible translucent area, details of iris slightly obscured: 2
- Nacreous area, no details of iris visible, size of pupil barely discernible: 3
- Opaque cornea, iris not discernible through the opacity: 4

Iris:
- Normal: 0
- Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia,
or injection, any of these or combination of any thereof, iris still reacting to light
(sluggish reaction is positive): 1
- No reaction to light, haemorrhage, gross destruction (any or all of these): 2

Conjunctivae: Redness (refers to palpebral and bulbar conjunctivae)
- Blood vessels normal: 0
- Some blood vessels definitely hyperaemic (injected): 1
- Diffuse, crimson colour, individual vessels not easily discernible: 2
- Diffuse beefy red: 3

Edema: Lids and/or nictitating membranes
- No swelling: 0
- Slight swelling (includes nictitating membranes): 1
- Obvious swelling with partial eversion of lids: 2
- Swelling with lids about half-closed: 3
- Swelling with lids more than half-closed: 4

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

4

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

4

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

4

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

4

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

4

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

4

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Remarks on result:

not determinable

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Remarks on result:

not determinable

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Remarks on result:

not determinable

Irritant / corrosive response data:

Reversibility of any observed effect: Changes not fully reversible within 21 days

Other effects:

It was not possible to assess the effects of the test substance on the iris due to the opacity of the cornea.

Table 1: Acute eye irritation.

Animal Nº.	REGION OF EYE		1 h	24 h	48 h	72 h	7d	14 d	21 d
1	Cornea	Density	3	4	4	4	4	3	4
	Iris		0	-	-	-	-	-	-
	Conjunctiva	Hyperaemia	2	*	*	*	3	2	2
		Edema	4	4	4	4	4	3	3
2	Cornea	Density	3	4	4	4	4	4	4
	Iris		0	-	-	-	-	-	-
	Conjunctiva	Hyperaemia	2	*	3	3	3	2	2
		Edema	4	4	4	4	4	4	3
3	Cornea	Density	3	4	4	4	4	4	4
	Iris		0	-	-	-	-	-	-
	Conjunctiva	Hyperaemia	2	*	*	3	3	2	2
		Edema	4	4	4	3	3	2	2

-: Not possible (corneal opacity)

*: Not possible (white corneal opacity and suppuration)

Interpretation of results:

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

Remarks:

CLP Implementation.

Conclusions:

The scope of this experimental study was to evaluate the degree of eye irritation or corrosion caused by Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) after a single application to the surface of 3 rabbit's eye. Observations to obtain current results were recorded until 21 days after administration. Results showed that the substance is causing severe ocular damage.

Executive summary:

The aim of this study was to determine the degree of eye irritation or corrosion caused N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE)

(LAE) after a single application to the surface of the rabbit's eye.

A total of 3 albino rabbits were tested by a single application of 0.1 ml (99% purity of the test substance) to one of the eye, the untreated eye serves as the control. The degree of eye irritation/corrosion was evaluated by scoring lesions of conjunctiva, cornea and iris at specific intervals.

The animal behavior was observed immediately after instillation of the product and then were returned to their cages and checked at regular intervals.The degree of ocular irritation was evaluated in each rabbit at 1, 24, 48 and 72 hours after treatment, following the criteria set out below. Additional observations were made at 7, 14 and 21 days after administration to assess the reversibility or irreversibility of the same.

After assessment and evaluation of the alterations according to the OECD GUIDELINE FOR TESTING OF CHEMICALS, protocol no. 405, of 24^thFebruary 1987 and the EEC Directive of the 31^stMy 1992 (92/69/EEC) Annex, PartB, Method B.5, it is considered to cause SEVERE OCULAR IRRITATION.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 6, 2006 to September 6, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A guideline study with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Granja Cunícola San Bernardo, S. L., (Tulebras, Navarra, Spain).
- Age at study initiation: 13-14 weeks
- Weight at study initiation: 3.0-3.1 Kg
- Housing: Individually in steel cages (52x58x43 cm) with a grille floor
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 ºC
- Humidity (%): 45-70 %
- Photoperiod (hrs dark / hrs light): 12 hours each 24 hours.

IN-LIFE DATES: From: May 29, 2006; To: June 12, 2006

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml

Observation period (in vivo):

7 days for 2 animals
14 days for 1 animal

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM:
Cornea Opacity: Degree of density (area most dense taken for reading)
- No ulceration or opacity: 0
- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre),
details of iris clearly visible: 1
- Easily discernible translucent area, details of iris slightly obscured: 2
- Nacreous area, no details of iris visible, size of pupil barely discernible: 3
- Opaque cornea, iris not discernible through the opacity: 4

Iris:
- Normal: 0
- Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia,
or injection, any of these or combination of any thereof, iris still reacting to light
(sluggish reaction is positive): 1
- No reaction to light, haemorrhage, gross destruction (any or all of these): 2

Conjunctivae: Redness (refers to palpebral and bulbar conjunctivae)
- Blood vessels normal: 0
- Some blood vessels definitely hyperaemic (injected): 1
- Diffuse, crimson colour, individual vessels not easily discernible: 2
- Diffuse beefy red: 3

Edema: Lids and/or nictitating membranes
- No swelling: 0
- Slight swelling (includes nictitating membranes): 1
- Obvious swelling with partial eversion of lids: 2
- Swelling with lids about half-closed: 3
- Swelling with lids more than half-closed: 4

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 h

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible within: 14 d

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible within: 7 d

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.66

Max. score:

1

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 h

Table 1: Acute eye irritation results:

Animal Nº.	REGION OF EYE		1 h	24 h	48 h	72 h	7d	14 d
1	Cornea	Density	0	0	0	0	0	0
	Iris		0	0	0	0	0	0
	Conjunctiva	Hyperaemia	2	2	1	1	1	0
		Edema	0	0	0	0	0	0
2	Cornea	Density	0	1	0	0	0	-
	Iris		0	0	0	0	0	-
	Conjunctiva	Hyperaemia	2	2	1	1	0	-
		Edema	2	1	1	0	0	-
3	Cornea	Density	0	0	0	0	0	-
	Iris		0	0	0	0	0	-
	Conjunctiva	Hyperaemia	2	2	1	0	0	-
		Edema	1	1	0	0	0	-

"-": Animals sacrified within 14 days of administration as no eye lesions were observed.

Interpretation of results:

GHS criteria not met

Remarks:

CLP Implementation.

Conclusions:

The alterations induced by the test substance were evaluated in 7 and 14 days for 2 and 1 New Zealand White rabbit respectevely. In view of the results obtained, the test item is considered to be NON IRRITANT TO THE EYE.

Executive summary:

The alterations induced by the test item were evaluated according to the OECD Guideline for Testing of Chemicals, Guideline 405, 24 April 2002 and the Commission Directive 2004/73/EC of 29 April 2004, Annex 2E. Method B.5. In view of the results obtained, the test item is considered to be NON IRRITANT TO THE EYE.

The degree of primary eye irritation caused by the test item was determined after applicationto the surface of the eye of the rabbit.

 

The test item was administered to one animal in the initial test. According to the ocular lesions observed in this animal, another two animals were administered in a confirmation test.

The mean values taking into account the values obtained in the examinations at 24, 48 and 72 hours following administration, for the three administered animals were:

-Corneal opacity: 0.11

-Lesions in the iris: 0.00

-Hyperaemia:1.22

-Edema 0.33

Therefore, the test substance was considered to cause NO OCULAR IRRITATION and it is not classified for ocular irritation/corrosion according to CLP Regulation.

Reference 4

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 5, 1996 to February 18, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: B&K Universal GJ, S. L.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 2.4 Kg
- Housing: Individually in steel cages (48x59x37 cm)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22 ºC
- Humidity (%): 45-80 %
- Photoperiod (hrs dark / hrs light): 12 hours each 24 hours.

IN-LIFE DATES: From: November 11, 1996; To: December 2, 1996

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.1 mL

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM:
Cornea Opacity: Degree of density (area most dense taken for reading)
- No ulceration or opacity: 0
- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre),
details of iris clearly visible: 1
- Easily discernible translucent area, details of iris slightly obscured: 2
- Nacreous area, no details of iris visible, size of pupil barely discernible: 3
- Opaque cornea, iris not discernible through the opacity: 4

Iris:
- Normal: 0
- Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia,
or injection, any of these or combination of any thereof, iris still reacting to light
(sluggish reaction is positive): 1
- No reaction to light, haemorrhage, gross destruction (any or all of these): 2

Conjunctivae: Redness (refers to palpebral and bulbar conjunctivae)
- Blood vessels normal: 0
- Some blood vessels definitely hyperaemic (injected): 1
- Diffuse, crimson colour, individual vessels not easily discernible: 2
- Diffuse beefy red: 3

Edema: Lids and/or nictitating membranes
- No swelling: 0
- Slight swelling (includes nictitating membranes): 1
- Obvious swelling with partial eversion of lids: 2
- Swelling with lids about half-closed: 3
- Swelling with lids more than half-closed: 4

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not reversible

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not reversible

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

not fully reversible within: 21d

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.7

Max. score:

1

Reversibility:

fully reversible within: 21 d

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.34

Max. score:

1

Reversibility:

fully reversible within: 21 d

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 21d

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.33

Max. score:

3

Reversibility:

not fully reversible within: 21d

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.33

Max. score:

3

Reversibility:

not fully reversible within: 21d

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: 21 d

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 21 d

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

fully reversible within: 14 d

Table 1: Acute eye irritation results:

Animal Nº.	REGION OF EYE		1 h	24 h	48 h	72 h	7d	14 d	21 d
1	Cornea	Density	1	2	2	2	2	2	2
	Iris		0	0	0	0	0	0	0
	Conjunctiva	Hyperaemia	2	2	3	3	2	1	0
		Edema	3	3	3	3	2	1	1
2	Cornea	Density	1	2	2	2	2	2	2
	Iris		0	0	1	1	1	0	0
	Conjunctiva	Hyperaemia	2	2	2	3	2	1	1
		Edema	3	2	2	2	2	1	0
3	Cornea	Density	2	2	1	1	2	1	1
	Iris		0	0	0	1	-	0	0
	Conjunctiva	Hyperaemia	2	2	2	3	3	1	1
		Edema	3	3	3	3	2	0	0

"-": Animals sacrified within 14 days of administration as no eye lesions were observed.

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

CLP Implementation.

Conclusions:

The degree of eye irritation or corrosion caused by the test substance was assessed after a single application to the surface of 3 albino rabbits' eyes. The degree of eye irritation/corrosion was evaluated until 21 days observations after treatment by scoring: lesions of conjunctiva, cornea and iris at specific intervals. Results showed that the substance is causing severe ocular damage.

Executive summary:

The aim of this study was to determine the degree of eye irritation or corrosion caused by N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) caused by the test substance after a single application to the surface of the rabbit's eye.

A total of 3 albino rabbits were tested by a single application of 0.1 ml (20% purity of the test substance) to one of the eye, the untreated eye serves as the control. The degree of eye irritation/corrosion was evaluated by scoring lesions of conjunctiva, cornea and iris at specific intervals.

The animal behavior was observed immediately after instillation of the product and then were returned to their cages and checked at regular intervals.The degree of ocular irritation was evaluated in each rabbit at 1, 24,48 and 72 hours after treatment, following the criteria set out below. Additional observations were made at 7, 14 and 21days after administration to assess the reversibility or irreversibility of the same.

The eye irritation test was performed according to OECD 405 guideline and EU method B.5. The substance causes severe ocular damage and is classified in Category 1: Irreversible effects on the eye, according to CLP Regulation.

Reference 5

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 19, 2000 to October 3, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A guideline study with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: B&K Universal GJ, S. L.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 2.6-3.4 Kg
- Housing: Individually in steel cages (45x59x37 or 52x58x43 cm)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25 ºC
- Humidity (%): 55-80 %
- Photoperiod (hrs dark / hrs light): 12 hours each 24 hours.

IN-LIFE DATES: From: July 17, 2000; To: August 1, 2000

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.1 mL

Observation period (in vivo):

14 days

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM:
Cornea Opacity: Degree of density (area most dense taken for reading)
- No ulceration or opacity: 0
- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre),
details of iris clearly visible: 1
- Easily discernible translucent area, details of iris slightly obscured: 2
- Nacreous area, no details of iris visible, size of pupil barely discernible: 3
- Opaque cornea, iris not discernible through the opacity: 4

Iris:
- Normal: 0
- Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia,
or injection, any of these or combination of any thereof, iris still reacting to light
(sluggish reaction is positive): 1
- No reaction to light, haemorrhage, gross destruction (any or all of these): 2

Conjunctivae: Redness (refers to palpebral and bulbar conjunctivae)
- Blood vessels normal: 0
- Some blood vessels definitely hyperaemic (injected): 1
- Diffuse, crimson colour, individual vessels not easily discernible: 2
- Diffuse beefy red: 3

Edema: Lids and/or nictitating membranes
- No swelling: 0
- Slight swelling (includes nictitating membranes): 1
- Obvious swelling with partial eversion of lids: 2
- Swelling with lids about half-closed: 3
- Swelling with lids more than half-closed: 4

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible within: 7 d

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible within: 14 d

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

1

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 d

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Table 1: Acute eye irritation results:

Animal Nº.	REGION OF EYE		1 h	24 h	48 h	72 h	7d
1	Cornea	Density	0	0	0	0	0
	Iris		0	0	0	0	0
	Conjunctiva	Hyperaemia	2	2	1	1	0
		Edema	3	1	1	0	0
2	Cornea	Density	0	0	0	0	0
	Iris		1	0	0	0	0
	Conjunctiva	Hyperaemia	2	2	1	1	1
		Edema	3	1	1	1	0
3	Cornea	Density	0	0	0	0	-
	Iris		1	0	0	0	-
	Conjunctiva	Hyperaemia	2	1	0	0	-
		Edema	1	0	0	0	-

"-": Animals sacrified within 14 days of administration as no eye lesions were observed.

Interpretation of results:

GHS criteria not met

Remarks:

CLP Implementation.

Conclusions:

The test substance was administered for the study of primary eye irritation. After assessment and evaluation of the alterations produced in 3 New Zealand White rabbits after 14 days of observational period. Results obtained indicate that the substance evaluated did not produce ocular irritation.

Executive summary:

The aim of this Study was to determine the degree of eye irritation or corrosion caused by the test substance after a single application to the surface of the rabbit's eye after 14 days of observational period.

 

The New Zealand White rabbit was chosen as a test species because it is an animal normally accepted as suitable for the study of primary eye irritation and there is sufficient background information to justify its use.

 

The application was topical as this is the most likely route for an accidental exposure of the human eye.

 The test substance was administered for the study of primary eye irritation. After assessment and evaluation of the alterations which it induced, according to the OECD GUIDELINE FOR TESTING OF CHEMICALS, protocol Nº405, of 24^th February 1987 and the EEC Directive of the 31^stJuly 1992 (92/69/EEC) Annex, PartB, MethodB.5, it is considered to cause NO OCULAR IRRITATION, and it is not classified for eye irritation/corrosion according to CLP Regulation.

Reference 6

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 19, 2000 to October 3, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A guideline study with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: B&K Universal GJ, S. L.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 2.7-3.5 Kg
- Housing: Individually in steel cages (45x59x37 or 52x58x43 cm)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25 ºC
- Humidity (%): 55-80 %
- Photoperiod (hrs dark / hrs light): 12 hours each 24 hours.

IN-LIFE DATES: From: July 17, 2000; To: July 24, 2000

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.1 mL

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM:
Cornea Opacity: Degree of density (area most dense taken for reading)
- No ulceration or opacity: 0
- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre),
details of iris clearly visible: 1
- Easily discernible translucent area, details of iris slightly obscured: 2
- Nacreous area, no details of iris visible, size of pupil barely discernible: 3
- Opaque cornea, iris not discernible through the opacity: 4

Iris:
- Normal: 0
- Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia,
or injection, any of these or combination of any thereof, iris still reacting to light
(sluggish reaction is positive): 1
- No reaction to light, haemorrhage, gross destruction (any or all of these): 2

Conjunctivae: Redness (refers to palpebral and bulbar conjunctivae)
- Blood vessels normal: 0
- Some blood vessels definitely hyperaemic (injected): 1
- Diffuse, crimson colour, individual vessels not easily discernible: 2
- Diffuse beefy red: 3

Edema: Lids and/or nictitating membranes
- No swelling: 0
- Slight swelling (includes nictitating membranes): 1
- Obvious swelling with partial eversion of lids: 2
- Swelling with lids about half-closed: 3
- Swelling with lids more than half-closed: 4

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 h

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 d

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 h

Table 1: Acute eye irritation results:

Animal Nº.	REGION OF EYE		1 h	24 h	48 h	72 h	7d
1	Cornea	Density	0	1	0	0	0
	Iris		0	0	0	0	0
	Conjunctiva	Hyperaemia	1	1	1	1	0
		Edema	0	0	0	0	0
2	Cornea	Density	0	0	0	0	-
	Iris		0	0	0	0	-
	Conjunctiva	Hyperaemia	1	0	1	0	-
		Edema	1	0	0	0	-
3	Cornea	Density	0	0	0	0	-
	Iris		0	0	0	0	-
	Conjunctiva	Hyperaemia	2	1	0	0	-
		Edema	1	1	0	0	-

"-": Animals sacrified within 14 days of administration as no eye lesions were observed.

Interpretation of results:

GHS criteria not met

Remarks:

CLP Implementation.

Conclusions:

The test substance was administered in order to study primary eye irritation alterations produced in 3 New Zealand White rabbits after 7 days of observational period. After assessment and evaluation of the alterations which it induced, it was concluded that it is considered to cause NO OCULAR IRRITATION.

Executive summary:

The aim of this Study was to determine the degree of eye irritation or corrosion caused by The test substance after a single application to the surface of therabbit's eye.

 

The New Zealand White rabbit was chosen as a test species because it is an animal normally accepted as suitable for the study of primary eye irritation and there is sufficient background information to justify its use.

 

The application was topical as this is the most likely route for an accidental exposure of the human eye.

 

The test substance was administered for the study of primary eye irritation. After assessment and evaluation of the alterations which it induced, according to the OECD GUIDELINE FOR TESTING OF CHEMICALS, protocol Nº405, of 24^th February 1987 and the EEC Directive of the 31^stJuly 1992 (92/69/EEC) Annex, PartB, MethodB.5, the test substance is considered to cause NO OCULAR IRRITATION and it is not classified for ocular irritation/corrosion according to CLP Regulation.

Reference 7

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 19, 2000 to October 3, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A guideline study with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: B&K Universal GJ, S. L.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 2.5-3.5 Kg
- Housing: Individually in steel cages (45x59x37 or 52x58x43 cm)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25 ºC
- Humidity (%): 55-80 %
- Photoperiod (hrs dark / hrs light): 12 hours each 24 hours.

IN-LIFE DATES: From: July 17, 2000; To: July 25, 2000

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.1 mL

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM:
Cornea Opacity: Degree of density (area most dense taken for reading)
- No ulceration or opacity: 0
- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre),
details of iris clearly visible: 1
- Easily discernible translucent area, details of iris slightly obscured: 2
- Nacreous area, no details of iris visible, size of pupil barely discernible: 3
- Opaque cornea, iris not discernible through the opacity: 4

Iris:
- Normal: 0
- Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia,
or injection, any of these or combination of any thereof, iris still reacting to light
(sluggish reaction is positive): 1
- No reaction to light, haemorrhage, gross destruction (any or all of these): 2

Conjunctivae: Redness (refers to palpebral and bulbar conjunctivae)
- Blood vessels normal: 0
- Some blood vessels definitely hyperaemic (injected): 1
- Diffuse, crimson colour, individual vessels not easily discernible: 2
- Diffuse beefy red: 3

Edema: Lids and/or nictitating membranes
- No swelling: 0
- Slight swelling (includes nictitating membranes): 1
- Obvious swelling with partial eversion of lids: 2
- Swelling with lids about half-closed: 3
- Swelling with lids more than half-closed: 4

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

1

Reversibility:

fully reversible within: 7 d

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Table 1: Acute eye irritation results:

Animal Nº.	REGION OF EYE		1 h	24 h	48 h	72 h	7d
1	Cornea	Density	0	0	0	0	-
	Iris		0	0	0	0	-
	Conjunctiva	Hyperaemia	1	0	0	0	-
		Edema	1	0	0	0	-
2	Cornea	Density	0	0	0	0	-
	Iris		0	0	0	0	-
	Conjunctiva	Hyperaemia	1	0	0	0	-
		Edema	0	1	0	0	-
3	Cornea	Density	0	0	0	0	0
	Iris		0	0	0	0	0
	Conjunctiva	Hyperaemia	1	1	0	1	0
		Edema	0	0	0	0	0

"-": Animals sacrified within 14 days of administration as no eye lesions were observed.

Interpretation of results:

GHS criteria not met

Remarks:

CLP Implementation.

Conclusions:

The test substance was administered to study the primary eye irritation of 3 New Zealand rabbits. After assessment and evaluation of the alterations which it induced, the substance is considered to cause NO OCULAR IRRITATION.

Executive summary:

The aim of this Study was to determine the degree of eye irritation or corrosion caused by The test substance after a single application to the surface of the rabbit's eye.

 

The New Zealand White rabbit was chosen as a test species because it is an animal normally accepted as suitable for the study of primary eye irritation and there is sufficient background information to justify its use.

 

The application was topical as this is the most likely route for an accidental exposure of the human eye.

 

The test substance was administered for the study of primary eye irritation.After assessment and evaluation of the alterations which it induced, according to the OECD GUIDELINE FOR TESTING OF CHEMICALS, protocol Nº405, of 24^th February 1987 and the EEC Directive of the 31^stJuly 1992 (92/69/EEC) Annex, PartB, MethodB.5, the test subtance was considered to cause NO OCULAR IRRITATION and it is not classified for ocular irritation/corrosion according to CLP Regulation,

Reference 8

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 19, 2000 to October 3, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A guideline study with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: B&K Universal GJ, S. L.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 2.7-3.3 Kg
- Housing: Individually in steel cages (45x59x37 or 52x58x43 cm)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25 ºC
- Humidity (%): 55-80 %
- Photoperiod (hrs dark / hrs light): 12 hours each 24 hours.

IN-LIFE DATES: From: July 17, 2000; To: July 24, 2000

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.1 mL

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM:
Cornea Opacity: Degree of density (area most dense taken for reading)
- No ulceration or opacity: 0
- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre),
details of iris clearly visible: 1
- Easily discernible translucent area, details of iris slightly obscured: 2
- Nacreous area, no details of iris visible, size of pupil barely discernible: 3
- Opaque cornea, iris not discernible through the opacity: 4

Iris:
- Normal: 0
- Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia,
or injection, any of these or combination of any thereof, iris still reacting to light
(sluggish reaction is positive): 1
- No reaction to light, haemorrhage, gross destruction (any or all of these): 2

Conjunctivae: Redness (refers to palpebral and bulbar conjunctivae)
- Blood vessels normal: 0
- Some blood vessels definitely hyperaemic (injected): 1
- Diffuse, crimson colour, individual vessels not easily discernible: 2
- Diffuse beefy red: 3

Edema: Lids and/or nictitating membranes
- No swelling: 0
- Slight swelling (includes nictitating membranes): 1
- Obvious swelling with partial eversion of lids: 2
- Swelling with lids about half-closed: 3
- Swelling with lids more than half-closed: 4

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 h

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

1

Reversibility:

fully reversible within: 7 d

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Table 1: Acute eye irritation results:

Animal Nº.	REGION OF EYE		1 h	24 h	48 h	72 h	7d
1	Cornea	Density	0	0	0	0	0
	Iris		0	0	0	0	0
	Conjunctiva	Hyperaemia	1	1	0	1	0
		Edema	0	0	0	0	0
2	Cornea	Density	0	1	0	0	-
	Iris		0	0	0	0	-
	Conjunctiva	Hyperaemia	1	0	0	0	-
		Edema	1	1	0	0	-
3	Cornea	Density	0	0	0	0	-
	Iris		0	0	0	0	-
	Conjunctiva	Hyperaemia	1	0	0	0	-
		Edema	0	0	0	0	-

"-": Animals sacrified within 14 days of administration as no eye lesions were observed.

Interpretation of results:

GHS criteria not met

Remarks:

CLP Implementation.

Conclusions:

The test substance was administered to study the primary eye irritation of 3 New Zealand rabbits after an observational period of 7 days. After assessment and evaluation of the alterations which it induced, the test substance is considered to cause NO OCULAR IRRITATION.

Executive summary:

The aim of this Study was to determine the degree of eye irritation or corrosion caused by the above-mentioned substance after a single application to the surface of the rabbit's eye.

 

The New Zealand White rabbit was chosen as a test species because it is an animal normally accepted as suitable for the study of primary eye irritation and there is sufficient background information to justify its use.

 

The application was topical as this is the most likely route for an accidental exposure of the human eye.

 

The test substance was administered for the study of primary eye irritation. After assessment and evaluation of the alterations which it induced, according to the OECD GUIDELINE FOR TESTING OF CHEMICALS, protocol Nº405, of 24^th February 1987 and the EEC Directive of the 31^stJuly 1992 (92/69/EEC) Annex, PartB, MethodB.5, the test substance was considered to cause NO OCULAR IRRITATION and it is not classified for ocular irritation/corrosion according to CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The study of skin irritation was performed according to OECD guideline protocol no. 404, of 24^thFebruary 1987 and the EEC Directive of the 31^stMy 1992 (92/69/EEC) Annex, Part B, Method B.4. The substance is not irritating to skin.

The eye irritation test was performed according to the OECD guideline, protocol no. 405, of 24^th February 1987 and the EEC Directive of the 31^stMy 1992 (92/69/EEC) Annex, PartB, Method B.5. LAE causes severe ocular damage. Tests assessed with different concentrations showed that there is no risk of damage to eyes in the range of concentrations tested which corresponds to 0.02%-0.82% LAE.

Justification for classification or non-classification

According to CLP Regulation:

Skin irritation: not classified.

Eye irreversible effects, Category 1.