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

Endpoint summary

Description of key information

The oral LD50 for the Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) is greater than 2000 mg/kg/bw.

The dermal LD50 for the Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) is greater than 2000 mg/kg/bw.

The inhalation LC50 for the Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) is greater than 5883 mg/m3 air

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 16, 2000 to July 27, 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:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

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

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan U. K. Ltd., Bicester, Oxon, England
- Age at study initiation: 5-7 weeks
- Weight at study initiation: 90-118 g
- Fasting period before study: overnight
- Housing: Groups of 3 rats of the same sex in metal cages within the treatment group
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C):18.5-20
- Humidity (%):34-55%
- Photoperiod (hrs dark / hrs light): 12 h dark/ 12 h artificial light

Route of administration:

oral: gavage

Vehicle:

other: 1% w/v aqueous methylcellulose

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 20% w/v

DOSAGE PREPARATION (if unusual): on the day of dosing

Doses:

2000 mg/kg

No. of animals per sex per dose:

3 Female, Dose 2000 mg/kg
3 Male, Dose 2000 mg/kg

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Weight Record: Days 1, 8 and 15; Rats were checked daily at least for mortalities.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Mortality:

Male: 2000 mg/kg bw; Number of animals: 3; Number of deaths: 0
Female: 2000 mg/kg bw; Number of animals: 3; Number of deaths: 0

Clinical signs:

Signs of toxicity related to dose levels: Clinical signs of reaction to treatment comprised of piloerection, salivation and hunched posture, seen in all animals on Day 1. In addition, abnormal gait and wet fur were observed in all females on Day 1. There were no other signs of reaction to treatment and recovery was complete in all males by Day 3 and in all females by Day 4.

Body weight:

All animals were considered to have achieved satisfactory bodyweight gains throughout the study.

Gross pathology:

No abnormalities were revealed at the macroscopic examination at study termination on Day 15.

Table 1: Mortality data:

Dose level (mg/Kg)	Sex	Nº of animals treated	Deaths
2000	Female	3	0
2000	Male	3	0

Interpretation of results:

GHS criteria not met

Remarks:

CLP Implementation.

Conclusions:

The acute toxicity study was realized according to OECD 423 guideline and EU method B.1 tris. The LD50 for the LAE is greater than 2000 mg/kg/bw.

Executive summary:

According to OECD 423 guideline and EU method B.1 tris, this study was performed with the purpose of ranging LD50 for the test substance: N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE).

With this objective, a group of 6 fasted rats (three males and three females) received a single oral gavage dose of the test substance, formulated in 1%w/v aqueous methylcellulose and administered at a dose level of 2000 mg/kg bodyweight.

All animals were considered to have achieved satisfactory bodyweight gains throughout the study.

No abnormalities were revealed in any of the animals at the macroscopic examination at study termination on Day 15.

The acute lethal oral dose to rats of LAE was demonstrated in this study to be greater than 2000 mg/kg bodyweight.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From May 22, 1995 to July 17, 1995

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.1 (Acute Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

Substance: Mirenat
Batch number: 00000003
Expiry date: November 5, 1995.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Olac Ltd, Bicester, Oxon, England
- Age at study initiation: 4-7 weeks of age prior to dosing
- Weight at study initiation: 104-124 g
- Fasting period before study: Overnight
- Housing: In groups up to 5 rats of the same sex in metal cages with wire mesh floors
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3ºC
- Humidity (%):30-70% RH
- Air changes (per hr): 10 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light in each 24 hours period

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Doses:

2.0 g/kg bw, contains 25% of LAE.

No. of animals per sex per dose:

5 Females
5 Males

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observatios at least twice daily for any mortalities; Bodyweight record: day 1, 8 and 15
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight and macroscopic examination.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 500 mg/kg bw

Remarks on result:

other: Dose of pure L.A.E.

Mortality:

Male: Number of animals: 5; Number of deaths: 0
Female: Number of animals: 5; Number of deaths: 0

Clinical signs:

Piloerection was observed in all rats within minutes of dosing and throughout the remainder of Day 1. There were no other clinical signs and recovery, as judged by external appearance and behaviour, was complete by Day 2.

Body weight:

All rats were considered to have achieved satisfactory bodyweight gains throughout the study.

Gross pathology:

No abnormalities were revealed at the macroscopic examination at study termination on Day 15.

No mortalities were observed.

Interpretation of results:

study cannot be used for classification

Conclusions:

According to the study performed to Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (L.A.E), the LD50 was found to be >500 mg/kg bw.

Executive summary:

The acute oral toxicity was determined according to EU B.1 method. The LD₅₀ for N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (L.A.E) was found to be greater than 500 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Quality of whole database:

Klimisch 1. This study was carried out in accordance with internationally valid GLP principles.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 17, 2005 to March 17, 2006

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 403 (Acute Inhalation Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

Substance: L.A.E. in ethanol
Batch number: LI-631 as of Oct. 19, 2005, content 0.634% of LAE

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Adult SPF bred Wistar Unilever rats, strain Hsd Cpb:WU from the experimental breeder Harlan-Winkelmann GmbH, Borchen (Germany) and Harlan Nederland (The Netherlands)
- Age at study initiation: 2 months old
- Weight at study initiation: Variation of individual weight did not exceed ±10% of the mean for each sex. Females: 173-187 g; males 176-201 g
- Housing: Singly in conventional Makrolon® Type IIIH cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days prior to start

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2ºC
- Humidity (%): 40-70%
- Air changes (per hr): 10 air changes per hour approximately
- Photoperiod (hrs dark / hrs light): 12 hours artificial light each 24 hours

IN-LIFE DATES: No mortalities

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposure tubes applying a directed-flow nose-only exposure principle.
- Exposure chamber volume: 50 L
- Source and rate of air: 20 l/min
- Method of conditioning air: Calibrated flowmeter/Soap bubble meter
- System of generating particulates/aerosols: Magnetic piston equipped with two electronic clocks
- Method of particle size determination: TSI-Laser velocimeter APS
- Treatment of exhaust air: Exhaust air was purified via cotton-wool/HEPA filters
- Temperature, humidity, pressure in air chamber: 21.9-23.2 ºC, humidity values were lower than recommended due to the use of conditioned, dry air for dispersion of the test substance.

TEST ATMOSPHERE
- Brief description of analytical method used: Gas chromatographic method (for propellants); HPLC (for the active ingredient)
- Samples taken from breathing zone: yes, in the vicinity of the breathing zone of the animals

VEHICLE
- Composition of vehicle (if applicable): Undiluted

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: Adequate to reach all potential target structures of the respiratory tract
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 4.6 um / 1.92

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Target concentration: 25000 mg/m3 (nominal 42000mg/m3).

No. of animals per sex per dose:

Group 1: Target concentration 0 mg/m3
5 male per dose
5 female per dose

Group 2: Target concentration: 25.000 mg/m3
5 male per dose
5 female per dose

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Weighing record: before exposure, day 3, 7 and 14; Observations: Several times on the day of exposure, at least once daily thereafter.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight and reflexes.

Statistics:

Statistical Evaluation of data:

Necropsy findings: In case of findings: Fisher test
Body weights: One-way ANOVA (vida infra)
Physiological data: Data of rectal temperature measurements are statistically evaluated using the ANOVA procedure (vida infra)
LC50: Rosello et al.; Schaper et al.
Analysis of variance: ANOVA (p= 0.05)

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 28 150 mg/m³ air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: LAE in ethanol

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5 883 mg/m³ air (analytical)

Based on:

act. ingr.

Exp. duration:

4 h

Remarks on result:

other: LAE

Mortality:

Mortality was not observed.

Clinical signs:

other: Group 1: All rats tolerated the exposure without specific signs. Group 2/Males: Bradypnea, labored breathing patterns, irregular breathing patterns, high-legged gait, piloerection. Group 2/Females: Labored breathing patterns, irregular breathing patterns,

Body weight:

Comparisons did not reveal changes in body weights considered to be of toxicological significance.

Gross pathology:

Animals sacrified at the end of the observation period: The macroscopic findings were indistinguishable amongst the groups.

Results obtained after exposure of rats for 4 hours:

Group/sex	Target concentration (mg/m^3)	Toxicological result(*)	Onset and duration signs	Onset mortality
1 / m	0	0/0/5	-	-
2 / m	25000	0/4/5	0 d - 3 d	-
1 / f	0	0/0/5	-	-
2 / f	25000	0/1/5	0 d - 4 d	-

(*) Number of dead animals/Number of animals with signs after cessation of exposure/Number of animals exposed.

Interpretation of results:

GHS criteria not met

Remarks:

CLP Implementation.

Conclusions:

Combined experimental evidence suggest that the test substance has a mild respiratory tract irritation potential if exposure is sufficiently high. Thus, the LC50 is greater than 28150 mg/m3 (LAE in ethanol). The LC50 for LAE is greater than 5883 mg/m3 (analytical concentration).

Executive summary:

The test conditions applied fulfilled recommendations of current test guidelines according to the OECD Guideline Nº 403 and EU method B.2, with regard to attainment of steady-state concentration, respirability of particles and the limit concentration tested. The aerosolized test substance proved to have virtually no acute inhalation toxicity to rats and mortality did not occurred even at levels of exposure exceeding significantly the limit concentration called for by the current testing guidelines applied. Combined experimental evidence, however, would suggest that the test substance has a mild respiratory tract irritation potential, if exposure to the aerosol is sufficiently high. Thus, for the test substance the LC₅₀ is greater than 28150 mg/m³ (LAE in ethanol). The LC50 for LAE is greater than 5883 mg/m³ (analytical concentration).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating conc.

Value:

5 883 mg/m³

Quality of whole database:

Klimisch 1. This study was carried out in accordance with internationally valid GLP principles.

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 16, 2000 to July 27, 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:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

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

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hsd: Sprague-Dawley obtained from Harlan U.K. Ltd., Bicester, Oxon, England
- Age at study initiation: 8-12 weeks prior to dosing
- Weight at study initiation: 206-256 g
- Housing: Individually in metal cages fitted with grid floors to ensure rapid removal of waste material to undertrays.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5-20ºC
- Humidity (%): 42-56%
- Photoperiod (hrs dark / hrs light): 12 h artificial light each 24 hour period

Type of coverage:

occlusive

Vehicle:

other: 1% w/v aqueous methylcellulose

Details on dermal exposure:

TEST SITE
- Area of exposure: 50 mmx50 mm
- % coverage: 10%
- Type of wrap if used: Porous gauze with a non-irritating dressing, further covered by a waterproof dressing encircled firmly around the trunk of the animal.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): With warm water (30-40ºC)
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 3.0 ml/kg bw
- Concentration (if solution): 667 mg/ml in 1% w/v methylcellulose

Duration of exposure:

24 h

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

Male: 2000 mg/kg bw; Number of animals: 5; Number of deaths: 0
Female: 2000 mg/kg bw; Number of animals: 5; Number of deaths: 0

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations twice daily; The bodyweight of each rat was recorded on Days 1, 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, macroscopic appearance

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

Male: 2000 mg/kg bw; Number of animals: 5; Number of deaths: 0
Female: 2000 mg/kg bw; Number of animals: 5; Number of deaths: 0

Clinical signs:

Signs of toxicity related to dose levels:
There was no evidence of a systemic response in any animal throughout the study.


All animals were considered to have achieved satisfactory
bodyweight gains throughout the study.

Body weight:

All animals were considered to have achieved satisfactory bodyweight gains throughout the study.

Gross pathology:

Effects on organs:
No macroscopic abnormalities were observed for animals killed at study termination on Day 15, with the exception of scabbing on the dose sites of three rats, plus thickening of the tissues on one.

Other findings:

Signs of toxicity (local):
There were no deaths and no clinical signs of reaction to treatment observed in any animal throughout the study.

Well-defined irritation (erythema and oedema up to Grade 2) was notable in all rats following removal of the dressings on Day 2 and persistent at this level throughout the following days before resolving in all but two instances by Day 9. In two rats a dermal response persisted through to Day 12 or 14 before resolving. Associated with the dermal irritation were reactions characterised by blanching of the skin, localised spots and/or scabbing and/or thickening of the skin and desquamation. These responses had resolved in all but three instances by Day 15.

Interpretation of results:

GHS criteria not met

Remarks:

CLP Implementation.

Conclusions:

Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (L.A.E) was demonstrated to be greater than 2000 mg/kg bw.

Executive summary:

According to the OECD Guideline Nº 402 and EU method B.3, a group of 10 rats, 5 males and 5 females, received a single topical application of the test substance formulated in 1% w/v aqueous methylcellulose and administered at a dosage of 2000 mg/kg bodyweight.

There were no clinical signs of reaction to N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (L.A.E) treatment observed in any animal throughout the study.

Well-defined irritation (erythema and oedema up to Grade 2) was notable in all rats following removal of the dressings on Day 2 and persistent at this level throughout the following days before resolving in all but two instances by Day 9. In two rats a dermal response persisted through to Day 12 or 14 before resolving. Associated with the dermal irritation were reactions characterised by blanching of the skin, localised spots and/or scabbing and/or thickening of the skin and desquamation. These responses had resolved in all but three instances by Day 15.

No macroscopies abnormalities were observed for animals killed at study termination on Day 15, with the exception of scabbing on the dose sites of three rats, plus thickened tissues on one.

The acute lethal dermal dose to rats of L.A.E. was demonstrated to be greater than 2000 mg/kg bodyweight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Quality of whole database:

Klimisch 1. This study was carried out in accordance with internationally valid GLP principles.

Additional information

The acute toxicity (oral) study was realized according to OECD 423 guideline and EU method B.1 tris.

- Key study 1: According to OECD 423 guideline and EU method B.1 tris, this study was performed with the purpose of ranging LD50 for the test substance: N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE). The acute lethal oral dose to rats of LAE was demonstrated in this study to be greater than 2000 mg/kg bodyweight.

The acute toxicity (inhalation) study was realized according to OECD 403 guideline and EU method B.2.

- Key study 1: The test conditions applied fulfilled recommendations of current test guidelines according to the OECD Guideline Nº 403 and EU method B.2, with regard to attainment of steady-state concentration, respirability of particles and the limit concentration tested. Concluding that for N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) the LC50 is greater than 5883 mg/m3.

The acute toxicity (dermal) study was realized according to OECD 402 guideline and EU method B.3.

- Key study 1: According to the OECD Guideline Nº 402 and EU method B.3, the test substance was administered at a dosage of 2000 mg/kg bodyweight. The acute lethal dermal dose to rats of L.A.E. was demonstrated to be greater than 2000 mg/kg bodyweight.

Justification for classification or non-classification

Observations and results obtained under the Guidelines mentioned above show that dehydrated N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) has no acute toxic effects via oral, dermal or by inhalation.

The substance is not classified for acute toxicity in accordance to CLP Regulation.