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

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 20, 2005 to December 21, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

GLP

Justification for type of information:

Comparable to a guideline study with GLP.

Objective of study:

toxicokinetics

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.36 (Toxicokinetics)

Qualifier:

according to guideline

Guideline:

other: Guidelines for Food Aditives Evaluations

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Substance: L.A.E.
Purity: 91.87%
Batch number: 12547
Expiry date June 16, 2006.

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK, Ltd, Margate, UK
- Age at study initiation: 7-10 weeks
- Weight at study initiation: 213-228 g
- Housing: Separately for each sex in groups of up to 6 in stainless steel battery cages suspended mesh floors
- Individual metabolism cages: no
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2ºC
- Humidity (%): 55±15%
- Air changes (per hr): c.a 15 per hour
- Photoperiod (hrs dark / hrs light): 12-hour light to dark

IN-LIFE DATES: From: August 23, 2005 To: August 26, 2005

Route of administration:

oral: gavage

Vehicle:

other: water, propylene glycol/water or glycerol/water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: All doses with propylene glycol and glycerol were prepared at a ratio of 1:3.5 Lauric arginate to solvent and then made up to volume with distilled water, such that the volume of the dose formulation administered was at an nominal rate of 5 ml/kg bw.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Propylene glycol (Group 2, 3 and 4); Glycerol (Group 5); distilled water (Group 6)
- Concentration in vehicle: Ratio of LAE:vehicle: 1:3.52 (Group 2); 1:3.63 (Group 3); 1:3.48 (Group 4); 1:3.49 (Group 5); - (Group 6)

Duration and frequency of treatment / exposure:

Single dose oral exposure
Observations up to 8 hours post-dose

Dose / conc.:

40 mg/kg bw (total dose)

Remarks:

Formulated in propylene glycol/water

Dose / conc.:

120 mg/kg bw (total dose)

Remarks:

Formulated in propylene glycol/water

Dose / conc.:

320 mg/kg bw (total dose)

Remarks:

Formulated in propylene glycol/water

Dose / conc.:

120 mg/kg bw (total dose)

Remarks:

Formulated in glycerol/water

Dose / conc.:

120 mg/kg bw (total dose)

Remarks:

Formulated in water

No. of animals per sex per dose / concentration:

4 males per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose level for the pilot experiment was selected as one which would result in obtaining sufficient data for selection of suitable plasma times for the main experiments. Therefore the dose level selected was 40 mg/kg bw. bodyweight of lauric arginate (LAE).
- Rationale for animal assignment (if not random): Male and female rats were studied and if no sex difference found, then only male would be used in the main study.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood
- Time and frequency of sampling: 30, 60, 90, 120, 240 and 480 minutes
- Other: Method type(s) for identification: LC-MS/MS

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: blood
- Time and frequency of sampling: 30, 60, 90, 120, 240 and 480 minutes
- Method type(s) for identification: LC-MS/MS (metabolite LAS)

Preliminary studies:

The results from the pilot experiment were used to base the dose levels, sampling regime and sex of animals to be used for the main experiment. There were no noticeable differences between male and female animals so only males were used in the main experiment.

Based on initial results, the main experiment was conducted at 3 dose levels of 40, 120 and 320 mg/kg bw, with the sampling times adjusted to reflect the absorption of LAE and LAS.

Type:

absorption

Results:

The time at which the maximum plasma concentration occurred (Tmax) indicated that absorption was generally rapid. Similar absorption is observed whether propylene glycol or glycerol are added to aid solubility in water

Details on absorption:

Propylene glycol/water formulation:
The time at which the maximum plasma concentration of LAE occurred (Tmax) was generally 0.5 or 1 hour post-dose at the dose levels of 40 and 120 mg/kg bw and in the range 0.5 to 4 hours at the dose level of 320 mg/kg bw, indicating that absorption was generally rapid. Tmax tended to be later at the highest dose level indicating that an increase in the oral dose tended to prolong the absorption phase. Tmax for LAS generally occurred at about the same time as for LAE.

Effect of formulation at a dose level of 120 mg/kg bw:
The time at which the maximum plasma concentration of LAE occurred (Tmax) was generally 0.5 or 1 hour post-dose, indicating that absorption was generally rapid, and did not appear to be greatly influenced by the formulation. Tmax for LAS generally occurred at about the same time as for LAE.

Metabolites identified:

yes

Details on metabolites:

Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) is rapidly hydrolysed to LAS in the gastrointestinal tract and by the tissue and plasma esterases. The low levels detected in this study are a consequence of this rapid metabolism and do not give a good indication of the extent of absorption of lauric arginate.

The present study gives no information about the absolute bioavailability of lauric arginate, but suggests that similar absorption is observed whether propylene glycol or glycerol are added to aid solubility in water.

Conclusions:

The present study has been performed to obtain further information on the plasma pharmacokinetics of Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) and its metabolite (N-α-lauroyl-L-arginine, LAS) in rats (4 males per dose) after oral gavage administration under different dose levels and formulation conditions that consisted on 3 different vehicles. Results concluded that the absorption is similar for all three of the vehicles tested.

Executive summary:

The objective of the study was to obtain further information on the plasma pharmacokinetics of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) and its metabolite (N^α-lauroyl-L-arginine, LAS) in rats after oral gavage administration under different dose level and formulation conditions.

An initial pilot experiment carried out at a dose level of 40 mg/kg bw formulated in propylene glycol/water to both male and female rats provided the basis for the dose levels and sampling regime for the main experiment. There were no noticeable differences in the results obtained between male and female rats therefore only male rats were used for the main experiment.

After administration of lauric arginate the rate and extent of systemic exposure of male rats to LAE was low and there did not appear to be a consistent relationship of plasma LAE with dose following a single oral (gavage) administration of a propylene glycol/water formulation at dose levels of 40, 120 and 320 mg/kg bw. This is related to the rapid hydrolysis of LAE to LAS. However, the extent of systemic exposure to the metabolite LAS appeared to be characterised by non-linear (dose-dependent) kinetics over the dose range 40 to 320 mg/kg bw, and increasing the dose of lauric arginate (LAE) above 40 mg/kg bw. resulted in a lower systemic exposure than would be predicted from a linear relationship, which is consistent with the possibility of an increase in plasma clearance at higher dose levels.

Systemic exposure to LAE, following administration at a dose level of 120 mg/kg bw., was also low after the glycerol/water and water formulations at around 10 ng/ml at 0.5 - 1 hour after administration, but was higher than after the propylene glycol/water formulation. This difference was much less marked for the C_maxvalues of LAS, and the AUC_gvalues for LAS were similar after all three formulations administered. Because of the rapid metabolism of LAE to LAS in the gastrointestinal tract and by tissue and plasma esterases only low plasma levels of LAE are observed. The differences observed in the Cmax values for LAE may not be of toxicological significance because of its transient presence in blood. Plasma levels of LAS, and especially the AUC for LAS, give a better indication of the absorption of lauric arginate.

The similarity in the values for the AUC of LAS show that the absorption is similar for all three of the vehicles tested (water, propylene glycol/water and glycerol/water).

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 11, 2000 to May 8, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

GLP

Justification for type of information:

Equivalent to a guideline study with GLP.

Objective of study:

distribution

excretion

metabolism

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

GLP compliance:

yes

Specific details on test material used for the study:

Substance: L.A.E.
Purity: <99%
Batch number: NPEL/LMA001/65
Expiry date: December 28, 2000.

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Weight at study initiation: 202-227 g
- Housing: Stainless steel with suspended mesh floors
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2ºC
- Humidity (%): 55±15%
- Photoperiod (hrs dark / hrs light): 12 hours light/12 ours dark

IN-LIFE DATES: From: August 23, 2000 To: August 23, 2000

Route of administration:

oral: gavage

Vehicle:

other:

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The target dose to be administered to rats was a nominal 200 mg/kg bodyweight. 0.7356 g of non-radiolabelled LAE was dissolved in an aliquot (14.8 ml) of [arginine-[U-14C]]LAE stock solution. No correction for the purity of the non-radiolabelled LAE was made. The material was dissolved and the final volume was adjusted to 20 ml by addition of methanol. The specific activity of the radiodiluted [arginine-[U-14C]]LAE was determined by
radioassay of triplicate aliquots (0.01 ml) of the solution.

An aliquot (15.8 ml) of the above solution was concentrated under a steam of nitrogen in a glass vial. This solution was stored at <-15ºC prior to dose preparation. The formulated dose was prepared on the day of dose administration. The concentrated solution was transferred to a Potter homogeniser where the remaining solvent was removed using nitrogen.

An aliquot (8 ml) of 1% methyl cellulose was added to the test substance in the homogeniser and mixed to form a homogenous suspension at a nominal concentration of 80 mg/ml. The volume of dose (rounded to the nearest 0.05 ml) was calculated, based on the mean bodyweight and a rate of administration of 2.7 ml/kg bodyweight. Rats were administered the dose by gastric intubation. To accurately determine the quantity of radioactivity administered to each rat, further aliquots (0.57 ml) of the dose suspension were similarly mixed with methanol in a volumetric flask (20 ml). The final volume was made up to 20 ml with methanol. The radiochemical purity of the test substance administered was determined by HPLC and was >99%

VEHICLE
The dose vehicle was prepared by dissolving 1 g of methyl cellulose in water 100 ml.

Duration and frequency of treatment / exposure:

IN VIVO: Single oral dose

Dose / conc.:

200 mg/kg bw (total dose)

Remarks:

IN VIVO

No. of animals per sex per dose / concentration:

IN VIVO: 6 males

Control animals:

no

Details on study design:

In vivo study:
The target dose to be administered to rats was a nominal 200 mg/kg body weight.

In vitro study:
PREPARATION OF S9 LIVER HOMOGENATE
A single male rat was lightly anaesthetised and a blood sample taken by cardiac puncture. The rat was then sacrificed and the liver removed and placed in 100 mM sodium phosphate buffer pH 7.4 on ice. In a room maintained at c.a 4°C the weight of the liver was recorded. The liver was scissor minced and homogenised with sodium phosphate buffer (36 ml) in a "potter" homogeniser using a Teflon® motor driven pestle. The homogenate was centrifuged at 9000 G for 20 minutes (L8-70M Ultracentrifuge, Beckman Coulter Bioresearch, USA). The temperature was maintained at ca 4°C. The supernatant or S9 fraction was removed and aliquots (4 ml) dispensed into separate tubes for storage at <-70°C.

TREATMENT OF S9 LIVER FRACTION
An aliquot (1 ml) of [arginine-[U-14C]]LAE stock solution was concentrated to near dryness under a stream of nitrogen and re-dissolved in methanol (5 ml). Duplicate aliquots (40 ul) were taken for radioassay. The concentration of 14C-LAE in solution was 0.978 mg/ml. Aliquots (4 ml) of the S9 liver fraction were treated with an aliquot (40 µl) of 14C-LAE to give a nominal treatment rate of 10 ug/ml.

TREATMENT OF CONTROL PLASMA
An aliquot (1 ml) of the solution of [Arginine-[U-14C]]LAE, prepared for treatment of the S9 liver fraction, was taken to dryness under a stream of nitrogen. The test substance was re-dissolved in sodium chloride solution (0.9% w/v). Duplicate aliquots (50 µl) were taken for radioassay. The concentration of LAE in solution was 0.89 mg/ml. An aliquot (5 ml) of control plasma was treated with an aliquot (55 µl) of the 14C-LAE to give a nominal treatment rate of 10 µg/ml.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: plasma
- Time and frequency of sampling: by cardiac puncture at 0.5, 1 and 4 hours after dose administration
- Other: Blood from an untreated rat used for the in vitro liver experiments together with a sample from three additional untreated rats was also collected.
- Method type(s) for identification: HPLC

Metabolites identified:

yes

Details on metabolites:

Arginine, ornithine, LAS (Nα-Lauroyl-N-Arginine) and unchanged LAE were identified by HPLC co-chromatography of reference substances with plasma extracts.

In vitro experiments using the S9 liver fraction:

Unchanged LAE, N-a-Lauroyl-L-arginine (LAS), arginine ethyl ester, arginine, ornithine and urea were identified in the S9 treated samples. The naturally occurring amino acid, ornithine was the major metabolite. In a control incubation in the absence of the S9 liver fraction no significant degradation of LAE was observed.

In vivo study:

Concentrations in plasma rose rapidly to a mean of 118 ug equivalents LAE/ml 4 hours after dosing. Extraction of radioactivity from plasma declined from a mean of 74.8% total radioactive residue (TRR) at 0.5 hours to a mean of 19.7% TRR 4 hours after dosing.

Analysis of the plasma extracts showed qualitatively similar profiles to those seen in the S9 liver fractions. LAE accounted for less than 10% TRR. The major metabolite was arginine which accounted for a mean maximum of 48% TRR in the 0.5 hour samples. Ornithine accounted for a mean of 7.7% TRR at the same time. An unretained polar fraction which was not identified and accounted for a mean maximum of 17% TRR. Treatment of control plasma with LAE at a nominal concentration of 10 and incubation at 37 ºC for up to 4 hours showed that esterase activity in the plasma rapidly hydrolyses the LAE to LAS and arginine. Arginine was further metabolised to ornithine. Extraction of radioactivity declined from 99.5% at zero-time to 86.4% at 4 hours. This suggests that some binding of radioactivity to plasma proteins does occur in vitro as well as in vivo.

This study has shown that LAE is rapidly metabolised by hydrolysis of the ethyl ester and lauroyl arnide to arginine which subsequently enters the naturally occurring urea cycle where it is hydrolysed to ornithine and urea. This work, together with that previously reported on the in vivo metabolism of LAE in the rat, demonstrates that LAE is metabolised primarily to naturally occurring amino acids. These in turn are incorporated into endogenous products and finally degraded to carbon dioxide through the normal mammalian biochemical pathways.

 

Conclusions:

In vitro incubation of [arginine-[U-14C]]LAE with preparations of S9 rat liver fractions and control rat plasma have enabled the in vivo biotransformation pathway to be elucidated. LAE is rapidly metabolised by hydrolysis of the ethyl ester and lauroyl amide to the naturally occurring amino acid arginine which undergoes further catabolism to ornithine and urea.

In vitro experiments with 14C-LAE in plasma demonstrated that LAE and N-α-lauroyl-L-argine could be quantitatively recovered from plasma by extraction with methanol. This suggests that the increase in the non-extractable radioactivity observed in vivo and to a lesser extent in vitro is probably due to binding of the LAE and/or its metabolites to plasma proteins and/or natural incorporation.

Given the rapid hydrolytic degradation of LAE and LAS in both liver and plasma in vitro and the results from analysis of in vivo plasma samples it can be concluded that exposure to these compounds in vivo is likely to be very short.

This experiment together with work previously reported on the in vivo metabolism of LAE in the rat demonstrates that LAE is primarily and rapidly metabolised to naturally occurring amino acids, mainly arginine and ornithine.

Executive summary:

As part of a programme to study the toxicity and metabolism of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) in mammals, the metabolite profiles in plasma and liver in vivo and in vitro were investigated. LAE radiolabeled with carbon-14 in the arginine carbons was used.

An untreated liver from a single rat was homogenised with phosphate buffer. Portions of the supernatant (S9) fraction were exposed to the radiolabeled LAE for up to 24 hours at 37°C. The S9 fractions were extracted with methanol and analysed by TLC, HPLC and LC-MS. Unchanged LAE, N^α-Lauroyl-L-arginine (LAS), arginine ethyl ester, arginine, ornithine and urea were identified in the S9 treated samples. Then naturally occurring amino acid, ornithine was the major metabolite. In a control incubation in the absence of the S9 liver fraction no significant degradation of LAE was observed.

Analysis of the plasma extracts showed qualitatively similar profiles to those seen in the S9 liver fractions. LAE accounted for less than 10% TRR. The major metabolite was arginine which accounted for a mean maximum of 48% TRR in the 0.5 hour samples. Ornithine accounted for a mean of 7.7% TRR at the same time. An unretained polar fraction which was not identified and accounted for a mean maximum of 17% TRR. Treatment of control plasma with LAE at a nominal concentration of 10µg/ml and incubation at 37°C for up to 4 hours showed that esterase activity in the plasma rapidly hydrolyses the LAE to LAS and arginine. Arginine was further metabolised to ornithine. Extraction of radioactivity declined from 99.5% at zero-time to 86.4% at 4 hours. This suggests that some binding of radioactivity to plasma proteins does occur in vitro as well as in vivo.This study has shown that LAE is rapidly metabolised by hydrolysis of the ethyl ester and lauroyl amide to arginine which subsequently enters the naturally occurring urea cycle where it is hydrolysed to ornithine and urea.

In vitro incubation of [Arginine-[U- C]]LAE with preparations of S9 rat liver fractions and control rat plasma have enabled the in vivo biotransformation pathway to be elucidated. LAE is rapidly metabolised by hydrolysis of the ethyl ester and lauroyl amide to the naturally occurring amino acid arginine which undergoes further catabolism to ornithine and urea.

In vitro experiments with¹⁴C-LAE in plasma demonstrated that LAE and N^α-lauroyl-L-argine could be quantitatively recovered from plasma by extraction with methanol. This suggests that the increase in the non-extractable radioactivity observedin vivoand to a lesser extentin vitro is probably due to binding of the LAE and/or its metabolites to plasma proteins and/or natural incorporation.

Given the rapid hydrolytic degradation of LAE and LAS in both liver and plasma in vitro and the results from analysis of in vivo plasma samples it can be concluded that exposure to these compounds in vivo is likely to be very short.

This experiment together with work previously reported on the in vivo metabolism of LAE in the rat demonstrates that LAE is primarily and rapidly metabolised to naturally occurring aminoacids, mainly arginine and ornithine.

Reference 3

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 23, 1998 to August 26, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

GLP

Justification for type of information:

Equivalent to a guideline study with GLP.

Objective of study:

absorption

excretion

metabolism

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

GLP compliance:

yes

Specific details on test material used for the study:

Substance: L.A.E.
Purity: <97%
Batch number: NPE/LMA001/65
Expiry date: Decem,ber, 1998.

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Margate, Kent, UK
- Age at study initiation: 7 - 9 weeks
- Weight at study initiation: 222-240 g
- Housing: Individually in glass metabowls (with urine/faeces separators)
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25ºC
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

IN-LIFE DATES: From: May 7, 1998 To: May 12, 1998

Route of administration:

oral: gavage

Vehicle:

other: methyl cellulose in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
0.5922 g of non-radiolabelled LAE was dissolved in an aliquot (12 ml) of [arginine-[U-14C]]LAE (batch NPE/LMA001/65) stock solution. The material was dissolved by addition of methanol (4 ml). The final volume was adjusted to 10 ml by removing solvent using a stream of nitrogen.
An aliquot 8.7 ml of the above solution was concentrated under a stream of nitrogen in a glass vial. This solution was stored at <-15°C prior to dose preparation. The concentrated solution was transferred to a Potter homogeniser where the remaining solvent was removed using nitrogen. The glass vial was rinsed with methanol (2 x 1 ml) and the rinsings added to the homogeniser and solvent evaporated using nitrogen.
An aliquot (7 ml) of 1% methyl cellulose was added to the test substance in the homogeniser and mixed using a motor driven teflon pestle to form a homogenous suspension at a nominal concentration of 80 mg/ml.
The dose suspension was mixed with methanol and the contents transferred to a volumetric flask (100 ml). The vial was rinsed with further aliquots of methanol and the rinsings added to the flask. The final volume was made up to 100 ml with methanol.
The actual dose received by each rat was in the range 177-180 mg/kg bodyweight
The radiochemical purity was 99.4%.

VEHICLE
- Concentration in vehicle: 10.048 mg/ml, prepared by dissolving 0.5024 g of methyl cellulose in water.

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
The specific activity and concentration of the test substance in solution was determined as:
0.014 MBq/mg
0.38 uCi/mg
8.4645 x 10s dpm/mg
64.7 mg/ml
The radiochemical purity was 99.4%.

Duration and frequency of treatment / exposure:

120 hours

Dose / conc.:

180 mg/kg bw (total dose)

Remarks:

1 dose at a nominal concentration in a range under 177-180 mg/kg bw

No. of animals per sex per dose / concentration:

4 males per one nominal concentration of 180 mg/kg bw

Control animals:

no

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, excretion)
- Tissues and body fluids sampled: urine, faeces, tissues, cage washes.
- Time and frequency of sampling: Separately for each animal at:
Urine and expired air: 0-8; 8-24 and 24 h up to 5 days after dosing.
Faeces: at 24 h up to intervals to 5 days.
Each cage was washed out with water at 24 h intervals up to 5 days after dosing.
Expired air was trapped in 2-ethoxyethanol:ethanolamina (3:1 v/v)

Type:

metabolism

Type:

absorption

Type:

excretion

Details on absorption:

Radioactivity was well absorbed and eliminated slowly. The results indicated almost complete absorption of LAE at the dose level used in the study.

Details on distribution in tissues:

The largest proportion of radioactivity, a mean of 46.4% dose was retained in the carcass at sacrifice, 5 days after administration. Of this mean 2.0% (45.8 ug equivalent LAE/g tissue net weight) and 3.4% dose (120 ug equivalent LAE/g tissue net weight) was measured in the gastrointestinal tract and liver, respectively.

Details on excretion:

The pattern and rate of excretion by each individual animal was very similar.

The major route of excretion was as carbon dioxide in expired air. A mean of 23.9% dose was eliminated via this route in the 24 hours following administration. Over 5 days a mean total of 36.6% dose was measured.

Excretion in urine was low. A mean 11.8% dose was excreted over 5 days.

Excretion of radioactivity in the faeces was a mean 4.3% dose. This indicates the test substance was almost completely absorbed.

Metabolites identified:

yes

Details on metabolites:

Analysis of the urine (0 - 24 hours) by HPLC showed a single peak with a retention time of two minutes. No radioactivity with the retention time of unchanged LAE was detected. This peak represented a mean 9% dose.

As the material was not retained well by the HPLC column used, the possibility of co-eluting metabolites could not be ruled out. Urine was therefore analysed by TLC in solvent systems C, D and E.

Solvent system C separated up to 7 radioactive components. The major component represented 85.6% of the sample radioactivity or a mean 7.7% dose. All the other components present represented ≤0.2% dose and were more polar than the major component.

The major urine metabolite was shown to co-chromatograph with the reference substance, 14C-urea in solvent system C, D and E.

Table 1: Excretion of Radioactivity by Rats after an oral Dose of test substance to rats at 180 mg/kg bw:

	Excretion
	Mean (% dose)	SD (% dose)
Urine
0 - 8 h	5.3	0.4
8 - 24 h	3.7	0.2
24 - 48 h	1.2	0.2
48 - 72 h	0.8	0.1
72 - 96 h	0.5	0.1
96 - 120 h	0.4	0.1
Subtotal	11.8	0.9
Faeces
0 -24 h	2.3	1.0
24 - 48 h	1.0	0.2
48 - 72 h	0.6	0.1
72 - 96 h	0.4	0.3
96 - 120 h	0.2	0.1
Subtotal	4.3	1.2
Expired Air
0 - 8 h	15.7	3.1
8 - 24 h	8.2	1.4
24 - 48 h	4.9	0.4
48 - 72 h	3.5	0.6
72 - 96 h	2.6	0.9
96 - 120 h	1.8	0.4
Subtotal	36.6	5.4
Cage wash	0.5	0.3
Tissues
GIT	2.0	0.1
Liver	3.4	0.4
Carcass	41.0	6.2
Total Recovery	99.5	1.8

SD: Standard deviation

NS: No sample

GIT: Gastrointestinal tract including contents

Conclusions:

A possible route by which these terminal metabolites might be formed, is that following metabolism of LAE to arginine, the arginine itself enters the urea cycle where it is degraded to ornithine and urea. The ornithine could then either be degraded further to citrulline and other amino acids in the urea cycle or to the metabolic intermediate α-ketoglutarate. The α-ketoglutarate could then enter the citric acid cycle with subsequent metabolism to carbon dioxide (Voet and Voet, 1990).

Incorporation of the radiolabel into all the amino acids of the urea cycle would subsequently lead to a large number of endogenous products in the body. This would explain the high level of retention of radioactivity in the carcass of the rats five days after dosing.

The high levels of radioactivity in liver might be expected as this organ is the main site in the body for the enzymes of the urea cycle. The liver also has a major function as a producer of proteins and bile and therefore radioactive amino acids formed as a result of LAE metabolism and catabolism would be likely to accumutate here.

Since the results indicate that the compound is converted to arginine, it is likely that the toxicology would be similar to that of arginine.

Executive summary:

To study the toxicity and metabolism of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) in mammals, four male rats were administered an oral dose of LAE at a nominal 180 mg/kg bodyweight. This was far greater than the expected human exposure. LAE radiolabelled with carbon-14 in the arginine carbons was used.

The patterns of metabolism, excretion and retention of radioactivity were studied.

During 5 days after dosing a mean of 36.6% dose was excreted as carbon dioxide in expired air, 11.8% dose in urine and 4.3% dose in faeces. A mean of 46.4% dose was retained in the carcass at sacrifice. Of this a mean 3.4% dose was measured in the liver. This indicates almost complete absorption of LAE at this dose level. The mean recovery of the administered radioactivity was 99.5%.

Analysis of the urine showed that the major radioactive component co-chromatographed with urea although the identity of this metabolite was not confirmed. Urea represented a mean of 7.7% dose in urine excreted up to 24 hours after administration. Up to 6 other minor radioactive components were detected in urine, none of which represented >0.2% dose.

Thus, it is concluded that LAE is likely to have been rapidly metabolised to the amino acid arginine which then underwent natural amino acid catabolism via the urea and citric acid cycles resulting in the formation of the observed terminal metabolites, urea and carbon dioxide. Entry into these biochemical pathways would result in the formation of other radiolabelled amino acids and subsequent incorporation into many endogenous products. This would explain the high retention of radioactivity after 5 days.

Reference 4

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 17, 2010 to November 5, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

According to guidelines and offical methods with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Environmental Health and Safety Publications, Series on Testing and Assessment Nº 28. Guidance document for the conduct of skin absorption studies (March 2004)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: COLIPA Guidelines for Percutaneous Absorption/Penetration (August 2007)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SCCS/1358/10 Basic Criteria for the in vitro Assessment of Dermal Absorption of Cosmetic Ingredients, adopted by the SCCS during the seventh plenary meeting of 22 June 2010

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Substance: L.A.E.
Purity: ca. 99%
Batch number: 3766CJW008-10

Radiolabelling:

yes

Remarks:

14C

Details on study design:

PREPARATION OF TEST SUBSTANCE SOLUTIONS
The test solutions of 0.4% [Arginine-U-14C]LAE·HCl were prepared by dissolving an appropriate amount of [Arginine-U-14C]LAE·HCl in MQ. The activity of this solution was determined by liquid scintillation counting and a final activity of 5.1 MBq/mL was achieved by dilution with MQ. The final concentration of [Arginine-U-14C]LAE·HCl was 4035 mg/L.

The test solution of 0.8% [Arginine-U-14C]LAE·HCl was prepared by dissolving an appropriate amount of [Arginine-U-14C]LAE·HCl in MQ. The activity of this solution was determined by liquid scintillation counting and a final activity of 10.2 MBq/mL was achieved by dilution with MQ. The final concentration was 7992 mg/L [Arginine-U-14C]LAE·HCl.

HOMOGENEITY AND STABILITY OF THE TEST SOLUTIONS
The homogeneity of the test solutions was checked by taking three samples (from the top, the bottom and the middle of the vial) and determining the activity of these samples in Ultima Gold LSC cocktail by liquid scintillation counting. The Relative Standard Deviation (RSD) of the three measurements should be < 3%, and it was 1.6% for the test solution of 0.4% and 1.4% for the test solution of 0.8%.

The stability of the test solutions for the duration of the study (24 hours) was checked by analyzing an aliquot of both test solutions with concentrations of 0.4% and 0.8% [Arginine-U- 14C]LAE·HCl immediately after preparation and after 24 h using the LC-UV-RAD method described in the study.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Biopredic International, Rennes, France
- Ethical approval if human skin: Human skin is a recommended test for skin absorption studies
- Type of skin: Human
- Preparative technique: Frozen dermatomed human skin discs
- Thickness of skin (in mm): c.a 0.5 mm
- Storage conditions: dry ice and stored at <=-15ºC

SKIN INTEGRITY TEST
Skin membranes were thawed, mounted in the diffusion cell and the skin integrity was tested by permeation of tritiated water. 200 μL saline containing tritiated water (3.7 kBq) were applied to the donor compartment of the flow-through cells. The receptor fluid consisted of saline and was collected every hour up to 3 hours after application. At the end of the experiment, the tritiated water remaining at the donor compartment was removed with a pipet and the skin was dried with two cotton swabs (Etos BV, Beverwijk, The Netherlands). The receptor fluid samples were analyzed by LSC.

PRINCIPLES OF ASSAY
- Receptor fluid: Saline supplemented with 0.5% BSA
- Solubility of test substance in receptor fluid: Soluble (94.5% recovery)
- Flow-through system: Receptor fluid was pumped at a flow rate of 1.5 ml/h
- Test temperature: 32±1ºC
- Humidity: ambient humidity
- Occlusion:No

Absorption in different matrices:

The percentages absorbed were lower for the concentration of 0.8% compared to the concentration of 0.4% [Arginine-U-14C]LAE·HCl. The average percentage absorbed was 0.81 ± 0.78% for the 0.8% concentration and 2.1 ± 0.9% for the 0.4% concentration.

Total recovery:

- Total recovery: The recoveries obtained in the experiments are an average of 95.1 ± 2.6% for the 0.4% [Arginine-U-14C]LAE·HCl test solution and 95.3 ± 4.0% for the 0.8% [Arginine-U-14C]LAE·HCl test solution. The total recovery of radioactivity for the individual skin samples was 100 ±10%,
which is according to the OECD guidelines.
- Limit of detection (LOD): ±2.6 (0.4%); ±3.9 (0.8%);

Key result

Dose:

0.4%

Parameter:

percentage

Absorption:

>= 1.2 - <= 3 %

Remarks on result:

other: 24 h

Key result

Dose:

0.8%

Parameter:

percentage

Absorption:

>= 0.04 - <= 1.6 %

Remarks on result:

other: 24 h

Skin integrity test

The permeability coefficients for water for the skin discs used in this study were within the acceptability criteria (Kp ≤ 4.5 x 10-3 cm/h).

Stability of [Arginine-U-14C]LAE·HCl test solutions

No decrease in the peak area of [Arginine-U-14C]LAE·HCl and no formation of additional degradation products after 24 h was

observed, indicating that [Arginine-U-14C]LAE·HCl was stable for the duration of the experiment.

Conclusions:

The percentage of [Arginine-U-14C]LAE·HCl absorbed was assessed in 10 skin discs of human donors according to OECD 428 Guideline. Results showed a lower at a concentration of 0.8% than at a concentration of 0.4%. The average percentage absorbed was 0.81 ±0.78% for the 0.8% concentration and 2.1±0.9% for the 0.4% concentration.

Executive summary:

According to the Guideline 428, the present study has been designed to provide information on absorption of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (L.A.E) applied to excised skin.

With that objective, dermal absorption of [Arginine-U-¹⁴C]LAE·HCl was investigated in human skin in vitro.

Human skin discs from 4 donors were exposed to a concentration of 0.4% (4035 mg/l) and human skin discs from 5 donors were exposed to a concentration of 0.8% (7992 mg/l) [Arginine-U-¹⁴C]LAE·HCl in water for 24 hours.

The integrity of each skin disc was checked by determination of the permeation of titrated water and was within the acceptability criteria.

[Arginine-U-¹⁴C]LAE·HCl test solutions (0.4 and 0.8%) were stable for the durationof the experiment (24 h) and [Arginine-U-¹⁴C]LAE·HCl test solutions in receptor fluid were also stable for 24 h.

The percentage of [Arginine-U-¹⁴C]LAE·HCl absorbed was lower at a concentration of 0.8% than at a concentration of 0.4%. The average percentage absorbed was 0.81 ±0.78% for the 0.8% concentration and 2.1±0.9% for the 0.4% concentration.

Reference 5

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2005-12-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: An expert review.

Justification for type of information:

Well documented scientific rewiev.

Principles of method if other than guideline:

A scientific review.

GLP compliance:

yes

Conclusions:

No bioaccumulation potential:
An overview of previous toxicokinetics studies has been prepared. It has been validated the rat as a good model for the toxicological assessment of Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE). Consequently, it has been assessed that LAE is primarily and rapidly metabolised to naturally occurring amino acids, mainly arginine and ornithine.

Executive summary:

As part of the safety evaluation of lauric arginate (LAE, N^α-Lauroyl-L-arginine ethyl ester monohydrochloride) it was recognised that knowledge of its metabolism could play a very important role particularly since the molecule consists ofa combination of two endogenous compounds, namely lauric acid and arginine with the addition of ethanol to form an ethyl ester. In addition if the metabolism can be compared between laboratory animals and humans it provides the opportunity for validatingthe relevance of the species used for toxicological assessment and to explain the very low toxicity of LAE. Accordingly a sequential series of specifically designed experiments have been conducted to maximise an understanding of the fate of the compound in animals.

The in vivo metabolic pathway of LAE was assessed. It has been established that in both rat and human LAE is sequentially metabolised to LAS and then arginine by hydrolysis of the ester and amide functions respectively. The latter reaction also results in the formation of lauric acid, a naturally-occurring fatty acid of no toxicological concern. The primary metabolic products of oral doses of LAE are therefore those encountered from intake of normal dietary constituents. The human data suggests that since molar concentrations of arginine in plasma are more than 10-fold higher than LAS the major portion of the dose was degraded by peptidases prior to absorption. It is expected that LAE would be hydrolysed to LAS as the first step. Any LAE that is absorbed would be hydrolysed in plasma to LAS. Furthermore any LAS that is absorbed or formed in plasma is clearly rapidly metabolised systemically as shown by its short half-life. When LAE was administered to human volunteers as bolus doses equivalent to a total daily intake of 1.5 to 2.5 mg/kg concentrations could be expected to be considerably higher than during exposure from food. However they were still below the limits of detection by 24 hours.

Thus, it has been assessed, that the metabolism of LAE in rat and human has been shown to be the same validating the rat as a good model for the toxicological assessment of LAE. LAE is primarily and rapidly metabolised to naturally occurring amino acids, mainly arginine and ornithine.

Description of key information

Short description of key information on bioaccumulation potential result:

LAE is primarily and rapidly metabolised to naturally occurring amino acids, mainly arginine and ornithine.

Short description of key information on dermal absorption rate:

The percentage of [Arginine-U-14C]LAE·HCl absorbed was lower at a concentration of 0.8% than at a concentration of 0.4%. The average percentage absorbed was 0.81 ±0.78% for the 0.8% concentration and 2.1±0.9% for the 0.4% concentration.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Several studies have been assessed to gather information on the behavior of the test substance N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) in Absorption, Distribution, Metabolism and Excretion, concluding:

-      LAE is primarily and rapidly metabolised to naturally occurring amino acids, mainly arginine and ornithine.

-      The dermal absorption of [Arginine-U-14C]LAE·HCl was investigated using human skin according to OECD guideline 428. The percentage of absorbed substance was lower at a concentration of 0.8% than at a concentration of 0.4%. The average percentage absorbed was 0.81 ±0.78% for the 0.8% concentration and 2.1±0.9% for the 0.4% concentration.

Discussion on bioaccumulation potential result:

Several studies have been assessed in order to study the potential bioaccumulation of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE):

- Key study 1: Results indicate that the similarity in the values for the AUC of LAS show that the absorption is similar for all three of the vehicles of the substance tested (water, propylene glycol/water and glycerol/water).

- Key study 2: This experiment together with work previously reported on the in vivo metabolism of LAE in the rat demonstrates that LAE is primarily and rapidly metabolised to naturally occurring amino acids, mainly arginine and ornithine.

- Key study 3: Since the results indicate that the compound is converted to arginine, it is likely that the toxicology would be similar to that of arginine.

- Supporting study 1: The metabolism of LAE in rat and human has been shown to be the same, validating the rat as a good model for the toxicological assessment of LAE.

Discussion on absorption rate:

- Key study 1: The dermal absorption of [Arginine-U-¹⁴C]LAE·HCl was investigated using human skin discs according to OECD guideline 428. The % of [Arginine-U-¹⁴C]LAE·HCl absorbed was lower at a concentration of 0.8% than at a concentration of 0.4%. The average percentage absorbed was 0.81 ±0.78% for the 0.8% concentration and 2.1±0.9% for the 0.4% concentration.