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

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 3, 2003 to November 9, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 9, 2000 to March 28, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: Range-finding study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Toxicological Principles for the Safety Assessment of Direct Food Additives and Colour Additives used in food - Red Book 1 (1982).

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Han Wistar

Details on species / strain selection:

The Han Wistar rat was used because of the historical control data available in the laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: 39-43 days
- Weight at study initiation: <±20% of the weight at arrival date: 72 to 89 g for males and 64 to 86 g for females two days after arrival.
- Housing: 5 same sex/cage in stainless steel body with a stainless steel mesh lid and floor
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days prior to study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 ºC
- Humidity (%): 40-70%
- Air changes (per hr): 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light: 12 hour dark cycle

IN-LIFE DATES: From: June 27, 2000 To: September 29, 2000

Route of administration:

oral: feed

Vehicle:

other: Ground diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dietary administration. LAE was incorporated into the ground diet to provide the required concentrations by initial preparation of a premix at a concentration of 270.000 ppm. The test substance was mixed with sieved basal diet until half the premix weight was achieved. The mixture was then ground in an electrical grinder, made up to the required weight by addition of further basal diet and subsequently mixed in a Turbular mixer. The final concentrations were achieved by diluting aliquots of the premix with an appropriate amount of basal diet and mixing in a Turbular mixer. Batches of each test diet were prepared and issued weekly to the animal unit. The unused diet, at the end of the week was discarded.
A conversion factor was applied for LAE batch No. 3036 to take into account a water content of 4.1%.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in Week 1 and Week 12 of treatment were analysed for achieved concentration of the test substance. The method of analysis was an adaptation of the method supplied by Lamirsa. The results of these assays confirmed that LAE was homogeneously distributed and stable in the diet for at least 22 days at ambient temperature.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Dosing regime: 7 days/week

Dose / conc.:

0 ppm

Dose / conc.:

5 000 ppm

Dose / conc.:

15 000 ppm

Dose / conc.:

50 000 ppm

No. of animals per sex per dose:

Male: 20 animals at a nominal dose of 0 ppm (actual ingested dose 0 mg/kg bw/day )
Male: 20 animals at a nominal dose of 5000 ppm (actual ingested dose 384 mg/kg bw/day )
Male: 20 animals at a nominal dose of 15000 ppm (actual ingested dose 1143 mg/kg bw/day )
Male: 20 animals at a nominal dose of 50000 ppm (actual ingested dose 3714 mg/kg bw/day )
Female: 20 animals at a nominal dose of 0 ppm (actual ingested dose 0 mg/kg bw/day )
Female: 20 animals at a nominal dose of 5000 ppm (actual ingested dose 445 mg/kg bw/day )
Female: 20 animals at a nominal dose of 15000 ppm (actual ingested dose 1286 mg/kg bw/day )
Female: 20 animals at a nominal dose of 50000 ppm (actual ingested dose 3915 mg/kg bw/day)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary route was selected to simulate the conditions of potential human exposure. Levels of 5.000, 15.000 and 50.000 ppm were based on results from a preliminary study (28-day study). In this preliminary study administration of LAE in the diet at levels of 50.000 ppm for four weeks was shown to be palatable and tolerated.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected at least twice daily for evidence of reaction to treatment or ill-health. Any deviations from normal were recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. In addition, a more detailed physical weekly exarnination was performed on each animal.
- Cage side observations were included: Cages and cage-trays were inspected daily for evidence of animal ill-health, such as blood or loose faeces.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed during the acclimatisation period, on the day that treatment commenced, weekly throughout the treatment period and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for each week throughout the treatment period. From these records the mean weekly consumption per animal was calculated for each cage.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption: Yes. Group mean food conversion efficiencies were calculated for each week of treatment.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Daily by visual observation. No treatment-related effect was suspected and, therefore no quantitative measurements were performed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before commencement of treatment both eyes of all animals were examined by means of an indirect ophthalmoscope, after the instillation of 0.5% tropicamide (Mydracil). During Week 13 of treatment ten males and ten females with the lowest animal numbers assigned to Groups 1 and 4 were similarly examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 13, blood samples were obtained from ten males and ten females with the lowest animal numbers from each group.
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes. Overnight.
- Parameters checked: Haematocrit (Hct); Haemoglobin concentration (Hb); Erythrocyte count (RBC); Total and differential leucocyte count (WBC)
Platelet count (Plt); Mean cell haemoglobin concentration (MCHC); Mean cell haemoglobin (MCH); Mean cell volume (MCV); Prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Week 13, blood samples were obtained from ten males and ten females with the lowest animal numbers from each group.
- Animals fasted: Yes. Overnight
- Parameters checked: Alkaline phosphatase activity (Alkphos); Alanine amino-transferase activity (ALT); Aspartate amino-transferase activity (AST); Gamma-glutamyl transpeptidase activity (gGT); Glucose concentration (Gluc); Total bilirubin concentration (Bili.Total); Total cholesterol concentration (Chol.Total); Total triglyceride concentration (Trig); Creatinine concentration (Creat); Urea concentration (Urea); Total protein concentration (Total prot); Albumin concentration (Alb); Albumin/globulin ratio (A/G Ratio); Sodium (Na), potassium (K) and chloride (CI); Calcium concentration (Ca Total); Inorganic phosphorus (Phos).

URINALYSIS: Yes
- Time schedule for collection of urine: During Week 13 of treatment overnight urine samples were collected from ten males and ten females with the lowest animal numbers from each group.
- Metabolism cages used for collection of urine: Yes. At approximately 16.30 hours each animal was placed in an individual metabolism cage without food or water, and urine was collected until approximately 07.40 hours the following day. The individual samples were examined in respect of:
- Animals fasted: Yes. Overnight.
- Parameters checked: Appearance (App); Volume (Vol); pH - by pH meter; Specific Gravity (SG); Protein (Prot); Glucose (Glue); Ketones (Keto); Bilirubin; Blood.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Ten males and ten females with the highest animal numbers for each group/sex were subjected to the procedures detailed below on the specified occasions. The functional observational battery was performed at the same time of day on each occasion and the observer was unaware of the experimental group to which the animal belonged. The animals were not necessarily all tested on the same day but the number of animals were balanced across the groups on each day of testing. Any deviation from normal was recorded with respect to nature and, where appropriate, degree of severity. Further details on test procedures and definitions and results are documented in the Functional Observational Battery Report.
In the hand and standard arena observations
- Battery of functions tested: sensory activity / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
All animals were subjected to a detailed necropsy. The necropsy procedure included a review of the history of each animal and a detailed examination of the external features and orifices, the neck and associated tissues and the cranial, thoracic, abdominal and pelvic cavities and their viscera. The requisite organs were weighed and the external and cut surfaces of the organs and tissues were examined as appropriate. Abnormalities and interactions were noted and the required tissue samples preserved in fixative.

HISTOPATHOLOGY: Yes
Samples of the following tissues were preserved in 10% neutral buffered formalin, with the exception of testes and epididymides that were initially placed in Bouin's fluid and subsequently retained in 70% industrial methylated spirit and eyes that were preserved in Davidson's fluid: Adrenals, Aorta, Brain, Caecum, Colon, Duodenum, Epididymides, Eyes, Femur with joint Heart, Ileum (including peyers patch), Jejunum, Kidneys, Liver, Lungs , including bronchi) Lymph nodes - mandibular - mesenteric, Mammary area, Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands, Sciatic nerve (one only), Seminal vesicles, Skin, Spinal cord, Spleen, Sternum, Stomach, Testes, Thymus, Thyroid with parathyroids, Trachea, Urinary bladder, Uterus and cervix, Vagina.

Other examinations:

Histology: Tissue samples preserved for histopathology from the animals specified below were dehydrated, embedded in paraffin wax, sectioned at approximately four to five micron thickness and stained with haematoxylin and eosin except the testes which were stained using a standard PAS method: Adrenals, Brain, Femur with joint, Heart, Ileum, Kidneys, Liver, Lungs, Spinal cord, Stomach, Thyroid, Uterus.
Microscopic examination was performed as follows: (i).On the tissue sections listed above, taken from all animals of Groups 1 and 4; (ii). The stomach, which showed a treatment-related change at the high concentration, was examined for all anhnals of Groups 2 and 3; (iii). Tissues reported at macroscopic examination as being abnormal were examined for all animals of Groups 2 and 3.

Organ weights: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus and cervix.

Statistics:

For organ weights and bodyweight changes, homogeneity of variance was tested using Bartlett's test. Whenever this was found to be statistically significant a Behrens-Fisher test was used to perform pairwise comparisons, otherwise a Dunnett's test was used.

Inter-group differences in macroscopic pathology and histopathology were assessed using Fisher's Exact test.

For clinical pathology data, if the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%), the proportion of values different from the mode was analysed (Fisher, 1950) followed by a test for a trend in proportion (Mantel, 1963). Otherwise, a test for heterogeneity of variance between treatments was applied (Bartlett, 1937). If significant heterogeneity was found at the 1% level, a logarithmic transformation was tried to see if a more stable variance structure could be obtained. If no significant heterogeneity was detected ( or if a satisfactory transformation was found), and more than two groups were being compared, group means were compared using Williams' test for a dose-related response (Williams, 1971-72), or if there was evidence for a non-monotonic response, Dunnett's test (Dunnett, 1955, 1964).

For separate two-group comparisons, a Student's t test was used. If significant heterogeneity of variance was present (and could not be removed by a logarithmic transformation), groups were compared using Shirley's non-parametric test for a dose-related response (Shirley, 1977), or if there was evidence for a non-monotonic response, Dunn's test (Dunn 1964). For separate two-group comparisons, a Wilcoxon rank sum test (Wilcoxon 1945) was used.

Unless stated, group mean values or incidences for the treated groups were not significantly different from those of the Controls (p>0.05).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

(There were no deaths, but clinical signs were observed for the groups of higher doses).

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(Marked bodyweight losses observed for the group of highest dose).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

(Markedly low for the group of highest dose)

Food efficiency:

no effects observed

Description (incidence and severity):

Slightly higher than control group

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

(treatment-related microscopic changes restricted to the stomach of animal receiving 15000 or 50000 ppm).

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths. Ungroomed coat was observed for the majority of males and females receiving 50.000 ppm (3.714 and 3.915 mg/kg/day respectively) and two females receiving 15,000 ppm (1286 mg/kg/day).

A high incidence of brown staining on the muzzle was also observed for animals receiving 50.000 ppm and for males receiving 15.000 ppm (1143 mg/kg/day).

BODY WEIGHT AND WEIGHT GAIN
Marked bodyweight losses were observed for animals receiving 50.000 ppm during the first week of treatment (-16% and -13% of initial bodyweight for males and females respectively). Bodyweight gains were also significantly lower that those of the Controls for animals receiving 15.000 ppm and for males receiving 5,000 ppm (384 mg/kg/day). From Week 2, bodyweight gains for treated animals were similar to, or higher than, those of the Controls.

Overall, bodyweights did not fully recover with total gains of 79 and 83% of Controls for males and females receiving 50.000 ppm respectively and 89% of Controls for males receiving 15.000 ppm.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was markedly low during the first week of treatment for animals receiving 50.000 ppm. Food consumption for animals receiving 15.000 ppm and males receiving 5.000 ppm was also slightly lower than that of the Controls. Food consumption remained low during subsequent weeks for animals receiving 50,000 ppm, but to a lesser degree.

FOOD EFFICIENCY
Food conversion efficiency during the first week of treatment was incalculable for animals receiving 50.000 ppm because of the bodyweight losses seen this week. Thereafter, food conversion efficiency was slightly higher than, or similar to that of the Controls.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related ophtalmic findings

NEUROBEHAVIOUR
Motor activity scores were unaffected by treatment. There were no treatment related ophthalmic findings.

ORGAN WEIGHTS
There were no organ weight changes which were clearly attributable to treatment. A number of inter-group differences attained statistical significance, but these were considered to be associated with the large disparity in bodyweight and directly related to treatment.

HAEMATOLOGY
Haematology investigations during Week 13 of treatment revealed slightly high mean cell haemoglobin, mean cell haemoglobin concentration and mean cell volume and slightly low total white blood cell and lymphocyte counts for males receiving 50,000 ppm. The changes were, however, marginal
in degree and were not evident in females.

CLINICAL CHEMISTRY
Blood chemistry investigations revealed, when compared with the Controls, low total protein concentration for animals receiving 50.000 ppm and slightly low albumin concentration for animals receiving 50.000 ppm and females receiving 15,000 ppm. Slightly low cholesterol concentrations were also apparent for females receiving 50.000 ppm.

Apparent low gamma-glutamyl transferase activity for treated males were due to unusually high values for two Control animals and was unrelated to treatment. A few other inter-group differences attained significance, in particular, slightly low glucose concentration for males receiving 15.000 or 50.000 ppm and all treated female groups. These differences from the Controls were minor, individual values were within normal range expected for this species and, therefore, they were not considered of toxicological significance.

URINALYSIS
Urinalysis investigations during Week 13 of treatment revealed a low pH for males receiving 15.000 ppm or 50.000 ppm. This was not evident for females.

GROSS PATHOLOGY
Macroscopic examination of animals killed at completion of the treatment period did not reveal any treatment related findings.

HISTOPATHOLOGY
The only changes that were considered to be related to treatment were seen in the non-glandular region of the stomach, specifically in the area adjacent to the entry of the oesophagus.

The predominant change was parakeratosis which was present in the majority of males and females which received 50000 ppm and in a single female that received 15000 ppm.

Ulceration was seen in a single male and two females which received 50000 ppm and in a single male which received 15000 ppm. In addition, erosions and epithelial hyperplasia were seen in females which received 50000 ppm. These findings suggest an irritant action of the test material on the mucosal tissue, although it is unusual that the changes were restricted to a specific area of the stomach.

OTHER FINDINGS:

ARENA OBSERVATIONS
Animals receiving 50.000 ppm showed an increased incidence of slightly flattened gait (males) or slightly elevated gait (females), on several occasions during the 13 weeks of treatment. The severity gradings were, however, the lowest in all cases, the incidence was not consistent from week to week, the same gait abnormalities were also observed in some Control animals, and the incidence tended to decrease over the last few weeks of the study. Furthermore, the nature of these slight gait abnormalities only related to body position during locomotion, with no evidence of lack of co-ordination or ataxia. These slight or transient gait abnormalities were not, therefore, considered to be of neurotoxicological significance. Similar changes in body posture during locomotion are sometimes observed in animals experiencing abdominal discomfort due to gastrointestinal irritation.


Compared with Controls, activity and rearing scores for males receiving 50.000 ppm were markedly increased on several occasions during the first half of the treatment period. Females receiving 50.000 ppm also showed increased activity and rearing scores, but the differences were much less marked than in the males. These differences, however, decreased during the course of the study and the more comprehensive, automated assessmentof motor activity during Week 12 of treatment failed to show any treatment-related changes. It is possible that these increased activity levels in the arena were associated with the decreased bodyweight gain and decreased food consumption recorded in animals receiving 50.000 ppm. Animals with reduced food intake, in this case probably due to the palatability of the test compound, may show higher than normal levels of
exploratory activity when placed in a new environment.

Key result

Dose descriptor:

NOAEL

Effect level:

384 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

food efficiency

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

445 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

food efficiency

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

food efficiency

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

15 000 ppm

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

The administration to Han Wistar rats of Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE), at levels of 5.000, 15.000 and 50.000 ppm in the diet for 13 weeks was generally well tolerated. Evidence of mild toxicity was observed in-life for animals which received 15.000 or 50.000 ppm with effects on appearance, bodyweight gain, food consumption, food conversion efficiency and clinical pathology parameters. The only histopathological findings considered to be related to treatment were restricted to the stomach of animals receiving 15000 or 50000 ppm and comprised parakeratosis, ulceration, erosions and epithelial hyperplasia.

The No-Observed-Adverse-Effect Level (NOAEL) as identified by this study is considered to be 5.000 ppm (384 mg/kg bw/day for male; 445 mg/kg bw/day for female )

Executive summary:

The objective of this study was to assess the systemic toxic potential of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) in a 13-week dietary study in Han Wistar rats. It was conducted in accordance with the requirements of the European Economic Communities, Organisation for Economic Co-operation and Development, Testing of Chemicals Guideline No. 408 (revised 1998) and the Topological Principles for the Safety Assessment of Direct Food Additives and Colour Additives used in food - Red Book 1 (1982).

Groups of 20 male and 20 female Han Wistar rats received LAE in the diet at concentrations of 5.000, 15.000 and 50.000 ppm during a 13-week treatment period. A similarly constituted Control group received the basal diet only.

At the end of the study there were no deaths.

 

Ungroomed coat was observed for the majority of animals receiving 50.000 ppm and two females receiving 15.000 ppm. This was associated with high incidences of yellow staining of the coat for animals receiving 50000 ppm. A high incidence of brown staining on the muzzle was also observed for animals receiving 50000 ppm and males receiving 15000 ppm.

 

There was no clear evidence of neurotoxicity during the weekly functional observational battery tests.

 

Marked bodyweight losses were observed during the first week of treatment for animals receiving 50.000 ppm and significantly low bodyweight gains were observed for animals receiving 15.000 ppm and males receiving 5000 ppm. From Week 2 onwards, bodyweight gains for treated animals were similar to, or higher than those of the Controls, however, overall bodyweights did not fully recover for animals receiving 50.000 ppm or males receiving 15.000 ppm after 13 weeks of treatment.

 

Food consumption was markedly low during the first week of treatment for animals receiving 50.000 ppm and slightly low for animals receiving 15.000 ppm and males receiving 5.000 ppm. Food consumption remained low during subsequent weeks for animals receiving 50.000 ppm.

 

Food conversion efficiency during the first week of treatment was incalculable for animals receiving 50.000 ppm because of bodyweight losses recorded for this week. During the remainder of the treatment period it was similar, or slightly superior, to that of the Controls.

 

Group mean dosages over the 13 weeks of treatment were 384, 1143, 3714 mg/kg bw/day for males receiving 5.000, 15.000 or 50.000 ppm respectively and 445, 1286 and 3915 mg/kg bw/day for the equivalent female groups.

The administration to Han Wistar rats of LAE, at levels of 5.000, 15.000 and 50.000 ppm in the diet for 13 weeks was generally well tolerated. Evidence of mild toxicity was observed in-life for animals which received 15000 or 50000 ppm with effects on appearance, bodyweight gain, food consumption, food conversion efficiency and clinical pathology parameters. The only histopathological findings considered to be related to treatment were restricted to the stomach of animals receiving 15000 or 50000 ppm and comprised parakeratosis, ulceration, erosions and epithelial hyperplasia.

 

The No-Observed-Adverse-Effect Level (NOAEL) as identified by this study is considered to be 384 and 445 mg/kg bw/day for the equivalent male and female groups.

 

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.30 (Chronic Toxicity Studies)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

434-630-6

EC Name:

-

Cas Number:

60372-77-2

Molecular formula:

Hill formula: C20H41N4O3Cl

IUPAC Name:

ethyl N2-dodecanoyl-l-argininate hydrochloride

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: Nα-Lauroyl-L-arginine ethyl ester monohydrochloride
- Physical state: White powder
- Lot/batch No.: 7446
- Analytical purity: 88.2%
- Stability under test conditions: c.a. 4ºC in the dark
- Expiration date of the lot/batch: 2003-02-27

Test animals

Species:

rat

Strain:

other: Crl:CD (SD) IGS BR

Details on species / strain selection:

Because of historical control data available in the laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: 50-57 days
- Weight at study initiation: 273 g (s.d. 16.0) for males and 200 g (s.d. 14.0) for females
- Housing: 4 same sex/cage in stainless steel body with a stainless steel mesh lid and floor
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 15 days prior to study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 ºC
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12-hour light: 12 hour dark cycle

IN-LIFE DATES: From: February 26, 2003 To: March 15, 2004

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: Ground diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was used as supplied. LAE was prepared for administration as a series of graded concentrations in the diet. The required amount of test substance was weighed out and an equal amount of plain diet added and stirred. Then an amount of plain diet that approximately equalled the weight of the mixture was added to the original mixture and stirred until apparently homogeneous then mechanically mixed. Plain diet was added using a doubling-up process until the required concentration was achieved.

DIET PREPARATION
Rate of preparation of diet (frequency): All formulated diets were prepared freshly every week. The formulations were stored at ambient temperature in the dark prior to use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in Weeks 1, 13, 26, 39 and 51 of treatment were analysed for achieved concentration of the test substance.

Duration of treatment / exposure:

Test duration: 52 weeks

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

2 000 ppm

Doses / concentrations 3

Dose / conc.:

6 000 ppm

Doses / concentrations 4

Dose / conc.:

18 000 ppm

No. of animals per sex per dose:

Actual ingested dose:
Male: 20 animals at a nominal dose of 0 ppm ( 0 mg/kg bw/day mean achieved intake)
Male: 20 animals at a nominal dose of 2000 ppm ( 106 mg/kg bw/day mean achieved intake)
Male: 20 animals at a nominal dose of 6000 ppm (307 mg/kg bw/day mean achieved intake )
Male: 20 animals at a nominal dose of 18000 ppm (907 mg/kg bw/day mean achieved intake)
Female: 20 animals at a nominal dose of 0 ppm (0 mg/kg bw/day mean achieved intake)
Female: 20 animals at a nominal dose of 2000 ppm (131 mg/kg bw/day mean achieved intake)
Female: 20 animals at a nominal dose of 6000 ppm (393 mg/kg bw/day mean achieved intake)
Female: 20 animals at a nominal dose of 18000 ppm (1128 mg/kg bw/day mean achieved intake)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The concentrations of test substance in the diet used in this study (0, 2000, 6000 and 18000 ppm) were selected in conjunction with the Sponsor. On the previous 13-week rat toxicity study in Han Wistar rats, concentrations of 0, 5000, 15000 and 50000 ppm were used. At 15000 ppm evidence of minor toxicity (low bodyweight gain, poor grooming and stomach lesions) was seen, whilst 5000 ppm was classed as the No Observable Adverse Effect Level (NOAEL).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.
- Cage side observations were included: Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatisation period observations of the animals and their cages were recorded once per day.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded one week before treatment commenced (Week -1), on the day that treatment commenced (Week 0), weekly throughout the treatment period for the first 26 weeks, thereafter once every 4 weeks, and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started (Week -1), and each week throughout the treatment period for the first 26 weeks. Thereafter, although the animals continued to be fed on a weekly basis the weight of food given was only recorded once every 4 weeks. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption: Yes. Group mean food conversion efficiencies were calculated for each week of treatment.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Daily by visual observation.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:Before treatment commenced, the eyes of all animals allocated to the study (including spare animals) were examined by means of a binocular indirect ophthalmoscope. During Week 51 of treatment the eyes of all animals of Groups 1 (Control) and 4 (18000 ppm) were similarly examined. Prior to each examination, the pupils of each animal were dilated using 0.5% tropicamide ophthalmic solution (Mydriacyl, Alcon Laboratories Ltd.). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Weeks 14, 26 and 52 of treatment, blood samples were obtained from first surviving 10 animals/sex/group after overnight food deprivation. Animals were held under light general anaesthesia and blood samples were withdrawn from the retro-orbital sinus.
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes. Overnight.
- Parameters checked: Haematocrit (Hct); Haemoglobin concentration (Hb); Erythrocyte count (RBC); Total and differential leucocyte count (WBC)
Platelet count (Plt); Mean cell haemoglobin concentration (MCHC); Mean cell haemoglobin (MCH); Mean cell volume (MCV); Differential WBC count Neutrophils (N) Lymphocytes (L) Eosinophils (E) Basophils (B) Monocytes (M) Large unstained cells (LUC), Prothrombin time (PT), Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Weeks 14, 26 and 52 of treatment, blood samples were obtained from first surviving 10 animals/sex/group after overnight food deprivation.
- Animals fasted: Yes. Overnight
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Sodium (Na), Potassium (K), Total protein (Total Prot), Albumin (Alb), Albumin/globulin ratio (A/G Ratio).

URINALYSIS: Yes
- Time schedule for collection of urine:During Weeks 12, 25 and 51 of treatment overnight urine samples were collected from all animals.
- Metabolism cages used for collection of urine: Yes.Animals were placed in an individual metabolism cage without food or water between approximately 15.00 and 16.30 hours; urine was collected until between approximately 07.00 and 10:00 hours the following day.
- Animals fasted: Yes. Overnight.
- Parameters checked: Appearance (App); Volume (Vol); pH - by pH meter; Specific Gravity (SG); Protein (Prot); Glucose (Glue); Ketones (Keto); Bilirubin; Blood, microscopic examination of the urine sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: Before treatment commenced and during each week of treatment detailed physical examination and arena observations were performed on the first surviving 10 animals in each group/sex. During Week 49 of treatment, sensory reactivity, grip strength and motor activity assessments were performed on 10 males and 10 females from each group, where possible the same animals used for physical and arena activity. Animals were not necessarily all tested in one day, but the numbers of animals and the time of testing were balanced across the groups. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers.
- Battery of functions tested: sensory activity /grip strenght / motor activity

OTHER:

- TOXICOKINETICS: During Week 52 of treatment blood samples were obtained from 18 male and 18 female animals in each treated group. Blood samples were centrifuged as soon as possible after being taken (nominally at 0°C) and/or when necessary kept cool (on water ice) prior centrifugation.
Plasma samples were immediately processed using the sample preparation procedure described in the bioanalytical method and the supernatants produced were stored at nominally +4°C. Bioanalysis was subject to the satisfactory validation of the bioanalytical method, details of which are documented in a separate Methodology Validation study (055). Samples from all treated groups were analysed. Samples from untreated control groups were retained for possible method calibration use.

- HAEMATOLOGY, BONE MARROW: Bone marrow samples were obtained from the tibia bone during necropsy of all animals killed. Smears prepared from these samples were air-dried, fixed in methanol and stained using a Romanowsky procedure.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. The requisite organs were weighed and external and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. The retained tissues were checked against the tissue preservation list before disposal of the carcass. Testes and epididymides were fixed in Bouin's solution prior to transfer to 70% industrial methylated spirit and eyes were fixed in Davidson's fluid. Samples (or the whole) of the other tissues from all animals were preserved in 10% neutral buffered formalin.

HISTOPATHOLOGY: Yes
Tissue samples were dehydrated, embedded in paraffin wax, sectioned at approximately four to five micron thickness and stained with haematoxylin and eosin, except the testes which were also stained using a standard periodic acid/Schiff (PAS) method. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required for microscopic pathology. Microscopic examination was performed as follows: All tissues, listed below, preserved for examination were examined for all animals of Groups 1 (Control) and 4 (18000 ppm) sacrificed on completion of the scheduled treatment period and for all animals killed or dying during the study: Adrenals, Aorta - thoracic, Brain, Caecum, Colon, Duodenum, Epididymides, Eyes, Femurs, Harderian glands, Head, Heart, Ileum, Jejunum, Kidneys, Lachrymal glands, Liver, Lungs, Lymph, nodes, Mammary area Oesophagus Optic nerves, Ovaries, Pancreas, Peyers patch, Pituitary, Prostate, Rectum, Salivary glands (Submandibular/Sublinguar) Sciatic nerves Seminal vesicles Skeletal muscle - thighs, Skin, Spinal cord, Spleen, Sternum, Stomach, Testes, Thymus, Thyroid with parathyroids, Tongue, Trachea, Urinary bladder, Uterus and cervix, Vagina.

Other examinations:

Organ weights: Adrenals, brain, kidneys, liver, ovaries, testes.

Statistics:

The following sequence of statistical tests was used for grip strength and motor activity, bodyweight, food consumption, organ weight and clinical pathology data:
If 75% of the data (across all groups) were the same value, then a frequency analysis was applied. Treatment groups were compared using a Mantel test for a trend in proportions (Mantel 1963) and also pairwise Fisher's Exact tests (Fisher 1973) for each dose group against the control both for i) values =c, and for ii) values <=c versus values >c, as applicable.

If Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level, then parametric analysis was applied. If the F1 test for monotonicity of dose-response (Healey 1999) was not significant at the 1% level, Williams' test for a monotonic trend (Williams 1971, 1972) was applied. If the F1 test was significant, suggesting that the dose-response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.

If Bartlett's test was significant at the 1% level, then logarithmic and square-root transformations were tried. If Bartlett's test was still significant, then non-parametric tests were applied. If the H1 test for monotonicity of dose-response (Healey 1999) was not significant at the 1% level, Shirley's test for a monotonic trend (Shirley 1977) was applied. If the H1 test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel 1959) was performed instead.

For organ weight data, analysis of covariance was initially performed using terminal bodyweight as covariate.

Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Females at 18000 ppm dose level showed consistently higher than control weekly incidences of brown fur staining. Males receiving 18000 ppm did not show any clear consistent differences from controls with regard to brown fur staining or ungroomed appearance. Females at 6000 ppm dose level also tended to show higher than control weekly incidences of brown fur staining but the weekly incidences were generally lower than at the highest dose level and was mainly confined to the head region. Males receiving 6000 ppm did not show any clear consistent differences from controls with regard to brown fur staining or ungroomed appearance. Both sexes receiving 2000 ppm did not show any clear consistent differences from controls with regard to brown fur staining or ungroomed appearance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were six unscheduled deaths during the study, none of these deaths were considered to be attributable to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males and females receiving 18000 ppm showed initial lower body weight than control mean bodyweight gains for the first 3 weeks of treatment (Week 0-3). Similar effects on bodyweight gain to those reported at 18000 ppm were seen for both sexes at 6000 ppm, although the magnitude of the initial differences from controls was less than those reported at 18000 ppm. The weight gain of both sexes receiving 2000 ppm was considered to be unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the first week of treatment both sexes at 18000 dose level ate notably less food in comparison with both the pre-dose data recorded for these animals and concurrent controls. Throughout the treatment period food intake for males and females at 6000 ppm concentration was slightly lower and lower respectively than concurrent controls values and that recorded pre-dose for these males. For 2000 ppm dose, there was considered to be no effect of treatment in either sex.

Food efficiency:

no effects observed

Description (incidence and severity):

For 18000 ppm and 6000ppm, in Week 1 food conversion efficiency for both sexes at this level was lower than controls but thereafter (Week 2-26) food conversion efficiency for these animals was similar to controls. For 2000 ppm, there were no effects of treatment on food conversion efficiency for either sex.

Water consumption and compound intake (if drinking water study):

not specified

Description (incidence and severity):

The achieved intake for each group of females in terms of "mg/kg bodyweight/day" was higher than the corresponding male group. Overall group mean achieved intakes at 2000, 6000 and 18000 ppm for the period Weeks 1 to 52 were 106, 307 and 907 mg/kg bodyweight/day for males and 131, 393 and 1128 mg/kg bodyweight/day for females.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment related ocular changes reported the duration of the study. The ophthalmic examination performed at pre-treatment and after 52 weeks of treatment revealed findings that were within the normal limits for the animals at their age and of this strain.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no clear effects of treatment on the bone marrow. All differences from control were minor, not dosage-related and/or inconsistent across both sex groups, and as such were minor, not dosage-related and/or inconsistent accross both sex groups, and as such considered not to be associated with treatment. There were no effects considered to be of toxicological significance on Haematology peripheral, there were no effects of treatment on the red blood cell or clotting parameters throughout the study.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Higher group mean urea values were noted for all groups of treated females in Week 52, a dosage-relationship was evident between 6000 and 18000 ppm, but not between 2000 and 6000 ppm. The mean value recorded for females at 18000 ppm in Week 52 achieved statistical significance in comparison with controls. There were no other differences considered to be of toxicological importance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The composition of the urine was considered unaffected by treatment. The inter-group differences in urinary volumes and mean SG value for the females in Week 51 were considered to be due to fluctuations commonly noted in laboratory kept animals. Therefore, in the absence of clear dosage related trends and/or consistency across both sex groups, these differences were considered not to be treatment related.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

NEUROBEHAVIOUR
No effects of treatment were noted.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no clear effects of treatment on organ weights. A lower than control mean absolute adrenal weight noted, thus the difference in adrenal weights was not considered to be attributable to treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Depressions on the epithelial aspect of the forestomach (in the oesophageal groove) were observed in 12/19 male rats and 9/19 female rats at 18000 ppm, 5/20 male rats and 6/20 female rats at 6000 ppm and 1/18 male rats and 5/19 female rats at 2000 ppm compared with 0/19 male control rats and 2/20 female control rats. Dosage related higher than control incidence of thickening of the uterine cervix was noted for all treated female groups.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

(treatment-related microscopic changes restricted to the stomach of animal receiving 18000 ppm).

The only toxicologically significant effect observed was on the forestomach in the 18000 ppm treated group. The lesions were consistent with local irritation of the mucosa with hyperplasia, erosions and ulceration, with evidence of healing and reepithelialisation. Although similar lesions were observed in all groups, including controls, they were statistically significantly increased in the top dose group only compared with the controls. The changes seen in the stomach were confined to a specific area of the forestomach, the oesophageal groove and are thought to represent a local response to irritation. Thus they are considered to be indicative of direct systemic toxicity. There was no correlation between the individual animals which showed lower bodyweight gain, poor grooming and/or brown fur staining and the presence of these stomach lesions. Nor was there any correlation between the animals which showed stomach lesions and those which exhibited white blood cell and/or biochemical disturbances. This further supports the hypothesis that the stomach lesions are not indicative of systemic toxicity.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

HISTOPATHOLOGY: NEOPLASTIC
Four cases of mammary adenocarcinoma were reported in females, one at 2000 ppm, another at 6000 ppm, and two at 18000 ppm. However, the relatively low incidence of these tumours, and the absence of any other treatment related changes in the mammary tissues, does not suggest any relation to the administration of the test substance. Historical control data show a spontaneous incidence of these tumours with 2 being observed in 179 control females.

Other effects:

not specified

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

307 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

393 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

3

Key result

Dose descriptor:

LOAEL

Effect level:

907 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on local irritant changes in the forestomach

4

Key result

Dose descriptor:

LOAEL

Effect level:

1 128 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on local irritant changes in the forestomach

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

18 000 ppm

System:

gastrointestinal tract

Organ:

other: Forestomach

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Formulation chemistry:

The mean concentration of lauric arginate in test diet formulations analysed for the study were within ± 7% of nominal concentrations confirming accurate formulation.

The homogeneity was confirmed for lauric arginate in R&M No.1 diet formulations at a nominal concentration of 2000 ppm. The stability was confirmed during ambient temperature storage for 22 days, representing the maximum time from preparation to completion of feeding.

Achieved dosage:

Overall group mean achieved intakes at 2000, 6000 and 18000 ppm for the period Weeks 1 to 52 were 106, 307 and 907 mg/kg bw/day for males and 131, 393 and 1128 mg/kg bw/day for females.

 

Applicant's summary and conclusion

Conclusions:

Based on the calculated intake data, the NOAEL (Chronic study, 52 weeks) was 6000 ppm equivalent to 307 mg/kg bw/day in the males and 393 mg/kg bw/day in the females. The corresponding LOAEL was 18000 ppm equivalent to 907 mg/kg bw/day and 1128 mg/kg bw/day for the males and females respectively, based on local irritant changes in the forestomach.

Executive summary:

According to Organisation for Economic Co-operation and Development, Testing Chemicals Guidelines No.452, Adopted may 1981.EC (1996) B.30 Chronic Toxicity Test. Annex V to Directive 67/548/EEC as amended in Directive 88/302/EEC (OJ No.L133, 1998):

The systemic toxic potential of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE), to Crl:CD®(SD)IGS BR rats by dietary administration was assessed over a period of 52 weeks. Three groups of twenty male and twenty female rats, a total of 90 male and 90 female rats with control group, received lauric arginate at concentrations in the diet of 2000, 6000 or 18000 ppm for 52 weeks. A similarly constituted Control group received untreated diet in the same way and at similar frequency to that of the treated animals. Overall group mean achieved intakes at 2000, 6000 and 18000 ppm for the period Week 1 to 52 were 106, 307 and 907 mg/kg bw/day for males and 131, 393 and 1128 mg/kg bw/day for females. Doses were selected on the previous 13-week rat toxicity study in Han Wistar rats.

During the study, clinical condition, bodyweight and food consumption were recorded for all animals. Ophthalmic examination, haematology (peripheral blood), blood chemistry and urinalysis were carried out at scheduled intervals. In addition, detailed physical examination.

Noteworthy findings in this study are listed below:

 

Toxicokinetics: The rate and extent of systemic exposure of rats to lauric arginate and its metabolite LAS appeared to be characterised by dose-independent (linear) kinetics over the dietary concentration 2000 to 18000 ppm during Week 52 of the 52-week toxicity study. There was some evidence that the kinetics of lauric arginate in female rats may have been non-linear (dose-dependent), however, this did not reach statistical significance. The study also provided evidence that in general there were no differences in the systemic exposure of male and female rats to lauric arginate or LAS.

 

Clinical observation in the period up to Week 13 females only showed higher weekly incidences of generalised brown fur staining and ungroomed coats in the 18000 ppm dose group and to a lesser extent in the 6000 ppm dose group than in the controls. Thereafter, no clear difference between groups was observed.

 

At 6000 and 18000 ppm both sexes showed low bodyweight gain and initial reduced food conversion efficiency in both sexes. The reduced food conversion efficiency was confined to the first week of treatment, and for males at both dose levels and for females at 18000 ppm, this coincided with lower than control food intake. However females receiving 6000 ppm also showed reduced food conversion efficiency in Week 1 without a concomitant effect on food intake. From approximately Weeks 26 of treatment lower bodyweight gains were again apparent for these females at 6000 or 18000 ppm. The group treated with 2000 ppm lauric arginate in the diet was not affected.

 

Higher high and low beam motor activity scores were noted for males only in Week 49 in the 18000 ppm dose group.

There were no clear effects of treatment on the bone marrow. All differences from controlwere minor, not dosage-related and/or inconsistent across both sex groups, and as such considered not to be associated with treatment.

In conclusion, treatment related effects other than the forestomach findings were noted at the 6000 and 18000 ppm dosage levels. These consisted of low bodyweight gain and initial reduced food consumption in both sexes. Although these bodyweight changes at both 6000 and 18000 were considered to be treatment-related, at 6000 ppm based on a combination of the short duration, the magnitude of difference from controls, lack of adverse effects on survival or general condition of the animals, they were not considered to be of toxicological importance. Animals receiving 18000 ppm showed a more pronounced effect on bodyweight gain and irritant effects of treatment on the forestomach with limited ulceration and signs of healing. No significant toxicological effects were observed in the animals receiving 6000 ppm or 2000 ppm lauric arginate in the diet.

Based on the calculated intake data, the NOAEL in this study was 6000 ppm equivalent to 307 mg/kg bw/day in the males and 393 mg/kg bw/day in the females. The corresponding LOAEL was 18000 ppm equivalent to 907 mg/kg bw/day and 1128 mg/kg bw/day for the males and females respectively, based on local irritant.