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

Endpoint summary

Description of key information

Several studies have been assessed for Oral Repeated dose toxicity with durations according to: 28 days, 90 days and 52 weeks. In general doses have been well tolerated.

However, the key study to assess a NOAEL effect was the Chronic study in rats developed during 52 weeks. The corresponding LOAEL was 18000 ppm equivalent to 907 mg/kg bw and 1128 mg/kg bw/day for the males and females respectively, based on local irritant changes in the forestomach. 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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.30 (Chronic Toxicity Studies)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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

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

Dose / conc.:

0 ppm

Dose / conc.:

2 000 ppm

Dose / conc.:

6 000 ppm

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).

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.

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

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

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

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

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

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

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.

 

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 6, 1996 to November 4, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl: CD(SD) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Manston (UK)
- Age at study initiation: 7 weeks
- Weight at study initiation: 209 to 278 g for males and 151 to 199 g for females.
- 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: 21 days prior to study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 ºC
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): 12-hour light: 12 hour dark cycle

IN-LIFE DATES: From: December 7, 1995 To: March 11, 1996

Route of administration:

oral: feed

Vehicle:

other: Ground diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
A pre-mix was prepared by adding SDS Rat and Mouse No. 1 maintenance diet to the test substance, thoroughly stirring by hand and mixing in a Turbula mixer for 5 minutes. A second premix was made by diluting the first pre-mix with more diet and mixing in a Turbula mixer for 5 minutes. The high dose level (50.000 ppm) was prepared by stirring the required amount of the first pre-mix into bulk plain diet followed by mixing for 5 minutes. Lower concentrations were prepared by diluting the most appropriate premix with plain diet and mixing in a Turbula mixer for 5 minutes.

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 the formulations for Weeks 1 and 10 were also analysed to check the accuracy of preparation. Contingency samples were taken in Week 13 but since good results were obtained in the Week 1 and 10 samples the contingency samples have been discarded.

Duration of treatment / exposure:

Test duration: 90 days

Frequency of treatment:

Dosing regime: 7 days/week

Dose / conc.:

0 ppm

Dose / conc.:

3 200 ppm

Remarks:

Equivalent to 220 and 262 mg/kg-bw/ day in male and female rats respectively.

Dose / conc.:

12 800 ppm

Remarks:

Equivalent to 904 and 1067 mg/kg-bw/day in male and female rats respectively.

Dose / conc.:

50 000 ppm

Remarks:

Equivalent to 3324 and 3927 mg/kg-bw/day in male and female rats respectively.

No. of animals per sex per dose:

Male: 10 animals at 0 ppm
Male: 10 animals at 3200 ppm
Male: 10 animals at 12800 ppm
Male: 10 animals at 50000 ppm
Female: 10 animals at 0 ppm Female: 10 animals at 3200 ppm
Female: 10 animals at 12800 ppm
Female: 10 animals at 50000 ppm

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 3.200, 12.800 and 50.000 ppm were based on results from a preliminary study (28-day study).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Individual animals were observed at least once daily for any signs of behavioural changes, reaction to treatment or ill health. A detailed palpation was performed on each week day for the First 4 weeks of the study. After 4 weeks of treatment detailed palpations were performed weekly. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals. Further checks were made early in each working day and again in the afternoon to look for dead or moribund animals. This allowed post mortem examination to be carried out during the working period of that day. On Saturdays, Sundays and public holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded at the time of allocation of animals to groups, on Week 1, on the day of commencement of treatment, and once a week thereafter including the day of sacrifice. Weights have been reported from Week 1.

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 quantity of food consumed by each cage of rats was recorded on a weekly basis. Food intake per rat (g/rat/week) was calculated using the amount of food given to and left by each cage in each group and the number of rats surviving in each cage.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. At weekly intervals the group mean achieved intake of test substance (mg/kg/day) was calculated from the group mean bodyweight and food consumption and the dietary inclusion levels of E6123. The "food consumed" was calculated as indicated in the Food consumption section and is not a mean of the individual cage means. The mid-week bodyweight was calculated from each individual animal and uses the mid-week bodyweight.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption: Yes. Food conversion ratios were calculated over the period Weeks 1 to 13, from the bodyweight and food consumption data as weight of food consumption per unit gain in bodyweight. The "food consumed" was calculated as indicated in the Food consumption section and is not a mean of the individual cage means. The 'bodyweight gain' was calculated from the gain of each animal and uses the mean gain in the formula.

WATER CONSUMPTION (if drinking water study): Yes
- Time schedule for examinations: Daily monitoring by visual appraisal of the water bottles was maintained throughout the study. Water consumption was measured accurately, by weight, for 7 consecutive days during Week 12 for all cages in all groups. The quantities of water offered were measured into a suitable container each day and recorded. The following morning any residue was removed, measured and recorded before fresh water was offered.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced the eyes of all animals were examined by means of a Keeler indirect ophthalmoscope. During Week 13 the eyes of all animals in the control and high dosage level groups were examined. Prior to examination, the pupils of each animal were dilated using a Tropicamide ophthalmic solution.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 13 samples of blood were withdrawn, from the orbital sinus of all rats from each group.
- Anaesthetic used for blood collection: Yes (ether anaesthesia).
- Animals fasted: Yes. Overnight.
- Parameters checked: Packed cell volume (PCV); % Haemoglobin (Hb); Red blood cell count (RBC); Absolute indices were calculated as follows:
Mean corpuscular haemoglobin concentration (MCHC); Mean corpuscular volume (MCV); Mean corpuscular haemoglobin (MCH); Total white cell count (WBC Total); Neutrophils (N); Lymphocytes (L); Eosinophils (E); Basophils (B); Monocytes (M); Large unstained cells (LUC); Platelet count (Pits); Thrombotest (TT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Week 13 samples of blood were withdrawn.
- Animals fasted: Yes. Overnight
- Parameters checked: Total protein; Albumin (Alb); Globulin (Glob): by subtraction; Total protein; minus Albumin; Urea nitrogen (Urea Nitr); Creatinine; Sodium (Na); Potassium (K); Calcium (Ca); Inorganic Phosphorus (P); Chloride (CI); Total cholesterol (Choi); Enzymatic assay); Alkaline phosphatase (AP); Total bilirubin (Bili-rubin); Glucose (hexokinase mediated assay); Glutamic-pyruvic transaminase (GPT) Glutamic oxaloacetic transaminase (GOT); γ Glutamyl transferase (γGT); Ornithine Carbamoyl transferase (OCT).

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.
- Animals fasted: Yes. Overnight.
- Parameters checked: Appearance (App); Volume (Vol); pH - by pH meter; Specific Gravity (SG); Protein (Prot).

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
MICROSCOPY:
- Time schedule: An aliquote of the urine sample was centrifugated and the deposit spread on a microscopic slide. The deposit was examined for the presence of: Epithelial cells, Polymorphonuclear leucocytes, Mononuclear leucocytes, Erythrocytes, Organisms, Renal tubule casts, Sperm, Other abnormal constituents.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
On completion of 13 weeks of treatment, all animals were killed and subjected to detailed necropsy procedure. All superficial tissues were examined visually and by palpation, the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. All subcutaneous tissues were examined. The condition of the thoracic viscera was noted, with due attention to the thymus, lymph nodes and heart. The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastrointestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver was sectioned at intervals of a few millimetres; the kidneys were incised and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intraabdominal lymph nodes and accessory reproductive organs were recorded. The following organs from all animals killed at the scheduled sacrifice were dissected free of fat and weighed: adrenals, liver, spleen, brain, ovaries, testes, epididymides, pituitary, thyroid, heart, prostate, uterus, kidneys, seminal vesicles. Samples of all tissues from all animals were preserved.

HISTOPATHOLOGY: Yes.
For testes and epididymides, tissues were embedded in paraffin wax and sections stained with PAS-haematoxylin. A transverse section of each testis and a full longitudinal section of each epididymis was cut at 2 micrometres. Microscopic assessment of the testes was made with reference to the stages of the cycle of the seminiferous epithelium. Other tissues were embedded in paraffin wax and sections cut at 4 micrometres were stained with haematoxylin and eosin. Frozen sections of liver, fixed in buffered formalin, were cut on a cryostat at 12 micrometres and stained for fat with Oil Red O (ORO). Sections of kidney were stained with ORO or Periodic Acid-Schiff reagent at the discretion of the pathologist.
The specified list of tissues including all macroscopically abnormal tissues from all animals from the control group and all animals from the high dosage level that were killed following 13 weeks of treatment. Any macroscopically abnormal tissue in any animal from the low and intermediate groups killed following 13 weeks of treatment, and from any animal that died during the study. Lungs, liver and kidneys from all animals in the low and intermediate dosage level groups.

Other examinations:

Organ weights: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, spleen, testes, thyroid, uterus.

Statistics:

All statistical analyses were carried out separately for males and females.

Data relating to food and water consumption were analysed on a cage basis. For all other parameters, the analyses were carried out using the individual animal as the basic experimental unit.

Food consumption data were analysed using cumulative cage totals and water consumption data were analysed as the total recorded intake over selected time periods, expressed on a weekly basis. Bodyweight data were analysed using weight gains.

The following sequence of statistical tests was used for food consumption, water consumption, bodyweight, clinical pathology and organ weight data:

If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%), the proportion of animals with values different from the mode was analysed, (Fisher, 1950 and Mantel, 1963). Otherwise:

A test was applied to test for heterogeneity of variance between treatments, (Bartlett, 1937). Where significant (at the 1 % level) heterogeneity was found, 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), a one-way analysis of variance (Snedecor and Cochran, 1967) was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, an analysis of ranks was used (Kruskal-Wallis, 1952/3) (Hollander and Wolfe, 1968).

Analyses of variance were followed by Student's t test and Williams' test (Williams, 1971/2) for a dose-related response, although only the one thought most appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of these tests (Shirley, 1977).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

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

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no treatment related clinical signs noted during the study. There was a slightly higher incidence of hair loss noted amongst females receiving 12.800 or 50.000 ppm but there was no obvious pattern or increase in severity with increasing dosage - as such this change is considered to be coincidental. There was one unscheduled death during the study. Animal no. 10 (Group 1 males) collapsed and died during Week 1, however, this death is not considered to have affected the evaluation of the toxicity of the test substance.

BODY WEIGHT AND WEIGHT GAIN
Overall mean bodyweight gains for all treated groups of males were similar to concurrent controls. The overall mean bodyweight gain for all treated groups of females was slightly lower than concurrent controls during the 13 weeks of treatment with statistical significance being attained for animals receiving 12.800 or 50.000 ppm, however there was no dosage-relationship. In the absence of a relationship to dosage and any effect in the males, the lower bodyweight gain in females is of uncertain relationship to treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The mean cumulative food intake of animals in treated groups was comparable with the controls for both sexes.

FOOD EFFICIENCY
The efficiency of food utilisation for females in all treated groups was marginally inferior to the concurrent controls, although there was no dosage relationship apparent. The efficiency of food utilisation of treated groups of males was considered to be comparable with the concurrent controls.

OPHTHALMOSCOPIC EXAMINATION
Examination during Week 13 of the eyes of control group animals and those receiving 50.000 ppm did not reveal any lesions considered to be attributable to treatment, with the only finding noted being within normal limits for animals of this age and strain (no lesions were noted prior to treatment).

HAEMATOLOGY
There was a slightly lower total white blood cell count amongst animals receiving 12.800 or 50.000 ppm when compared to the concurrent control groups with the values for females attaining statistical significance. However there was no consistency in the type of white blood cell contributing to the lower total cell count and as such these effects are of uncertain toxicological importance.

CLINICAL CHEMISTRY
Although there were slight differences in some biochemical parameters amongst animals receiving 12.800 or 50.000 ppm that occasionally attained statistical significance when compared with the concurrent controls, all differences were slight and inconsistent across the sexes and hence no relationship to treatment is suspected.

URINALYSIS
Amongst males receiving 50.000 ppm group mean urine volume was was slightly higher than the controls, being consistent with the increase in water intake recorded for these animals, although the difference from the controls did not attain statistical significance.

ORGAN WEIGHTS
There was a slightly higher group mean adjusted liver weight amongst females receiving 50.000 ppm when compared to the controls. No similar increase was apparent in the males at this dosage, and since there were no microscopic changes detected in the liver, this finding is of uncertain toxicological significance. Although there were occasional intergroup differences in other organ weights, there were no other changes that were considered attributable to treatment and no changes supported by findings at histopathological examination.

GROSS PATHOLOGY
The only changes at macroscopic post mortem examination was an increased incidence of alopecia amongst females receiving 12.800 or 50.000 ppm. In isolation this change is considered to be of uncertain biological significance.

HISTOPATHOLOGY
No treatment-related changes were noted in any of the tissues examined. Other minor changes reported were considered to be of no toxicological importance.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Results for the test substance LAE

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Results for the test substance LAE

Key result

Critical effects observed:

not specified

Analytical chemistry:

The formulations of the test substance in rodent diet were confirmed as homogeneous and stable for up to 15 days from preparation.

The analysed concentrations in diets for feeding in Weeks 1 and 10 were within acceptable limits, providing an indication of good accurary of formulation during the toxicity study.

Achieved intakes:

The achieved intakes followed the expected pattern for a fixed level dietary study, with exposure to the test substance being higher during the period of fastest growth and also higher for females than males. The intended interval in achieved intakes between the groups (approximately 4-fold) was well maintained throughout the study. Overall group mean water intake for males receiving 50.000 ppm was slightly higher than the concurrent controls. Overall group mean water intake was generally comparable for all other treated groups in comparison with concurrent controls.

 

Conclusions:

The experiment performed had the scope to assess the toxicity of the test substance when administered to groups of rats (each of 10 animals per sex) by the dietary route at inclusion levels of 0 (Control), 3.200, 12.800 or 50.000 ppm for a period of 13 weeks.Treatment with the test substance at 50.000 ppm was associated with slight changes evident in male and female rats when compared with untreated control animals, evident as an equivocal lower bodyweight gain of females, and an increase in water consumption and urine volume of males. There were no treatment-related changes in organ weight analysis or findings at macroscopic post mortem examination that were confirmed by histological examination.

There were considered to be no conclusive signs of toxicity amongst animals fed a diet containing 3.200 ppm (220 for male and 262 for female mg/kg bw/day) or 12.800 (904 for male and 1.067 for female mg/kg bw/day) ppm of test substance (175 for male and 207 for female mg/kg bw/day of LAE).

Within the context of this study, evidence of toxicity was noted amongst male and female rats receiving 50.000 ppm dose concentration (3.324 mg/kg bw/day for males and 3.927 mg/kg bw/day for females).

Executive summary:

The objective of this study was to assess the toxicity of the test substance to rats when administered in the diet over a period of 13 weeks was designed according to OECD Guideline for Testing of Chemicals, No. 408, "Subchronic Oral Toxicity - Rodent", dated 12 May 1981.

This report describes the experiment performed to assess the toxicity of the test substance when administered to groups of rats (each of 10 animals per sex) by the dietary route at inclusion levels of 0 (Control), 3.200, 12.800 or 50.000 ppm for a period of 13 weeks. The mean achieved intakes over the 13 weeks of treatment were 0 (Control), 220, 904 and 3.324 mg/kg/day for males and 0 (Control), 262, 1.067 and 3.927 mg/kg bw/day for females.

The dosages employed, 0, 3.200, 12.800 and 50.000 ppm were selected with reference to a 4-week preliminary study. The rat was the species of choice due to regulatory requirements and the strain was chosen taking into account the availability of background data. The oral route is an anticipated route of human exposure to the test substance.

During the experimental period clinical signs, bodyweight, food and water consumption were recorded and at the end of the study haematological, biochemical, urinalysis and ophthalmologic investigations were performed. All animals were killed after 13 weeks of treatment andexamined macroscopically. A range of organs was weighed and tissues preserved for subsequent histopathology.

Within the context of this study, evidence of toxicity was noted amongst male and female rats receiving 50.000 ppm dose concentration. Reduced bodyweight gains were noted for females of the highest dose treated groups however, since no similar finding was apparent for males and no other evidence of toxicity was apparent, it was concluded that the no-effect level for continuous administration of the test substance to rats for 13 weeks was likely to be 12.800 ppm (904 and 1067 mg/kg bw/day). According to the composition of the test substance, LAE in the test material, the achieved intake corresponds to 175 and 207 mg/kg bw/day.

Reference 3

Endpoint:

repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2004-10-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to Guidance for Industry on Bioanalytical Method Validation, FDA (CDER) 2001, with GLP.

Qualifier:

according to guideline

Guideline:

other: Guidance for Industry on Bioanalytical Method Validation, FDA (CDER) 2001.

GLP compliance:

yes

Critical effects observed:

not specified

Conclusions:

LC-MS/MS method for the measurement of 12C-LAE nad 12C-LAS in rat plasma has been validated for use. Due to the rapid loss of LAE in plasma, the need for rapid analysis should be particularly noted.

Executive summary:

A bioanalytical method for the determination of ¹²C-LAE and its metabolite ¹²C-LAS in rat plasma by a LC-MS/MS procedure has been validated.

The validation procedures followed during this study were based upon the Guidance for Industry on Bioanalytical Method Validation, FDA (CDER) 2001.

LAE was found to be very unstable in rat plasma so the extraction procedure described has to be closely followed in order to minimise the conversion of LAE to LAS. In particular, the plasma must be cooled on ice immedately after separation from blood, and the cooled extraction solvent added as soon as possible (within 5 minutes).

Aliquots of the reconstituted samples (20 µl) were injected onto an LC-MS/MS system for analysis and analytes were separated isocratically. The LC system was connected to the MS/MS system and each component detected by its characteristic ion transitions.

The relationship between the peak area ratio of each analyte to its internal standard and the analyte concentration in plasma was linear (R>0.998) over the chosen calibration ranges. The precision of the bioanalytical method was evaluated by determining the coeffciientes of variation of the measured concentrations of ¹²C-LAE and ¹²C-LAS following the analysis of quality control samples. The variation coefficient for within intrabatch analysis met the required acceptance criteria of <=15 %. The accuracy measurement, expressed in terms of relative error, was evaluated following the analysis of quality control samples. The relative errors derived from within intrabatch analysis met the required acceptance criteria of being within ±15%. The low limit of quantiifcation of the method was 1ng/ml for ¹²C-LAE and 5 ng/ml for ¹²C-LAS. At these concentrations the required precision and accuracy criteria were met.

In conclusion, a LC-MS/MS method for the measurement of ¹²C-LAE and ¹²C-LAS in rat plasma has been validated for use. Due to the rapid loss of LAE in plasma, the need for rapid analysis should be particularly noted.

Reference 4

Endpoint:

repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2008-01-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: An expert review.

Conclusions:

The reduction of mature white cells in the peripheral blood is almost certainly part of a normal physiological response to the changes seen in the stomach and associated tissues.

It has not been possible to consistently correlate the individual white blood cell counts with individual histological changes.

Executive summary:

This report is an interpretation of the white cell data contained in the reports of 5 separate studies performed over a 10 year period (1995 - 2005).

The studies examined were:

• Nº 2 - a 4 week study using LAE in formulation. Test substance: Diet levels 0, 3200, 12800, 50000 ppm
• Nº 030 - a 4 week study using LAE (no histopathology). LAE: Diet levels - 0, 3200, 12800, 50000 ppm
• Nº 3 - a 13 week study using LAE in formulation. Test substance: Diet levels - 0, 3200, 12800, 50000 ppm
• Nº 031 - a 13 week study using LAE. LAE: Diet levels - 0, 5000, 15000, 50000 ppm
• Nº 050 - a 52 week study using LAE. LAE: Diet levels - 0, 2000, 6000, 18000 ppm

In the present review, of the 5 studies involved, 3 (of 13 or 52 weeks duration) have shown slight to moderate reductions in total white cell counts, particularly in neutrophils or lymphocytes. Some statistical differences were also noted in monocytes and/or LUC's, but due to the very small numbers of these cells, it is difficult to ascribe any toxicological significance to these changes. In two of the studies (031 and 050), the reductions in white cells generally correlate with ulceration and/or irritation of the stomach lining and are therefore likely to be the result of these changes. In the 52-week study, where signs of repair of the initial insult are apparent in the histopathology, the lack of white cell changes in the latter stages reflect the improving situation.

 

The white cell count obtained at any given time accounts only for freely circulating cells. The dynamics of production and release from the bone marrow, and the egress from blood to tissues can influence these counts in conditions of both health and disease. The counts are basically influenced by one or more of the following mechanisms: (1) the rate of production, (2) the inflow rate of cells from the bone marrow, (3) the intravascular life span of the cells, and (4) the outflow rate of cells from blood to tissues. Thus a leucopenia occurs from a decreased intravascular survival rate, accelerated migration into tissue, reduced marrow release and/or reduced production of new cells.

 

In a situation where an increased number of specific cells are required, for example, neutrophils at the site of activity in cases of tissue inflammation, or red cells in the peripheral circulation in cases of acute anaemia, readily available cells are mobilised first, thus reducing the number of such cells in the circulation or bone marrow in the first instance. Whilst increased cell production starts fairly quickly, there is inevitably a "lag-phase" before this happens. Thus, in the early stages of an inflammatory reaction, a reduction in the number of white cells in the peripheral blood may be seen, whilst in an acute or severe anaemia, a marked release of late, and some intermediate, normoblasts from the marrow occurs. Either event may temporarily shift the M:E Ratio in the "wrong" direction (Brown, 1991).

 

In a 13-week study (Nº 3) using LAE in formulation, an apparent anomaly occurs, where there is a slight to moderate reduction in the white cell counts with only very minor visible damage to the stomach lining. However, this type of reaction is not uncommon in toxicological studies, nor in diagnostic work. For example, in a Cyanomolgus monkey study, the animals were bled twice a week for 8 weeks, as the bone marrow was likely to be the prime target organ. No treatment related changes were noted, but from week 4 onwards, on each occasion the animals were bled, one or two showed excessively high white counts, up to 40 - 60,000. These appeared at random, including controls, and subsequent samples invariably showed either normal results, or a return to normal (counts down to around 20,000). A pattern was discovered when the occurrence of these high counts was plotted against time, which showed that the first high count had occurred in an animal in a particular cagein the battery, and that the next ones were in adjacent cages (either laterally or vertically). This pattern was repeated over a 4 - 5 week period, ultimately affecting all animals, but the reason for these changes was never established (Brown, 1994 - from a case history used as part of the presentation). Similarly, in horses, a high Myeloid Left Shift Index (3.3 - 9.7) is normally diagnostic of a severe or acute infection, but one animal, with a Myeloid Left Shift Index of 15.3, showed no signs at all, either clinically, nor in any cultures (Brown, 1991).

Conversely, some animals show a reduced peripheral white cell count during infection (Chavatte et al, 1991). These cases illustrate the sensitivity of white cells in the peripheral blood (and/or in the marrow) to detect the beginnings of adverse changes (for example minor inflammation), either before they become clinically and/or pathologically identifiable, or if the stimulus is not sufficient. to induce further changes. In relation to the 13 -week study (Nº 3), these results suggest early pre-ulcerative or pre-irritation changes which, perhaps due to this particular formulation, have not progressed to positive damage. Since the only available bone marrow data (from the 52-week study) indicates that myeloid cell production was normal, any reduction in the numberof these cells in the peripheral blood was not due to the dynamics of production. The apparent lack of change or damage to the intra-cellular components of the myeloid cells, and no evidence of an increase in cell destruction, migration into the tissues appear to be the most likely cause of the reductions seen. It may prove difficult to demonstrate this point with another study, although taking marrow samples at the time of maximum white cell reduction may make things clearer.

It is clear that the experimental compound causes changes to the stomach and associated tissues, ranging from parakeratosis and epithelial hyperplasia to ulceration. Any of these conditions are likely to evoke a white cell response. Therefore it is not unreasonable to expect a correlation between white blood cell counts and histopathological lesions in the stomach and associated tissues. Review of the data from individual animals on the repeat dose toxicity studies does not however reveal a consistent correlation. This lack of correlation is considered to be due to the dynamic nature of such changes, which may present in different ways, depending on the severity of the insult as well as the time of onset compared with the time of blood sampling. However, ifthe interpretation of the results is based on a group basis, there is a better correlation between those levels where there are effects on the white blood cell counts and lesions in the stomach and associated tissues.

Thus, the white blood cell changes seen in these studies appear to be the result of a normal response to the changes induced to the stomach lining.

Reference 5

Endpoint:

repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2008-06-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: An expert review.

Conclusions:

Observed hematologic findings are not toxicologically significant. This conclusion is based on the following considerations:
1. The observed effects on the hemogram and particularly on the white blood cell parameters (both total and differential) are inconsistent both within and between studies.
2. Histopathological changes that would be expected if the hematologic changes represented toxicity were not observed in any study.

Executive summary:

Inconsistent and variable hematologic effects

The observed effects on the hemogram and particularly on the white blood cell parameters are inconsistent both within and between studies. Both the total white cell counts and the differential counts show this inconsistency. Furthermore, if any of the white blood cell effects observed in either of the 90-day studies were of toxicological importance, it would be expected that they would be more clearly reflected in the 52-week study and that was not the case.

In the Han Wistar rat 90-day study, the highest dose of ethyl lauroyl arginate was associated with a greater than 20% decrease in total white blood cells which could largely be explained by the decrease in lymphocytes. This effect was statistically significant in males but not in females. Changes of this degree of magnitude in various clinical pathology parameters have been observed in many National Toxicology Program toxicity studies with no clear explanation other than the inherent variability associated with hematology and clinical chemistry measurements, and by the fact that these measurements are based on small numbers of animals, usually a group size of 10.

In the 90-day Han Wistar study, there was considerable variability in the hemogram, with the mid-dose males showing elevated rather than decreased total white cell and lymphocyte counts. It is difficult to definitively ascribe a cause to the lowered white cell count in the high-dose males, but if it were a real toxicologic effect, it would be expected that a clear dose response would have been observed, which was certainly not the case in this study.

While the second 90-day study was conducted in Sprague-Dawley rats, it would be reasonable to expect a similar effect in high dose male rats as that seen in the Han Wistar study if the effect were a bona fide toxicologic response. This was not the case. Instead, the Sprague-Dawley males had a marginal (13%) and non-statistically significant decrease in total WBC count with reduced neutrophils rather than lymphocytes being mostly likely responsible. On the other hand, in the Sprague-Dawley study there was a statistically significant decrease in total WBC count in high dose females with decreased lymphocytes and monocytes contributing to this change. It is of interest to note that the variability in WBC counts in the control female rats is substantially lower thanthe variability in the treated groups of females.

In the 52-week chronic study conducted using Sprague-Dawley rats, there were two interim (14- and 26-week) hematology measurements in addition to those made at the conclusion of the study. Since toxicologic effects would be expected to be reflected in a temporal fashion that makes biological sense, this study design is important since it allows the monitoring of effects over time. There were no biologically, toxicologically, or statistically significant changes in total white cell counts in either males or females at the 14-week interim sampling. This time interval is similar to the Sprague-Dawley 90-day study hematology measurements and the doses in mg/kg/day were slightly higher in the 52-week study. One would thus have expected to observe a similar effect as that seen in the 90-day study. That was not the case. This inconsistency in response is noteworthy from a toxicological viewpoint.

What was evident in the 14-day interim hematology data, however, was a statistically significant reduction in monocytes and LUC (large unstained cells). In the absence of a significant shift in the total WBC count, the monocyte and LUC effect is not of particular concern, but I will address this more specifically in the following section of this report.

The 26-week interim hematology data from the 52-week chronic study showed a statistically significant decrease in total WBC count in all dosed males with the primary contribution attributable to a decrease in lymphocytes. A similar effect in the mid- and high-dose females was also present. This represents the most compelling effect in the hemogram among the studies with a clear dose response in the females. A decrease in monocytes and LUC is also evident although, given the relatively minor component of these in the total white blood cell differential, they really do not make a tangible contribution to the decreased total WBC count. While there is substantial variability in the WBC counts in each group, the variability is similar among the groups. However, as discussed below, this effect did not persist and, therefore, is unlikely to be toxicologically relevant.

The 52-week hematology data show a statistically significant decreased WBCcount in high dose males, primarily attributed to a decrease in neutrophils. The females have a non-statistically significant decrease in total white cell count, also primarily attributable to neutrophils. The variability in the neutrophil counts is exceedingly high when looking at the group standard deviations. This raises some concern about the validity of the neutrophil measurement. What is particularly noteworthy, however, is the fact that there is no clear dose response in the WBC count at 52 weeks aswas seen at 26 weeks. Furthermore, the decrease in the WBC counts at 26 weeks was attributable primarily to lymphocytes while the reduced WBC count at 52-weeks is primarily attributable to a highly variable neutrophil component. As in the earlier samplingtime points, in both genders there are statistical flags associated with decreases in monocytes and LUC, neither of which tangibly contribute to the white cell count.

Absence of toxicologically appropriate responses

If the hematologicalchanges observed in the various rat ethyl lauroyl arginate toxicity studies were toxicologically relevant, one should observe a clear dose-response, a consistent contributory cell type responsible for any change in total WBC count, and appropriate bone marrow changes indicative of compound-related hematotoxicity sufficient to reduce the WBC count (Weiss, Toxicologic Pathology 21:135-140,1993). However, the bone marrow histology was normal in both sexes, at all dose levels, and in all studies. Furthermore,bone marrow cytology conducted at the end of the 52-week study was normal and showed no treatment-associated differences. Thus, the lower but variable WBC counts cannot be attributed to hematotoxicity in the progenitor populations of most of the leukocytescomprising the white blood cell differential counts.

With respect to lymphocytes, any decrement in the circulating lymphocytes of toxicological significance would be expected to originate in the various lymphoid organs where lymphocytes are produced. There were no histopathological changes in the thymus, lymph nodes or spleen observed in either sex or at any dose in the anatomic pathology evaluations in these studies. Even if treatment-related stress attributable to ethyl lauroyl arginate or decreased foodconsumption were a factor, lymphocytolysis at least in the thymic cortex would be expected as it is the most sensitive metric for assessing central causes of decreased circulating lymphocytes. Furthermore, stress-related changes would have cause consistent alterations in the adrenal glands, a finding not seen in the anatomical pathology results from the various studies.

Ruling out decreased food consumption, which is known to affect WBC counts (Levine et al., Toxicologic Pathology 21: 1-14,1993), increased stress, which also can affect WBC counts, and bone marrow cytoxicity, two other possible explanations for the white blood cell hemogram changes are theoretically possible. One is viral infection, which is highly unlikely based on the study findings and the reputation of the laboratory conducting the studies and the long interval between the studies. The other possibility is that there was a decreased demand for leukocytes based upon a bactericidal reduction in microorganisms attributable to the biologicalproperties of ethyl lauroyl arginate. That is difficult to prove in the absence of good published literature on that topic but is plausible if we take our cues from the livestock and poultry industries where bacteriocidal and bacteriostatic antibiotic dietary supplementation enhances growth rates, presumably by reducing potentially harmful opportunistic microorganisms in the body. While the opinion provided in the SCCP report suggesting that the decreased white blood cell count was linked to forestomach lesions is possible, it is equally reasonable to expect that forestomach inflammation would produce an increased white blood cell count with an increased number of neutrophils.

As was pointed out above, in several sampling intervals there were decreases in monocytes and LUC. While the monocyte numbers contributing to the total white blood cell count are too low to have made a tangible contribution, their decrease of in

terest.

In reviewing the publications on veterinary hematology and results in the National Toxicology Program database, and after consultation with the current veterinary hematologist at the National Toxicology Program, it appears that there are no known instances where a decrease in circulating monocytes has been considereda toxicologically significant response in the absence of bone marrow depression or severe immunodepression. With respect to the decreases noted in large unstained cells (LUC), this measurement is a reflection of two methods related to how the hematology analyzer does differential cell determinations. One involves light scatter and nuclear complexity and the other involves peroxidase staining and cell size. Monocytes are weakly peroxidase positive and very large and are easily identified by the analyzer. Lymphocytes are peroxidase negative and are easily segregated and counted as small unstained cells. Neutrophils are peroxidase positive. Basophils segregate with lymphocytes but can be identified by the nuclear complexity method. The LUC are variant or atypical lymphocytes or blast cells. They are typically very low in number and if elevated, one should review the smear to identify atypical lymphocytes, reactive lymphocytes, or blast cells. Since the LUC results were not elevated but rather were depressed in the ethyl lauroyl arginate toxicity studies, the reduced numbers are not considered biologically relevant.

The high variability in observed hematological responses is characteristic of hematology results often seen in rodent toxicity studies and is typically attributed to the small numbers of animals sampled. Also, there is absence of associated histopathology in hematopoetic and lymphoreticular tissues to support a hematotoxic effect of ethyl lauroyl arginate in rats.

Reference 6

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 22, 2000 to July 27, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

GLP

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not applicable

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: 42 days
- Weight at study initiation: 100-138 g after 7 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: 14 days prior to study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca 21 ºC
- Humidity (%): 55%
- Air changes (per hr): 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light: 12 hour dark cycle

IN-LIFE DATES: From: March 22, 2000 To: May 1, 2000

Route of administration:

oral: feed

Vehicle:

other: ground diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The LAE was incorporated into the ground diet. 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 fine basal diet and subsequently mixed in a Turbula mixer. The final concentrations were achieved by diluting aliquots of the premix with an appropriate amount of basal diet and mixing in a Turbula mixer. The unused diet at the end of each week was discarded.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly to the animal unit.
- Mixing appropriate amounts with (Type of food): Mixed with sieved basal diet until half the premix weight was achieved.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Detailed records of substance usage were maintained. The amount of test substance necessary to prepare the test diets and the quantity actually used were determined on each occasion that diet was mixed. The difference between these amounts was checked before the diets were dispensed.

A record of the weight of each diet dispensed and the amount remaining after it had been supplied to the cages was made. The balance was compared with the predicted usage as a check that the diet had been administered correctly. No significant discrepancy was found.

Duration of treatment / exposure:

All animals were treated for at least 28 days

Frequency of treatment:

Daily

Dose / conc.:

25 000 ppm

Remarks:

Nominal in diet

Dose / conc.:

37 500 ppm

Remarks:

Nominal in diet

Dose / conc.:

50 000 ppm

Remarks:

Nominal in diet

No. of animals per sex per dose:

5 animals per sex per group (dose)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: To simulate the conditions of human exposure

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: inspected daily for evidence of animal ill-health, such as blood or loose faeces.

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: During the acclimatisation period, on the day that treatment commenced, at the end of Week 1, twice weekly for Weeks 2-4, and before necropsy. On Day 27 because of clinical pathology procedures on Day 28.

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. For each week of treatment.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 4 weeks of treatment
- Anaesthetic used for blood collection: Yes (Isoflurane anaesthesia)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked examined: Haematocrit (Hct); Haemoglobin concentration (Hb); Erythrocyte count (RBC); Total and differential leucocyte count (WBC); Platelet count (Pit); Mean cell haemoglobin (MCH); Mean cell volume (MCV); Mean cell haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 4 weeks of treatment
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: Alkaline phosphatase (Alk. Phos), Alanine amino-transferase (ALT), Aspartate amino-transferase (AST), Glucose (Gluc), Total bilirubin (Bili. Total), Total cholesterol (Chol Total), Urea , Total protein (Total Prot) , Electrophoretic protein fractions [Alb (Albumin), al Glob (a-one globulin), a2 Glob (a-two globulin), Beta Glob (P globulin) and Gamma Glob (y glo bulin)], Albumin/globulin ratio (A/G), Sodium (Na), Potassium (K) and Chloride (CI) concentrations , Calcium concentration (Ca Total), Inorganic phosphorus (Phos)

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. 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 external and cut surfaces of the organs and tissues were examined as appropriate. Abnormalities were noted and the required tissue samples preserved in fixative.

HISTOPATHOLOGY: Yes.
Tissues preserved for histopathology:
Adrenals, Brain, Caecum, Colon, Duodenum, Epididymides, Head, Heart, Ileum, Jejunum, Kidneys, Lungs (including bronchi), Lymph nodes (mandibular, mesenteric), Liver, Oesophagus, Ovaries, Rectum, Sciatic nerves, Spinal cord, Spleen, Stomach, Testes, Thymus, Thyroid with parathyroids, Trachea, Urinary bladder, Uterus with cervix.

Other examinations:

Organ weights: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Lungs (including bronchi), Ovaries, Spleen, Testes, Thymus, Thyroid with parathyroids, uterus with cervix.

Statistics:

The significance of inter-group differences in haematology and blood chemistry was assessed by Student's t-test using a pooled error variance. Statistical significances for eosinophil, basophil, monocyte and large unstained cell counts are not reported as these data are not normally distributed.

Bodyweight changes and organ weights were assessed by Student's t-test using a pooled error variance.

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

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):

(All females receiving 50.000 ppm had piloerection and an ungroomed coat. Salivation was seen in all females and most males receiving 50.000 ppm).

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(The overall gain (Days 0 - 27) for treated males was low compared with that of the Controls; males receiving 50.000 ppm were markedly affected. The overall gain for females was similar to or higher than that of the Controls.)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

(The overall intake was low for treated animals; males showed a dosage-related depression of food intake while females all had similar low intakes.)

Food efficiency:

no effects observed

Description (incidence and severity):

(The overall food conversion efficiencies for treated males were superior to that of the Controls.)

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
There were no deaths.
All females receiving 50.000 ppm had piloerection and an ungroomed coat; two females receiving 37.500 ppm also had an ungroomed coat. Salivation was seen in all females and most males receiving 50.000 ppm. Brown staining of the muzzle was evident for most animals in each treated group.

BODY WEIGHT AND WEIGHT GAIN
During the first week of treatment, animals receiving 50.000 ppm lost or did not gain any weight, while animals receiving 25.000 or 37.500 ppm gained significantly less weight than the Controls; dosage relationship was apparent. During the next three days i.e. up to Day 10, males receiving 37.500 or 5.000 ppm gained weight but this was still less than the Controls. Conversely, during the same period, females receiving these levels gained significantly more weight than their controls. The weight gain from Day 7 to 27 for treated males was similar to that of Controls and for treated females was higher than Control values; the gain for females given 37.500 or 50.000 ppm was markedly high. The overall gain (Days 0 - 27) for treated males however was low compared with that of the Controls; males receiving 50.000 ppm were markedly affected. The overall gain for females was similar to or higher than that of the Controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
During the first week of treatment, food consumption was markedly low for animals receiving 50.000 ppm and low for animals receiving 25.000 or 37.500 ppm; dosage-relationship was apparent for each sex. During the remaining three weeks of treatment, treated animals continued to eat less food than the Controls with the exception in Week 2 for treated females which consumed a similar amount of diet. The overall intake was low for treated animals; males showed a dosage-related depression of food intake while females all had similar low intakes.

FOOD EFFICIENCY
During the first week of treatment, food conversion efficiency was incalculable for males receiving 50.000 ppm as they lost weight, markedly low for females receiving this level and slightly low for males receiving 37.500 ppm. For the remaining three weeks of treatment, food conversion efficiencies for all treated animals were similar to or greater than the Controls; females receiving 50.000 ppm had distinctly superior efficiencies. With the exception of Week 1 data, the overall food conversion efficiencies for treated males were superior to that of the Controls.

HAEMATOLOGY
Haematology investigations after 4 weeks of treatment (just before necropsy) did not show any significant findings clearly related to treatment. The differences from Control group that were apparent were marginal and comprised slightly high haemoglobin concentration, mean cell haemoglobin and volume for females receiving 50000 ppm. There was however, considerable variation of individual values within each group. Other inter-group differences were considered minor and not related to treatment.

CLINICAL CHEMISTRY
Blood chemistry investigations after 4 weeks of treatment showed compared to Controls, slightly low total protein concentration for treated males, and low albumin and calcium concentrations for males receiving 37.500 or 50.000 ppm. Slightly high alkaline phosphotase, alanine amino-transferase and aspartate amino-transferase activities were evident for females receiving 50.000 ppm. Females receiving 37.500 ppm was had a slightly high aspartate amino-transferase and alanine amino¬transferase activities. These changes were, however, only evident for a few females of this group.

ORGAN WEIGHTS
Organ weight analysis for animals killed after 4 weeks of treatment did no reveal any findings related to treatment. The differences from Controls that were evident were considered to be related to the low bodyweights rather than to treatment.

GROSS PATHOLOGY
There were no macroscopic findings considered to be related to treatment.
Organ weight analysis for animals killed after 4 weeks of treatment did no reveal any findings related to treatment. The differences from Controls that were evident were considered to be related to the low bodyweights rather than to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

50 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose rated

Key result

Critical effects observed:

no

Conclusions:

A total of 25 males and 25 female Han Wistar rats received Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) in concentrations of: 25.000 ppm, 37.500 ppm and 50.000 ppm during a 4 week treatment period. No deaths were detected. Administration of LAE in the diet at levels of 50.000 ppm over a four-week period were palatable and tolerated. Marked effects on bodyweight gain, food consumption and food conversion efficiency were evident during Week 1 but recovery was seen during the remaining period of treatment. Clinical pathology changes were confined to slight effects in the liver. A high level of 50.000 ppm was considered suitable for the forthcoming 13-week study.

Executive summary:

The administration of LAE in the diet at concentrations of up to 50.000 ppm was generally well tolerated over a four-week treatment period.

 

There were marked depression/lack of bodyweight gain, food consumption and food conversion efficiencies during the first week of treatment for animals receiving 37.500 or 50.000 ppm. Scatter, which is indicative of animals finding the diet unpalatable, was also high during Week 1. This was, however only transient as from Week 2, a recovery in growth parameters was evident. Recovery over the remaining three weeks was not sufficient to influence the overall values that reflected the changes seen in Week 1. It is considered that full recovery would be evident over a period greater than four weeks. The piloerection and ungroomed coat are indicators of general poor health. The salivation is a sign considered to be associated with the unpleasant taste of the treated diet and the brown staining is almost certainly the dried salivation.

 

Clinical pathology findings were indicative of changes in the liver. These comprised the low protein and albumin concentrations formales receiving 37.500 or 50.000 ppm and high enzyme activities in females receiving 50.000 ppm. The high enzyme activities are a common indicator of liver damage, while the protein changes indicate an effect on function. There were however, no weight changes or findings at macroscopic examination of the liver.

 

The treated diets were clearly unpalatable during Week 1 as indicated by the low bodyweight gain, food intake and food conversion efficiency. From Week 2 there was recovery in these parameters but the two sexes showed a difference in that the males continued to gain less weight than their Controls but conversely females gained more weight. In a forthcoming 13-week study, a high level of 50.000 ppm would be considered acceptable, as over a longer term, this was palatable and tolerated.

Findings in this study were evident at all levels and a low level for the subsequent study would have to identify a No-Observed -Effect -Level (NOEL). Therefore based on the findings of this study, levels of 5.000, 15.000 and 50.000 ppm are suggested in the forthcoming study.

Reference 7

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.

 

Reference 8

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 7, 1995 to December 14, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD BR

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: 6 weeks
- Weight at study initiation: 173 g to 207 g for males and 137 g to 163 g for females
- Housing: 5 per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days prior to study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-23 ºC
- Humidity (%): 46-72%
- Photoperiod (hrs dark / hrs light): 12-hour light: 12 hour dark cycle

IN-LIFE DATES: From: June 30, 1995 To: July 27, 1995

Route of administration:

oral: feed

Vehicle:

other: ground diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
A pre-mix was prepared each week by griding the test substance directly into SDS Rat and Mouse No. 1 maintenance diet and mixing in a Turbula mixer for a miriimum period of 5 minutes. The required concentrations were then prepared by direct dilution of the pre-mix with further quantities of untreated diet; homogeneity being achieved by mixing in a Turbula mixer for a minimum period of 5 minutes.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly to the animal unit.
- Mixing appropriate amounts with (Type of food): Mixed with sieved basal diet until half the premix weight was achieved.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

All animals were treated for at least 28 days

Frequency of treatment:

Daily

Dose / conc.:

3 200 ppm

Dose / conc.:

12 800 ppm

Dose / conc.:

50 000 ppm

No. of animals per sex per dose:

5 animals per sex per group (dose)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: To simulate the conditions of human exposure

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Dated and signed records of all activities relating to the day by day running and maintenance of the study within the animal unit as well, as to the group observations and examinations outlined in this procedure, were recorded. In addition, observations relating to individual animals made throughout the study were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
Individual animals were observed at least once daily for any signs of behavioural changes, reaction to treatment or ill health. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals. Further checks were made early in each working day and again in the afternoon to look for dead or moribund animals. On Saturdays and Sundays a similar procedure was followed except that the final check was carried out at approximately mid-day.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded at the time of allocation of animals to groups, on the day of commencement of treatment and once a week thereafter.

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 quantity of food consumed by each cage of rats was recorded on a weekly basis. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by the cage in each group and the number of rats surviving in that cage.

FOOD EFFICIENCY:
- Body weight gain: Yes. Food consumption ratios were calculated, over the period Weeks 1 to 4 from the bodyweight and food consumption data as weight of food consumed per unit gain in bodyweight. Food conversion ratio = Food consumed/Bodyweight gain.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Compound intake Time schedule for examinations: At weekly intervals the group mean achieved intake of test substance (mg/kg/day) was calculated from the group mean bodyweight and food consumption and the dietary inclusion levels of the test substance.
- Water consumption time schedule for examinations: Daily monitoring by visual appraisal of the water bottles was maintained throughout the study. No apparent treatment-related effects were noted and, as such, accurate measurement of water consumption was not instigated.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 4 weeks of treatment
- Anaesthetic used for blood collection: Yes (Ether anaesthesia)
- Animals fasted: Yes. Overnight.
- How many animals: All animals
- Parameters checked examined: Packed cell volume (PCV); Haemoglobin (Hb); Red cell count (RBC); Mean corpuscular haemoglobin concentration (MCHC); Mean corpuscular volume (MCV); Mean corpuscular haemoglobin (MCH); Platelet count (Plts); Total White cell count (WBC total); Neutrophils (N); Lymphocytes (L); Eosinophils (E); Basophils (B); Monocytes (M); Large unstained cells (LUC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 4 weeks of treatment
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: Glucose (Hexokinase mediated assay); Total protein; Albumin (Alb); Globulin (Glob): by subtraction; Total protein minus Albumin; Urea nitrogen (Urea Nitr); Creatinine; Alkaline phosphatase (AP); Glutamic-pyruvic transaminase (GPT); Reaction temperature 30°C; Glutamic-oxaloacetic transaminase (GOT); Total bilirubin (Bili-rubin); Sodium (Na); Potassium (K); Calcium (Ca); Inorganic Phosphorus (P); Chloride (CI); Total cholesterol (Choi) - (enzymatic assay).

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted, with due attention to the thymus, lymph nodes and heart. The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastrointestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable Ulumination. The liver was sectioned at intervals of a few millimetres; the kidneys were incised and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intraabdominal lymph nodes and accessory reproductive organs were recorded.

HISTOPATHOLOGY: Yes.
Tissues preserved for histopathology: Samples of all the tissues listed from all animals were preserved:
adrenals; alimentary tract (oesophagus, stomach duodenum, jejunum, ileum, caecum, colon and rectum); aorta; brain (medullary, cerebellar
and cerebral sections) epididymides eyes; femur (with joint) Harderian gland head (to preserve nasal cavity, paranasal sinuses, oral cavity, nasopharynx, middle ear, teeth, lachrymal gland and Zymbal's gland); heart kidneys; larynx and pharynx liver; lung (all lobes and mainstem bronchi); lymph nodes (cervical and mesenteric); mammary gland; other macroscopically abnormal tissue; ovaries; pancreas; pituitary; prostate; salivary gland; sciatic nerve; seminal vesicles; skeletal muscle skin; spinal column (to preserve samples of spinal cord from the cervical level); spleen; sternum (for bone
and marrow) testes; thymus (where present) thyroids (with parathyroids) tongue trachea; urinary bladder; uterus (corpus and cervix); vagina.

Other examinations:

The following organs from all animals killed at the scheduled sacrifice were dissected free of fat and weighed, paired organs were weighed separately: adrenals brain, epididymides, heart, kidneys liver, ovaries, pituitary, prostate and seminal vesicles.

Organ weights: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, spleen, testes, thyroid, uterus.

Statistics:

The significance of inter-group differences in haematology and blood chemistry was assessed by Student's t-test using a pooled error variance. Statistical significances for eosinophil, basophil, monocyte and large unstained cell counts are not reported as these data are not normally distributed.

Bodyweight changes and organ weights were assessed by Student's t-test using a pooled error variance.

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

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:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY:
There were no unscheduled deaths during the study.
There were no treatment-related clinical signs noted throughout the study.

BODY WEIGHT AND WEIGHT GAIN
Overall mean bodyweight gain for all treated groups was comparable with or marginally higher than concurrent controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Cumulative mean food intake was generally similar for all treated groups in comparison with concurrent controls.

FOOD EFFICIENCY
Efficiency of food utilisation, as determined by food conversation ratios, for all treated groups was generally similar to concurrent controls.

HAEMATOLOGY
Haematological investigations performed during Week 4 did not reveal any differences between treated groups and concurrent controls considered to be of toxicological significance although statistical significance was attained in some instances.

CLINICAL CHEMISTRY
Biochemical analysis of specific parameters in blood was performed during Week 4; it did not reveal any differences between treated groups and concurrent controls considered to be of toxicological importance.

ORGAN WEIGHTS
Analysis of organ weight data after 4 weeks of treatment did not reveal any differences between the control and treated groups considered to be of toxicological significance, although occasional statistical significance was obtained.

GROSS PATHOLOGY
Macroscopic examination of animals sacrificed after 4 weeks of treatment did not reveal any lesions considered to be attributable to treatment with all findings falling within the expected background range of macroscopic changes.

Key result

Dose descriptor:

NOAEL

Effect level:

1 027.45 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: For the test substance: LAE

Key result

Dose descriptor:

NOAEL

Effect level:

1 139.97 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: For the test substance: LAE

Key result

Critical effects observed:

no

Conclusions:

The toxicity of the test substance was studied when administered to groups each of 5 male and 5 female of rats by dietary administration over a period of 4 weeks and to provide guidance in the choice of dosages for a subsequent 13-week toxicity study. There were no treatment-related findings at any dosage level and thus the no-observed effect level in this study of 4 weeks duration was 50.000 ppm, equivalent to an average intake of 5.269 mg/kg bw/day in males and 5.846 mg/kg bw/day in females (1027.45 mg/kg bw/day of LAE in males and 1139.97 mg/kg bw/day of LAE in females).

It was thus concluded that since 50.000 ppm is nominal maximum dietary inclusion level which can be used without compromising the nutritional quality of the diet, a dosage of 50.000 ppm would be acceptable for studies of longer duration in the rat.

Executive summary:

The objective of this study was to assess the toxicity of the test substance a food additive, to rats by dietary administration over a period of 4 weeks and to provide guidance in the choice of dosages for a subsequent 13-week toxicity study.

There were no treatment-related findings at any dosage level and thus the no-observed effect level in this study of 4 weeks duration was 50.000 ppm, equivalent to an average intake of 5.269 mg/kg bw/day of the test substance (1027.45 mg/kg bw/day of LAE) in males and 5.846 mg/kg bw/day (1139.97 mg/kg bw/day of LAE) in females.

 

It was thus concluded that since 50.000 ppm is the maximum dietary inclusion level which can be used without compromising the nutritional quality of the diet, a dosage of 50.000 ppm would be acceptable for studies of longer duration in the rat.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

307 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

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

System:

gastrointestinal tract

Organ:

stomach

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Studies with the test substance LAE:

- Subacute study: 28 days. Key study 1.

Administration of LAE in the diet at levels of 50.000 ppm over a four-week period were palatable and tolerated. Marked effects on bodyweight gain, food consumption and food conversion efficiency were evident during Week 1 but recovery was seen during the remaining period of treatment. Clinical pathology changes were confined to slight effects in the liver. A high level of 50.000 ppm was considered suitable for the forthcoming 13-week study.

- Subchronic study: 90 days. Key study 2.

According to OECD 408 Guideline, 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 (or 384, 1143, 3714 mg/kg/day for males receiving 5.000, 15.000 or 50.000 ppm respectively and 445, 1286 and 3915 mg/kg/day for the equivalent female groups).  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 15.000 or 50.000 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.

- Chronic study, 52 weeks. Key study 3.

Based on the calculated intake data, the NOAEL in this study was 6000 ppm equivalent to 307 mg/kg bw in the males and 393 mg/kg bw 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.

Studies with 19.5% LAE:

- Subacute study: 28 days. Key study 4. (19.5% LAE)

There were no treatment-related findings at any dosage level and thus the no-observed effect level in this study of 4 weeks duration was 50.000 ppm, equivalent to an average intake of 5.269 mg/kg bodyweight/day in males and 5.846 mg/kg bodyweight/day in females (1027.45 mg/kg bw/day in terms of LAE for males and 1139 mg/kg bw/day for females). 

- Subchronic study: 90 days. Key study 5. (19.4% LAE)

According to OECD 408 Guideline within the context of this study, evidence of toxicity was noted amongst male and female rats receiving 50.000 ppm dose concentration. Reduced bodyweight gains were noted for females of the highest dose treated groups however, since no similar finding was apparent for males and no other evidence of toxicity was apparent, it was concluded that the no-effect level for continuous administration to rats for 13 weeks was likely to be 12.800 ppm, which is equivalent to 904 mg/kg bw/day (175 mg/kg bw/day in terms of LAE for males) and 1067 mg/kg bw/day (207 mg/kg bw/day in terms of LAE for females)

Justification for classification or non-classification

According to CLP Regulation and the available data, the substance under study does not meet the criteria to be classified because Oral: NOAEL>100mg/kg bw/day.