D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C18-C18 (unsaturated) acyl] derivs.

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-11-21 to 2014-11-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(6E)-N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadec-6-enamide; N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadecanamide

EC Number:

940-373-5

Cas Number:

1591782-99-8

Molecular formula:

C25H49NO6 (C18 unsatd.) C25H51NO6 (C18 satd.)

IUPAC Name:

(6E)-N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadec-6-enamide; N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadecanamide

Test material form:

other: Waxy solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System:
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 8-9 weeks old, females: 8-9 weeks old.
Body weight at the allocation of the animals to the experimental groups:
males: 217 - 283 g (mean: 250.60 g, ± 20% = 200.48 – 300.72 g)
females: 154 - 183 g (mean: 170.83 g, ± 20% = 136.67 – 205.00 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on
Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions:
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0801)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological
controls at regular intervals)
- The animals were housed in groups (5 animals/sex/cages) in type IV cages, polysulphone cages on Altromin saw fibre bedding
(lot no. 030713 and 260913)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE.
- Adequate acclimatisation period (at least five days)

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. None of the animals showed
any adverse clinical signs. Before the first administration all animals used for the study were weighed and assigned to the experimental
groups with achieving a most homogenous variation in body weight throughout the groups of males and females.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The doses for this study were chosen by the study monitor based on repeated dose toxicity study with the analogue test substance.
The doses used for the study with analogue test substance were 100, 250 and 625 mg/kg body weight/ day.
Similar dose levels were chosen for this study after discussion. The following doses were selected for the 3 dose groups
(LD = low dose, MD = medium dose, HD = high dose). The animals were treated with the test item formulation or vehicle on 7 days per week
for a period of 28 days. 5 animals per gender and group were subjected to necropsy one day after the last administration (end of treatment period).
5 animals per gender of the C and of the HD group were subjected to necropsy 14 days after the last administration (end of recovery period).

Control: 0 mg/kg body weight
Low Dose: 125 mg/kg body weight
Medium Dose: 300 mg/kg body weight
High Dose: 650 mg/kg body weight

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence
of dose levels was selected with a view to demonstrate any dose related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume
as used for the high dose group.

Administration of Doses
The test item and control formulation were administered at a single dose to the animals by oral gavage at an application volume of
5 mL/kg bw. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For determination of the concentration of test item in dosing formulations, samples were retained from all groups once weekly during the
treatment period and stored between -15 and -35 °C.
Stability of the dosing formulations was tested once at the beginning of the treatment period. From the low and high dose group samples of dosing
formulations were frozen at 0 hours and 6 hours after the preparation and stored at -15 and -35 °C.
In the 1st and 4th week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared
high and low dose formulations and stored between -15 and -35 °C.
Each sample was retained twice (sample A, sample B, each of at least 5 mL).
At the end of the treatment period all A samples of dosing formulations were analysed at BSL BIOSERVICE Scientific Laboratories GmbH in
accordance with GLP under the reference number 136165.
The B samples were retained at BSL until the analysis had been performed, and will be discarded after completion of the final study report.

Duration of treatment / exposure:

The animals were treated with the test item or vehicle on 7 days per week for a period of 28 days.

Frequency of treatment:

The animals were treated once daily.

Doses / concentrations

Remarks:

Doses / Concentrations:
125 mg/kg bw, 300 mg/kg bw and 650 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

60 animals (30 males and 30 females) were included in the study (5 male and 5 female animals per group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: random based on body weight
- Section schedule rationale : alle male animals together and all female animals together

Examinations

Observations and examinations performed and frequency:

Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and
weekly during the treatment and recovery period.
Food consumption was measured weekly during the treatment and recovery period.

Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 28 days. The recovery animals were observed for an
additional period of 14 days following the last administration. General clinical observations were made once a day, preferably at the same
time each day after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and
mortality except on weekends and public holidays when observations were made once daily.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia,
vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made
outside the home cage in a standard arena once before the first administration and once a week thereafter.
Ophthalmological examination, using an ophthalmoscope was made on all animals before the first administration and in the last week of
the treatment period as well as at the end of the recovery period in the recovery animals.

Functional Observations
Once before the first exposure and once in the fourth week of exposure as well as in the last week of the recovery period multiple detailed
behavioural observations were made outside the home cage using a functional observational battery of tests. These tests were conducted
in all animals.

Haematology
Haematological parameters (haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV),
mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT),
white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso) and large unstained cells)
were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.

Blood Coagulation
Coagulation parameters (prothrombin time (PT), activated partial thromboplastin time (aPTT)) were examined at the end of the treatment
and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes.

Clinical Biochemistry
Parameters of clinical biochemistry (alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP),
creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc),
sodium (Na), potassium (K)) were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.

Urinalysis
A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals.
The following parameters (specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood,
leukocytes) were measured using qualitative indicators (Heiland Urine Stripes URI 10SL). Additionally, urine colour/ appearance were recorded.

Sacrifice and pathology:

Pathology
One day after the last administration (study day 29) all animals of the treatment period and 2 weeks after the last administration all animals
of the recovery period (study day 43) were sacrificed using anesthesia (ketamine, Pharmanovo, lot no: 24664, expiry date: 30/06/2015 and xylazin,
Serumwerk, lot no. 00512, expiry date: 07/2014) and subjected to a detailed gross necropsy which includes careful examination of the external
surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ Weight
The wet weight of the organs (liver, uterus with cervix, kidneys, thymus, adrenals, thyroid/ parathyroid glands, testes, spleen, epididymides,
brain, prostate, seminal vesicles and coagulating glands, pituitary gland, ovaries,heart) of all sacrificed animals was recorded as soon as possible.
Paired organs were weighed together.

The following tissues (brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), eye, uterus with cervix (females),
liver, vagina (females), kidneys, testes (males), adrenal glands, epididymides (males), stomach, prostate and seminal vesicles with
coagulating glands as a whole (males), small and large intestines (including Peyer´s patches), urinary bladder, thymus, lymphnodes
(mesentric and axillary), thyroid glands, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, spleen, sternum with bone marrow,
lung and trachea, pituitary gland, mammary glands, oesophagus, skin) from all animals were preserved in 10% neutral buffered formalin
except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were
transferred to 70% ethanol.

Histopathology
The afore-listed organs (see above) were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining
for the animals of the groups 1 and 4 sacrificed at the end of the treatment period.
Since microscopic changes that could be attributed to treatment with the test item were not observed in any animals of HD group in the
initial histopathologic examination, further histoprocessing and histopathology evaluation were not performed.
Any gross lesion macroscopically identified was examined microscopically in all animals.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath Services,
Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the
GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The Study
phase from test site 1 and 2 was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005
[SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed
according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of
compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a
pathology phase report to the study director upon the completion of the study.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical
biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the
main groups using a one-way ANOVA and a post-hoc Dunnett Test. Statistical comparisons of data acquired during the recovery period were
performed with a Student’s t-Test. These statistics were performed with GraphPad Prism V.6.01 software or IDBS E-WorkBook 9.4.0 software
(p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

Mortality:

mortality observed, treatment-related

Description (incidence):

no adverse effects

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

no relevance to test item treatment

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality
No mortality occurred in the control or any of the dose groups during the treatment and the recovery periods of this study.

Clinical Observations
All male and female animals of MD and HD groups showed signs of salivation and moving the bedding, which were considered to be due to test
item treatment. These clinical signs were only observed immediately post dose administration, which suggests no systemic effect but more likely
a local effect. There were also isolated signs of diarrhea (male=C 1/5, HD 2/5, RHD 1/5; female= HD 1/5, RC 1/5), nasal discharge
(male= LD 1/5, RC 1/5; female= C 1/5), piloerection (male= HD 1/5), eschar (male= C 2/5, RC 2/5; female= RHD 2/5) and alopecia (female= MD 1/5).
These clinical signs were transient in appearance and were considered to have no toxicological relevance.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.
There were no ophthalmoscopic findings in any of the animals of this study.

Functional Observation Battery
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.
There were no biologically relevant differences in body temperature between the groups.

Body Weight Development
In both males and females, there were no changes of toxicological relevance noted in test item treated groups when compared to the corresponding
control groups during the treatment and the recovery periods. There were no statistically significant changes between the test item treated and
the control groups. The mean body weight increased with the progress of the study in the C, LD, MD and HD groups.
There were several transient changes (increase or decrease) in body weight gain without showing statistical significance in all dose groups during
the treatment and recovery period when compared to the corresponding control groups. But these changes were not considered to have any
biological relevance.

Food Consumption
In males and females, there were no changes of toxicological relevance for food consumption in the test item treated groups during the treatment
and the recovery period.
In females, the food consumption was higher during the treatment days 1-8, 15-22, which affected the cumulative food consumption of treatment
days 1-28 in LD, MD and HD groups when compared to corresponding control. This difference in food consumption was transient and had no
relevance to treatment.

Haematology and Blood Coagulation
In both males and females, there were no statistically or biologically significant changes of toxicological relevance noted for the measured
haematological and blood coagulation parameters. In males at the end of treatment there were moderately higher mean eosinophil values in LD, MD
and HD groups when compared to control. These differences were not statistically significant and had no dose response relation. At the end of
recovery there were slightly lower, not statistically significant, mean neutrophil and eosinophil values in male HD group. These differences were
due to higher value in single isolated males (animal 24 for neutrophil and animal 22 for eosinophil) of recovery control group (aberrant control)
and thus were not considered to be toxicologically relevant.
In females, there was marginally higher mean WBC count noted in LD, MD and HD groups. These differences were statistically insignificant and in
addition, the mean WBC of HD group was comparable to the mean WBC of recovery control group. Therefore, the differences were considered to
have no biological relevance.

Clinical Biochemistry
In both males and females, there were no adverse changes of toxicological relevance noted for the measured clinical biochemistry parameters.
However, there was statistically significantly higher mean AP value in male LD group, but there was no dose- response relation noted. There was
also statistically significantly lower mean ASAT in female MD and HD groups and mean TP in female HD group when compared to the corresponding
control. The decrease in TP value of HD group correlated to the slightly but not statistically significantly lower mean ALB of HD group.
Furthermore, in males at the end of treatment there were marginally higher mean AP in MD and HD groups and mean TBA in LD, MD and HD groups,
compared to controls. These changes were statistically insignificant and had no dose- response relation. At the end of recovery there was a higher,
not statistically significant, mean TBA value in HD group when compared to corresponding control.
In females, at the end of treatment there was marginally higher and lower, not statistically significant, mean TBA and Na, respectively in HD group
when compared to controls. There was also higher mean AP value in Recovery HD group.
The ALAT, ASAT, AP, TBA, Crea and Na values in main group animals (control and dose groups) were lower when compared to recovery groups
(control and dose groups). Although there were differences in clinical biochemistry parameters (mentioned above), these were not considered to
have adverse toxicological relevance due to the absence of any associated macroscopic or microscopic findings in liver and kidney.

Urinalysis
In males at the end of recovery higher erythrocyte level were found in 2 males (no.s 28 and 30) of recovery HD group. There were marginally
higher leukocyte level in 2 males (no.s 26 and 30) and marginally higher bilirubin, ketamine and protein levels in 1 male (no. 26) of recovery HD
group. In females at the end of treatment there was higher erythrocyte level in 1 female (no. 41) of MD group. There were also marginally higher
ketamine, erythrocyte and leucocyte value in 1 female (no. 46) of HD group. These differences were in comparison to concurrent control group.
In the absence of any associated pathological signs, the differences in males and females were not considered to be relevant to treatment.
Besides, all urinary parameters were in the normal range of variation and no noticeable differences between test item treated and control group
were observed.

Pathology
There were no gross lesions that could be attributed to treatment with the test item.
At the end of treatment, yellow focus on the epididymis was found in 2/ 5 control and 2/5 LD group males. Fluid distended uterus/ cervix was
found in 1/5 LD and 2/5 MD group females. At the end of recovery dilated pelvis was found in 1/5 HD group males.
All gross lesions recorded were considered to be within the range of normal background alterations which may be recorded in animals of this
strain and age.

Organ Weight
There were no treatment related effects on organ weights (absolute and relative to brain and terminal body weight) at the end of treatment
and recovery periods. Although, there were statistically significantly higher mean absolute brain weight in male recovery HD group and relative
brain (to terminal body weight) weights in female LD groups when compared to controls. But these changes were not associated with any
macroscopic or microscopic findings and was considered incidental.
In males there were slightly lower (in dose response relation) mean absolute and relative pituitary gland weights in LD, MD and HD groups
(-4% to -20% Vs Control). In females the mean absolute and relative pituitary weights were higher in all dose groups when compared to c
orresponding control. The absolute pituitary weights of males of all dose and control groups were within the histrocal control range and in
females the mean and most individual absolute pituitary weights were slightly higher than the historical control range. In the absence of
macroscopical and histopathological findings no relevenace to test item treatment was considered.
Several other differences in mean absolute and/or relative organ weights (relative to terminal body weight and brain weight) between control and
treated groups were either statistically insignificant and/or there were no dose response relation. There were no macroscopic or microscopic
findings in any of the organs of treated and control groups. Therefore, the differences in organ weight data were not considered to have relevance
to test item treatment.

Histopathology
There were no microscopic lesions that could be attributed to treatment with the test item.
All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Dose Formulation Analysis
Concentration analysis of formulation samples was determined in study weeks 1, 2, 3 and 4 for all dose groups. The mean recoveries
observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 102.3%, 106.5% and 115.2% of the nominal concentration,
respectively. Nominal concentrations were confirmed for all dose groups, as mean of measured concentration did not differ from nominal
concentration by more than 20%.
Stability of formulation samples was investigated in study week 1 based on concentration in the LD and HD dose groups. After 6 hours storage
at room temperature recovery compared to starting value was 91.7% and 98.9%. All samples were stable, as concentration after storage did not
differ from start value by more than 20%.
Homogeneity of formulation samples was determined in study weeks 1 and 4 for the LD and HD dose groups. The mean recovery observed for
the LD dose group was 95.8% and 101.5% of the nominal value and 115.7% and 114.4% of the nominal value for HD dose group. The coefficients
of variation of the different sampling locations (top, middle, bottom) were 3.1% and 15.1% in LD dose group and 2.3% and 0.9% in HD dose group.
All samples were homogenous, as COV was below or equal 20%.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 650 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no adverse effects of C18/C18 unsatd.-Glucamide found up to a dose level of 650 mg/kg body weight/ day. The NOAEL of C18/C18 unsatd.-Glucamide in this study is considered to be 650 mg/kg body weight/ day.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

On the basis of this 28-Day Repeated Dose Oral Toxicity study with C18/C18 unsatd.-Glucamide in male and female Wistar rats with dose levels
of 125, 300, and 650 mg/kg body weight/ day, followed by a recovery period of 14 days, the following conclusions can be made:
There were slightly lower and higher mean pituitary gland weights (absolute and relative) in males and females, respectively in all dose groups.
In males, the absolute pituitary weights of all dose and control groups were within the histrocal control range and in females the mean and most
individual absolute pituitary weights were slightly higher than the historical control range. In the absence of macroscopical and histopathological
findings no relevenace to test item treatment was considered.
There were no adverse effects of C18/C18 unsatd.-Glucamide found up to a dose level of 650 mg/kg body weight/ day.
The NOAEL of C18/C18 unsatd.-Glucamide in this study is considered to be 650 mg/kg body weight/ day.

Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of C18/C18 unsatd.-Glucamide via

oral administration to rats over a period of 28 days.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 28 days. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study.

The 4 groups comprised 5 male and 5 female Wistar rats. The animals were housed in groups (5 animals/sex/ cages).

The following doses were evaluated:

Control:                           0         mg/kg body weight/ day

Low Dose:                    125     mg/kg body weight/ day

Medium Dose:              300    mg/kg body weight/ day

High Dose:                   650    mg/kg body weight/ day

The test item formulation was prepared freshly on each day of administration. The test item was suspended in corn oil and administered

daily during a 28-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly

body weight measurements. During the period of administration, the animals were observed precisely each day for signs of toxicity.

All animals were sacrificed and observed macroscopically. To detect possible delayed occurrence or persistence of, or recovery from toxic

effects, animals in the recovery group were observed for a period of 14 days following the last administration.

Body weight and food consumption were measured weekly.

At the conclusion of the treatment period, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was

taken and a set of organs/tissues was preserved. Haematological, blood coagulation and clinical biochemistry parameters were examined

from blood samples collected from an abdominal aorta of all terminally sacrificed animals.

Urinalysis was made on urine samples collected directly from the urinary bladder of all terminally sacrificed animals.

A full histopathological evaluation of the tissues was performed on high dose and control animals.Since microscopic changes that could

be attributed to treatment with the test item were not observed in any animals of HD group in the initial histopathologic examination,

further histoprocessing and histopathology evaluation were not performed.

Summary Results

No mortality occurred in the control or any of the dose groups during the treatment or recovery period of this study.

All male and female animals of MD and HD groups showed signs of salivation and moving the bedding, which were considered to be due to

test item treatment. These clinical signs were only observed immediately post dose administration, which suggests no systemic effect but

more likely a local effect. Diarrhea, nasal discharge, piloerection, eschar and alopecia were transient in appearance or also occurred in the

control group. There was no considerable difference between the genders.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.

There were no ophthalmoscopic findings in any of the animals of this study.

No significant differences were found in the body weight gain or food consumption of the dose groups of males and females when compared

to the corresponding control groups. No differences were found that were considered toxicologically relevantin the haematological and

blood coagulation parameters analysed in any of the treated groups when compared to the corresponding control group.

No toxicologically relevant effect was found in the clinical biochemistryparameters analysed in any of the treated groups when compared to

the corresponding control group. Statistically significant differences were not assumed to be biologically relevant.

There were no considerable test item-related differences in any of the urine parameters tested.

There were no gross lesions that could be attributed to treatment with the test item.

There were slightly lower and higher mean pituitary gland weights (absolute and relative) in males and females, respectively in all dose groups.

In males, the absolute pituitary weights of all dose and control groups were within the histrocal control range and in females the mean and

most individual absolute pituitary weights were slightly higher than the historical control range. In the absence of macroscopical and histopathological findings no relevenace to test item treatment was considered. There were no microscopic lesions that could be attributed

to treatment with the test item. All microscopic findings recorded were within the range of normal background lesions which may be recorded

in animals of this strain and age. Formulation analysis was performed on samples of all dose groups collected at various intervals during

the study. The concentration verification of samples of all dose groups was investigated in study weeks 1, 2, 5 and 4.

The mean recoveries in LD, MD and HD groups were 102.3%, 106.5% and 115.2% of the nominal concentration, respectively.

The stability of formulation samples of LD and HD dose groups was investigated in study week 1. After 6 hours storage at room temperature recovery compared to starting value was 91.7 (for LD dose) and 98.9% (for HD dose).All samples were stable, as concentration after storage

did not differ from start value by more than 20%.

The Homogeneity of formulation samples of LD and HD dose groups was determined in study week 1 and 4. The mean recoveries observed

for LD dose group was between 95.8 % and 101.5% of the nominal value and between 115.7% and 114.4% of the nominal value for HD dose group. The coefficients of variation of the different sampling levels (top, middle and bottom) were between 3.1% and 15.1% in LD dose group, between 2.3% and 0.9% in HD dose group.

Conclusion

On the basis of this 28-Day Repeated Dose Oral Toxicity study with C18/C18 unsatd.-Glucamide in male and female Wistar rats with dose levels

of 125, 300, and 650 mg/kgbody weight/ day, followed by a recovery period of 14 days, the following conclusions can be made:

There were slightly lower and higher mean pituitary gland weights (absolute and relative) in males and females, respectively in all dose groups.

In males, the absolute pituitary weights of all dose and control groups were within the histrocal control range and in females the mean and most individual absolute pituitary weights were slightly higher than the historical control range. In the absence of macroscopical and histopathological findings no relevenace to test item treatment was considered.

There were no adverse effects of C18/C18 unsatd.-Glucamide found up to a dose level of 650 mg/kg body weight/ day.

The NOAEL of C18/C18 unsatd.-Glucamide in this study is considered to be 650 mg/kg body weight/ day.