N-(2-hydroxypropyl)oleamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2016-January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

No major deviation during the course of the study

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

23 march 2017

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-hydroxypropyl)oleamide

EC Number:

203-828-2

EC Name:

N-(2-hydroxypropyl)oleamide

Cas Number:

111-05-7

Molecular formula:

C21H41NO2

IUPAC Name:

N-(2-hydroxypropyl)oleamide

Test material form:

solid

Remarks:

beige waxy solid

Details on test material:

LOT/BATCH: 160602013073 w/o solvent

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 160602013073 w/o Solvent
- Expiration date of the lot/batch: 2018/05/30
- Purity test date: 2016/06/22

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Immediately upon receipt, the test item was registered, then stored at room temperature in accordance with the Sponsor0s instructions. The complete description of the chemical and physical properties of the test item including stability is the responsibility of the Sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: the test item formulations at 20 mg/mL to 200 mg/mL in its vehicle are stable for 30 days at room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: Test item was warmed in water bath at approximately 50°C. To ensure good homogeneity, an appropriate amount of test item was weighed. Corn oil previously warmed in water bath at approximately 50°C was added up to obtain the appropriate amount. The formulation was mixed using magnetic stirrer in the water bath at approximately 50°C for at least 10 minutes then the formulation was mixed using magnetic stirrer at room temperature for at least 30 minutes
- Form of administration: The day of treatment, all formulations and vehicle were warmed using a water bath at approximately 50°C for at least 30 minutes, under magnetic stirring for the formulation at 20 and 60 mg/mL. Then all formulations were kept under magnetic stirring at ambient temperature for at least 30 minutes.
N-(2-hydroxypropyl) Oleamide was administered as a suspension in its vehicle. Control animals were given the vehicle under the same conditions as animals dosed with N-(2-hydroxypropyl) Oleamide. The test item formulations were prepared and kept at room temperature and used within one week after preparation.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

SPF (Specific Pathogen Free) Sprague-Dawley - Crl: OFA (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 L'ARBRESLE Cedex, France
- Females (if applicable) nulliparous and non-pregnant: yes/
- Age at study initiation: 7 weeks on the day of the first administration.
- Weight at study initiation: Between 184.2 and 231.9g in males and 125.5g and 171.4g in females. The weight variation of animals used did not exceed +/- 20% of the mean weight of each sex.
- Housing: Animals were housed (separated by sex) in cages of standard dimensions with sawdust bedding (or equivalent).
- Diet: RM1 (E)-SQC SDS/DIETEX feed (quality controlled/radiation sterilised) was available ad libitum except during the fasting experimental period. A certificate of analysis concerning this feed product is included to the study report. The criteria for acceptable levels of contaminants in the feed supply were within the limits of the analytical specifications established by the diet manufacturer.
- Water: Drinking water was available ad libitum in polycarbonate bottles with a stainless steel nipple. A specimen of water is obtained approximately every 6 months and sent to Laboratoire de la Touraine - ZA n°1 du Papillon - Rue de l0Aviation - 37210 Parçay-Meslay - France, for analysis. The certificates of analysis are included in the report. The criteria for acceptable levels of contaminants in the water supplied were within the limits of the analytical specifications
- Acclimation period: A minimum of five days in the laboratory animal house where the experiment took place. Only animals without any visible sign of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The room temperature was between 19°C and 24°C and was recorded at the beginning and at the end of all observations.
- Humidity (%) and air changes (per hr): The animals were placed in an air-conditioned (20-24°C) animal house kept at relative humidity between 45% and 65% (except during the cleaning slot) in which nonrecycled filtered air was changed approximately 10 times per hour. Between 28 Nov at 08.00 p.m. and 29 Nov 2016 at 11.00 a.m. (for about 15 hours) and between 16 Jan at 10.00 p.m and 17 Jan 2017 at 12.30 a.m, an abnormal decrease in hygrometry was noted in the animal room.
- Photoperiod (hrs dark / hrs light): The artificial day/night cycle involved 12 hours light and 12 hours darkness with light on at 7.30 a.m. except on 30 Oct 2016.

IN-LIFE DATES: From: 18 october 2016 To: 20 January 2017

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

N-(2-hydroxypropyl) Oleamide or its vehicle was administered once a day at approximately the same time (a maximum range of 4 hours between the start and the end of the daily treatment) at each chosen dose level, by the oral route for 13 weeks. A constant administration volume of 5 mL/kg body weight (except some animals) was used in accordance with the previous study (COMBINED REPEATED DOSE TOXICITY STUDY WITH THE REPRODUCTION/DEVELOPMENTAL TOXICITY SCREENING TEST BY ORAL ROUTE (GAVAGE) IN RATS - OECD 422 - No.39644 RSR).

Vehicle:

corn oil

Details on oral exposure:

- DIET PREPARATION no information available

- VEHICLE
Corn oil was used (Reference C8267)
- Lot/batch no.: Batch Nos. MKBS6944V and MKBW9504V, Expiry dates: 08 Oct 2020 and 06 Oct 2021 respectively

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of test item in formulations were checked during the first and last week of the study.
Each concentration level and the vehicle was checked.
The analytical method is validated within the range 85-115% of the theoretical concentration for formulations between 19.765 mg/mL and 198.120 mg/mL, with a precision better than 10%. The samples in corn oil must be analysed within the stability period (i.e. 30 days at room temperature) and must be within the range 85-115% to meet the acceptance criteria.
Formulation analysis was performed on one formulation prepared during the first and the last week of the study. The concentrations tested were 20 mg/mL, 60 mg/mL and 200 mg/mL. The concentrations found were within the range of acceptance (+/-15% of the intended concentration). The absence of test item was also confirmed in the vehicle samples. These results show that the formulations were properly prepared.

Duration of treatment / exposure:

Main phase: Minimum of 90 days (13 weeks)

Frequency of treatment:

Once a day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

The study was conducted on 4 groups of 10 males and 10 females including a control group (80 animals in the main group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Proposed doses are selected in agreement with the Sponsor. The choice is based on previous studies (COMBINED REPEATED DOSE TOXICITY STUDY WITH THE REPRODUCTION/DEVELOPMENTAL TOXICITY SCREENING TEST BY ORAL ROUTE (GAVAGE) IN RATS - OECD 422 - Study No. 39644 RSR). Moreover, the highest dose should reveal signs of toxicity and the lowest dose should represent a no-observed-adverse effects level.
- Allocation of each animal to treatment : randomly determined before the start of the study. Homogeneity of groups was validated on the criterion of body weight measured on the day of randomisation, separately for males and females.
- Justification of the number of animals per group: The number of animals per group is the minimum number enabling an accurate assessment of the studied toxicological effect and comparisons using the appropriate statistical tests.
- dose adjustement: Doses of test item were expressed as mg/kg. Doses were adjusted on the basis of body weight measured at the most recent weighing.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed in the cage before the first dosing and at least once a day except for some animals. The time of observation was between 21 minutes and 1h45 post dose. The general disposition, behaviour and activity were observed. Once a week except for one female on week 3, animals were submitted to a full clinical examination in a cage without sawdust according to a standardised observation battery for general clinical signs, neurobehavioural, neurovegetative or psychotropic signs or neurotoxic effects. The time of observation was at 1 hour (+/- 30 min) or 3h35 on D7 for Female No. 1602396 post dose.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on the following days:
• on the day of randomisation
• at predose
• then weekly during the study
• on the day of necropsy (not exsanguinated), this is the reference weight used in the calculation of relative organ weights

FOOD CONSUMPTION : Yes
Food consumption was measured for each treatment cage except on the day of fasting. Animals were fasted during the night before scheduled blood sampling for haematology/coagulation parameters, clinical chemistry and during urine collection for urinalysis.

WATER CONSUMPTION : No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination (Retinography) was performed under the responsability of a person who has followed a veterinary ophthalmological training on all animals before the first dosing and during the last week.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was taken from all animals during the last week. Samples were taken just before killing for moribund animals when possible.
- Anaesthetic used for blood collection: Yes (isoflurane inhalation)
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was taken from all animals during the last week. Samples were taken just before killing for moribund animals when possible.
- Animals fasted: Yes

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine: Urine was collected from all animals, during the last week. Urine collection was performed individually in metabolism cages for a period of about 16 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (Food and water were withheld during collection)

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations: Before the first dosing and on the last week, animals were observed according to a standardised observation battery for neurobehavioural, neurovegetative or psychotropic signs or neurotoxic effects. The method is based on an Irwin screen modified by suppressing the graduation of intensity of clinical signs. Animals were observed individually in a cage without sawdust in a quiet room.The time of observation was 1 hour (+/- 36 min) post dose. The rectal temperature was measured at the end of each observation.
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Hyperalertness, Decrease in visual placing, Passive response to finger approach, Passivity when touched, Absence of reactivity, Stereotyped movements, Aggressiveness/irritability, Increase/decrease in fear, Reduced/increased spontaneous locomotor activity, Increasing grooming, Staggering gait, Abnormal gait, Loss of righting reflex, Absence/increase of startle response, Insensitivity/Hyperreactivity to pinching of tail, Body sag, Decrease/Increase in limb tone , Decrease in grip strength, Decrease in body tone, Decrease in abdominal tone, Sitting up, Rearing, Virtually permanent reared position, Loss of pinna reflex, Loss of corneal reflex, Hind limb reflex, Pallor, Tachycardia, Bradycardia, Myosis, Mydriasis, Ptosis, Exophthalmos, Lacrimation, Liquid/soft faeces

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals dying during the course of the study and animals killed in a moribund condition were necropsied as quickly as possible and specimens as required by the study plan obtained whenever practically possible. All animals (including any found dead or moribund) were submitted to full necropsy procedures including an examination of:
• the external surface
• all orifices
• the cranial cavity
• the external surface of the brain and samples of the spinal cord
• the thoracic and abdominal cavities and organs
• the cervical tissues and organs
• the carcass
Paired organs were weighed together. From all animals, excluding those found dead, the organs were dissected free of fat and other contiguous tissues at the discretion of the prosector and weighed.

HISTOPATHOLOGY: Yes
Selected organs were weighed, fixed and preserved at necropsy and examined histopathologically
The organs and tissues sampled were fixed and preserved in 4% buffered formalin with the following exceptions:
• testes and epididymides were fixed in alcoholic Bouin0s fluid (about 5 days), then transferred to ethanol 95%
• eyes and optic nerves were fixed and preserved in Davidson0s fluid (about 3 days), then transferred to ethanol 70%

Organs for organ weight determination and for histopathological examination are presented in table below.

SACRIFICE: Main phase animals will be killed following 13 weeks of treatment.

Other examinations:

None

Statistics:

None

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In decedent animals, the day before or the day of death, there were the following clinical signs:
• At 100 mg/kg, in Male No.1602323, there was no clinical sign.
• At 300 mg/kg, there was an increased salivation and absence of spontaneous locomotor activity in Male No.1602335 on D87 (the day before death). In male No.1602340, there was blood around and in the mouth on D58 (the day before death).
• At 1000 mg/kg, there were increased salivation, chromodacryorrhoea, dyspnoea, bradypnoea, decrease then absence of locomotor activity and it was lying on its sides on D59, in Male No. 1602344. In Male No. 1602345, there was increased salivation. In Female No. 1602388, there was increased salivation.

In surviving males and females treated with test item whatever the dose, there was a dose dependent:
• increased salivation (8.8%, 19.8% and 41.7% in males and 0.8%, 3.2% and 22.6% in females respectively) from D26 up to the end of the study
• chromodacryorrhoea (2.1%, 6.1% and 8.8% in males and 0.1%, 3.4% and 9.1% in females respectively) from D28 up to the end of the study
There were also isolated clinical signs such as a decrease in spontaneous locomotor activity from D57 up to D60 in at most 3/10 males treated with test item at 300 and 1000 mg/kg and also in 1/8 males treated with test item at 1000 mg/kg on D91, a decrease in fear in 1/8 males treated at 1000 mg/kg or 1/10 females treated at 100 mg/kg, an increase in fear or insensitivity to pinching of the tail in 1/10 females at 100 mg/kg.

Mortality:

mortality observed, treatment-related

Description (incidence):

6 animals treated with test item were found dead between D59 and D91:
• at 100 mg/kg, Male No. 1602323 was killed early on D77
• at 300 mg/kg, Male No. 1602335 on D88 and Male No. 1602340 on D59 were found dead
• at 1000 mg/kg, Male No. 1602344 was moribund and was found dead during the isoflurane anaesthesia, just before exsanguination on D59, Male No. 1602345 was found dead on D80, Female No. 1602388 was found dead on D91.
There was no mortality in the control group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no change in body gain in animals found dead except for male No. 1602345 treated at 1000 mg/kg which had a body weight loss (-9.6%) between D63 and D70.
There was a statistically significant lower body weight gain in males treated with test item at 1000 mg/kg from D14 up to the end of the study (on D91, body weight gain was +71% vs +107% in the control group) and at 100 mg/kg from D70 up to the end of the study (on D91, body weight gain was +87% vs +107% in the control group).
In males treated at 300 mg/kg, based on the mean values, there was no difference when compared to the control group due to Male No. 1602331 which had very high body weight (649g vs 510g the mean values of the group). However, if considering the individual values, there was also a lower body weight gain in the other males treated at 300 mg/kg. When the values of this animal were excluded, the body weight gain was +97% vs +107% in the control group.
One male treated with test item at 100 mg/kg (No. 1602324) had a body weight loss between D84 and D91 (-5.4%).
There was no change in body weight gain in females.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

There was a slightly lower food consumption in all males treated with test item whatever the dose (consumption was approximately 17-18 g/animal/day vs 20 g/animal/day in the control group.
In cage 6 (with Male Nos. 1602324 and 1602325) of group 2, the food consumption was lower during the last week of the treatment period.
There was no marked change in food consumption in females whatever the dose.

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

not examined

Description (incidence and severity):

Not necessary

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There was no abnormality at the ophthalmological examination on D91 when compared to the records before the start of the treatment.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the decedent animals only the animal killed on D77 yielded data. A slight reduction of the white cell series was seen (total white cell count, neutrophil count, eaosinophil count, basophil count, lymphocyte
count).
At 1000 mg/kg, there was a higher total white blood cell count in males (+20%) and in females (+30%) on D92. In females, there was also a statistically significant higher lymphocyte count (+33%) and monocyte count (+58%) on D92.
There was also a statistically significant higher reticulocyte count in males treated with test item at 100 and 1000 mg/kg mainly due to two lower values in the control group (in male Nos. 1602312 and 1602320). Therefore this changes was not attributed to the test item.
At 300 and 100 mg/kg, there was no change in haematology parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In the decedent animals only the animal killed on D77 yielded data. A slight higher ALT, AST and ALP activities and slightly lower total bilirubin and albumin.
There was dose related statistically significant higher plasma ALT, AST and ALP activities in the males treated with test item at 300 and 1000 mg/kg and a statistically significant higher ALT activity (+45%) in females treated at 1000 mg/kg.
There was also a statistically significant higher A/G ratio in male treated at 1000 mg/kg due to lower globulins. This was considered to be of minor degree (+14%) and was not considered as toxicologically relevant.
At 100 mg/kg in surviving animals and at 300 mg/kg in females, there was no change in blood chemistry parameters.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There was no change in urinary volume and semi-quantitative parameters.
There was a higher creatinine level in urine in Male No. 1602324 treated with test item at 100 mg/kg.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was statistically significant changes at 1000 mg/kg (in absolute and/or relative values body or brain weight):
• higher liver weight in males and females
• higher adrenals weight in males
• lower thymus weight in males
• higher testes and epididymides weight in males
There was a statistically significant higher adrenals weight at 100 and 300 mg/kg in males (in relative values % body weight). This was low in amplitude (+18% when compared to the control relative values % body weight) and was not considered as toxicologically relevant.
There were also statistically significant lower (in absolute values) spleen, thymus, heart and brain weights related to the lower body weight in males treated at 1000 mg/kg and lower thymus weight in males treated at 100 mg/kg due to the low individual value in Male No. 1602324.
There was no other change in organ weight in animals treated at 300 or at 100 mg/kg.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In decedent animals, there were mainly dark organs (mucosa, liver, spleen, lung or heart) or mottled lung and soft brain. These observations were usually observed in animals found dead or in moribund state.
In male No. 1602323 treated at 100 mg/kg and male No. 1602335 treated at 300 mg/kg, there was blood in the mouth.
In males 1602335 and 1602340 treated at 300 mg/kg, there was liquid in the thoracic cavity and a white film in the thoracic cavity in Male No. 1602340. For male No. 1602344 treated at 1000 mg/kg, there was a subcutaneous hole under the left forelimb and a subcutaneous mass on the neck.
In surviving animals, there were isolated macroscopic findings found at a low incidence such as point change in the heart (1/8 in males treated at 300 mg/kg) or dark mesenteric lymph nodes (1/8 in males treated at 1000 mg/kg) or observations found at the same incidence in the control and test item dose groups, such as point changes in thymus or liver (1/10 control males vs 1/8 treated males at 300 mg/kg), dark or point changes in sub maxillary lymph node. In Male No. 1602350 treated at 1000 mg/kg, there was pale and enlarged lung with dark area.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In decedent animals, there were no lesions suggesting the cause of death in any of these animals.
In the male treated at 100 mg/kg and killed early on D77, there was only mild periacinar hepatocytic hypertrophy and minimal sternal lipid hyperplasia.
In Male No. 1602345 and in Female No. 1602388 treated at 1000 mg/kg, there was minimal femoral lipid hyperplasia and minimal periacinar hepatocytic hypertrophy respectively. In other decedent animals treated at 300 or 1000 mg/kg, there were no relevant changes.
In surviving animals, there were minor lesions in the thymus, liver and bone marrow related to the treatment.
Thymic atrophy was present in the majority of animals whatever the treatment, the degree was greatest in animals treated at 1000 mg/kg but the incidence and degree in other groups including the control group was sufficiently inconsistent to deny any true link to the test item.
Hepatic changes in males treated at 1000 mg/kg and females treated at 300 and 1000 mg/kg were biliary prominence and periacinar hypertrophy. These findings were present in the control group, at the lower incidence and degree. The changes were adaptative in nature and very slight in degree, different following the gender and were not deemed significant. The partial replacement of bone marrow by lipid vacuolation in the femur was present in males treated at 1000 mg/kg and in the sternum in males treated at 300 and 1000 mg/kg.

Conclusion
There were slight histopathological changes in the liver and the bone marrow in animals treated with test item at 1000 mg/kg.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There was statistically changes in mobile spermatozoa at 100 and 1000 mg/kg, midpiece anomaly at 100 mg/kg, cytoplasmie droplet anomaly at 1000 mg/kg. since there were no changes in % mobile spermatozoa or the variation was very low, these changes were not attributed to the test item. There was no change in sperm analysis.

Effect levels

1

Key result

Dose descriptor:

LOAEL

Effect level:

ca. 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

clinical signs

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Table 7.5.1/2: Clinical signs most important differences - male

Clinicalsigns	Vehicle	Testitem100 mg/kg	Testitem300 mg/kg	Testitem1000 mg/kg
Increased salivation Chromodacryorrhoea Increased salivation before treatment Decrease in spontaneous locomotor activity Test item reflux during the treatment Blood around or in the mouth Piloerection Absence of spontaneous locomotor activity Recumbent position or lying on side Bradypnoea Polypnoea Dyspnoea Noisybreathing	0.0 0.5 0.0 0.0   0.3 0.0 0.0 0.0   0.0 0.0 0.0 0.0 0.0	8.8 2.1 0.0 0.0   0.1 0.0 0.0 0.1   0.0 0.0 0.0 0.0 0.0	19.8 6.1 0.1 1.1   0.3 0.6 0.3 0.2   0.0 0.0 0.1 0.1 0.1	41.7 8.8 1.7 1.1   1.0 0.1 0.0 0.2   0.1 0.1 0.0 0.1 0.0

Table 7.5.1/3: Body weight - male (mean table)

Treatment

Predose

D7

D14

D21

D28

D35

D42

Vehicle

Mean SEM % N

259 5 NA 10

313 5 +21 10

345 7 +33 10

374 8 +44 10

397 8 +53 10

418 9 +61 10

434 9 +68 10

Testitem 100mg/kg

Mean SEM % N P

256 4 NA 10 NS

304 6 +19 10 NS

333 8 +30 10 NS

357 8 +39 10 NS

375 10 +46 10 NS

391 10 +53 10 NS

407 11 +59 10 NS

Testitem 300mg/kg

Mean SEM % N P

251 5 NA 10 NS

306 7 +22 10 NS

343 10 +37 10 NS

369 11 +47 10 NS

390 13 +55 10 NS

409 13 +63 10 NS

422 17 +68 10 NS

Testitem 1000mg/kg

Mean SEM % N P

251 6 NA 10 NS

287 7 +14 10 NS

303 9 +21 10 ?

322 10 +28 10 ??

338 9 +35 10 ??

355 9 +41 10 ??

365 10 +45 10 ??

Threshold

34

34

34

34

34

34

34

Treatment

D49

D56

D63

D70

D77

D84

D91

Vehicle

Mean SEM % N

451 10 +74 10

465 10 +80 10

479 11 +85 10

494 11 +91 10

507 13 +96 10

519 12 +100 10

536 12 +107 10

Testitem 100mg/kg

Mean SEM % N P

421 11 +64 10 NS

435 11 +70 10 NS

447 12 +75 10 NS

452 12 +77 10 ?

463 12 +81 9 ?

469 13 +83 9 ??

479 14 +87 9 ??

Testitem 300mg/kg

Mean SEM % N P

445 15 +77 10 NS

448 20 +78 10 NS

464 20 +85 9 NS

479 21 +91 9 NS

491 20 +96 9 NS

501 20 +100 9 NS

510 23 +103 8 NS

Testitem 1000mg/kg

Mean SEM % N P

375 9 +49 10 ??

395 9 +57 10 ??

400 12 +59 9 ??

400 10 +59 9 ??

409 12 +63 9 ??

419 15 +67 8 ??

429 16 +71 8 ??

Threshold

34

34

40

40

38

39

41

Resultsexpresseding

D:day

Vehicle:Cornoil

NS:P>0.05,?:P≤0.05,??:P≤0.01,when compared to control group

Analysis of variance for repeated measurements with Dunnett's test

NA:not applicable

%:variation expressed in percentage in relation to predose values

Table 7.5.1/4: Body weight - female (mean table)

Treatment

Predose

D7

D14

D21

D28

D35

D42

Vehicle

Mean SEM % N

172 3 NA 10

191 3 +11 10

208 3 +21 10

221 3 +28 10

234 5 +36 10

244 6 +42 10

249 5 +45 10

Testitem 100mg/kg

Mean SEM % N P

178 3 NA 10 NS

198 4 +11 10 NS

215 5 +21 10 NS

225 7 +26 10 NS

229 6 +29 10 NS

239 7 +34 10 NS

244 7 +37 10 NS

Testitem 300mg/kg

Mean SEM % N P

173 3 NA 10 NS

197 3 +14 10 NS

213 4 +23 10 NS

226 5 +31 10 NS

236 5 +36 10 NS

243 5 +40 10 NS

250 5 +45 10 NS

Testitem 1000mg/kg

Mean SEM % N P

179 3 NA 10 NS

199 5 +11 10 NS

214 4 +20 10 NS

228 5 +27 10 NS

236 4 +32 10 NS

245 4 +37 10 NS

253 5 +41 10 NS

Threshold

20

20

20

20

20

20

20

Treatment

D49

D56

D63

D70

D77

D84

D91

Vehicle

Mean SEM % N

259 4 +51 10

261 5 +52 10

271 6 +58 10

281 8 +63 10

282 6 +64 10

282 5 +64 10

288 7 +67 10

Testitem 100mg/kg

Mean SEM % N P

254 7 +43 10 NS

262 7 +47 10 NS

260 8 +46 10 NS

268 9 +51 10 NS

273 9 +53 10 NS

277 9 +56 10 NS

276 9 +55 10 NS

Testitem 300mg/kg

Mean SEM % N P

258 6 +49 10 NS

267 5 +54 10 NS

272 6 +57 10 NS

274 6 +58 10 NS

278 5 +61 10 NS

282 7 +63 10 NS

282 8 +63 10 NS

Testitem 1000mg/kg

Mean SEM % N P

262 7 +46 10 NS

267 6 +49 10 NS

275 6 +54 10 NS

277 6 +55 10 NS

280 5 +56 10 NS

282 6 +58 10 NS

287 6 +60 10 NS

Threshold

20

20

20

20

20

20

20

Results expressed in g

D: day

Vehicle: Corn oil

NS:P>0.05, when compared to control group

Analysis of variance for repeated measurements with Dunnett's test

NA: not applicable

%: variation expressed in percentage in relation to predose values

Table 7.5.1/5: haematology and coagulation - male - day 92 (mean table)

Treatment

WBC

NEUT

EOSI

BASO

LYMP

MONO

LUC

RBC

HGB

Vehicle

Mean SEM N

8.3 0.5 10

1.52 0.19 10

0.13 0.02 10

0.03 0.00 10

6.3 0.3 10

0.26 0.02 10

0.11 0.01 10

9.0 0.2 10

15.9 0.2 10

Testitem 100mg/kg

Mean SEM N % P

7.8 0.5 9 -6 NS

1.31 0.22 9 -14 NS

0.08 0.01 9 -38 NS

0.02 0.00 9 -33 NS

6.1 0.5 9 -3 NS

0.21 0.02 9 -19 NS

0.09 0.01 9 -18 NS

9.1 0.1 9 +1 NS

15.8 0.1 9 -1 NS

Testitem 300mg/kg

Mean SEM N % P

8.3 0.6 8 0 NS

1.43 0.17 8 -6 NS

0.12 0.02 8 -8 NS

0.02 0.00 8 -33 NS

6.4 0.4 8 +2 NS

0.26 0.04 8 0 NS

0.09 0.02 8 -18 NS

8.9 0.2 8 -1 NS

15.5 0.3 8 -3 NS

Testitem 1000mg/kg

Mean SEM N % P

10.0 0.7 8 +20 NS

1.67 0.45 8 +10 NS

0.17 0.05 8 +31 NS

0.03 0.01 8 0 NS

7.7 0.8 8 +22 NS

0.30 0.04 8 +15 NS

0.11 0.01 8 0 NS

9.5 0.3 8 +6 NS

15.9 0.3 8 0 NS

Threshold

1.9

0.90

0.08

0.01

1.8

0.10

0.04

0.7

0.8

Treatment

HCT

RET

RDW

MCV

MCH

MCHC

THR

MPV

APTT

PT

Vehicle

Mean SEM N

46 0 10

135 5 10

12.2 0.5 10

52 1 10

17.8 0.3 10

34 0 10

866 95 10

7.3 0.7 10

19.9 0.9 10

15.1 0.3 10

Testitem 100mg/kg

Mean SEM N % P

47 0 9 +2 NS

163 8 9 +21 ?

12.3 0.4 9 +1 NS

52 1 9 0 NS

17.3 0.3 9 -3 NS

34 0 9 0 NS

831 52 9 -4 NS

6.6 0.1 9 -10 NS

21.5 0.6 8 +8 NS

14.6 0.4 9 -3 NS

Testitem 300mg/kg

Mean SEM N % P

47 1 8 +2 NS

152 6 8 +13 NS

12.0 0.3 8 -2 NS

52 1 8 0 NS

17.4 0.3 8 -2 NS

34 0 8 0 NS

906 43 8 +5 NS

6.7 0.1 8 -8 NS

20.0 0.9 8 +1 NS

15.5 0.4 8 +3 NS

Testitem 1000mg/kg

Mean SEM N % P

47 1 8 +2 NS

163 11 8 +21 ?

12.3 0.5 8 +1 NS

50 1 8 -4 NS

16.9 0.4 8 -5 NS

34 0 8 0 NS

854 73 8 -1 NS

6.8 0.2 8 -7 NS

23.1 2.1 8 +16 NS

15.9 1.1 8 +5 NS

Threshold

3

26

1.5

2

1.1

1

249

1.5

4.2

2.0

Vehicle: Corn oil

NS:P>0.05, ?:P<0.05, when compared with control group

Analysis of variance with Dunnett0s test if P <0.05

Table 7.5.1/6: haematology and coagulation - female - day 92 (mean table)

Treatment

WBC

NEUT

EOSI

BASO

LYMP

MONO

LUC

RBC

HGB

Vehicle

Mean SEM N

4.6 0.3 10

0.73 0.05 10

0.10 0.01 10

0.01 0.00 10

3.6 0.3 10

0.12 0.01 10

0.06 0.02 10

8.4 0.1 10

14.7 0.2 10

Testitem 100mg/kg

Mean SEM N % P

4.8 0.4 10 +4 NS

0.87 0.14 10 +19 NS

0.09 0.01 10 -10 NS

0.01 0.00 10 0 NS

3.6 0.3 10 0 NS

0.13 0.02 10 +8 NS

0.05 0.01 10 -17 NS

8.2 0.1 10 -2 NS

14.7 0.1 10 0 NS

Testitem 300mg/kg

Mean SEM N % P

4.2 0.3 10 -9 NS

0.63 0.10 10 -14 NS

0.09 0.01 10 -10 NS

0.01 0.00 10 0 NS

3.3 0.3 10 -8 NS

0.12 0.01 10 0 NS

0.05 0.01 10 -17 NS

8.1 0.1 10 -4 NS

14.6 0.1 10 -1 NS

Testitem 1000mg/kg

Mean SEM N % P

6.0 0.4 9 +30 ?

0.76 0.12 9 +4 NS

0.09 0.01 9 -10 NS

0.02 0.00 9 +100 NS

4.8 0.4 9 +33 ?

0.19 0.02 9 +58 ??

0.07 0.01 9 +17 NS

8.5 0.1 9 +1 NS

14.3 0.3 9 -3 NS

Threshold

1.2

0.36

0.04

0.01

1.1

0.06

0.04

0.3

0.6

Treatment

HCT

RET

RDW

MCV

MCH

MCHC

THR

MPV

APTT

PT

Vehicle

Mean SEM N

44 0 10

152 8 10

10.9 0.2 10

52 0 10

17.5 0.2 10

34 0 10

935 59 10

6.9 0.1 10

19.9 0.7 10

16.3 0.6 10

Testitem 100mg/kg

Mean SEM N % P

43 0 10 -2 NS

164 8 10 +8 NS

10.7 0.1 10 -2 NS

53 0 10 +2 NS

18.0 0.2 10 +3 NS

34 0 10 0 NS

1047 40 10 +12 NS

6.6 0.1 10 -4 NS

18.4 0.8 10 -8 NS

15.9 0.3 10 -2 NS

Testitem 300mg/kg

Mean SEM N % P

43 0 10 -2 NS

126 8 10 -17 NS

11.6 0.4 10 +6 NS

53 1 10 +2 NS

18.0 0.3 10 +3 NS

34 0 10 0 NS

885 80 10 -5 NS

6.9 0.1 10 0 NS

18.6 0.8 10 -7 NS

15.8 0.4 10 -3 NS

Testitem 1000mg/kg

Mean SEM N % P

44 1 9 0 NS

172 17 9 +13 NS

11.4 0.1 9 +5 NS

52 1 9 0 NS

16.8 0.4 9 -4 NS

33 0 9 -3 NS

1013 37 9 +8 NS

6.9 0.1 9 0 NS

19.0 0.4 9 -5 NS

15.1 0.2 9 -7 NS

Threshold

1

36

0.9

2

1.0

1

197

0.4

2.4

1.5

Vehicle: Corn oil

NS:P>0.05, ?:P<0.05, ??:P<0.01, when compared with control group

Analysis of variance with Dunnett0s test if P <0.05

Table 7.5.1/7: clinical chemistry - male - day 92 (mean table)

Treatment

ALT

AST

ALP

TBIL

PROT

ALB

CREA

Vehicle

Mean SEM N

35 2 10

60 3 10

125 9 10

1.98 0.13 10

60 1 10

29 0 10

32 2 10

Testitem 100mg/kg

Mean SEM N % P

37 2 9 +6 NS

66 4 9 +10 NS

127 9 9 +2 NS

2.07 0.09 9 +5 NS

58 1 9 -3 NS

29 0 9 0 NS

32 1 9 0 NS

Testitem 300mg/kg

Mean SEM N % P

42 2 8 +20 NS

77 3 8 +28 ??

165 12 8 +32 ?

2.18 0.18 8 +10 NS

60 2 8 0 NS

29 1 8 0 NS

38 2 8 +19 NS

Testitem 1000mg/kg

Mean SEM N % P

70 10 7 +100 ??

88 4 7 +47 ??

224 14 7 +79 ??

2.02 0.13 8 +2 NS

57 2 8 -5 NS

29 1 8 0 NS

35 2 8 +9 NS

Threshold

16

13

38

0.46

6

2

7

Treatment

UREA

GLU

CHOL

Cl

K

Na

GLOB

AG

Vehicle

Mean SEM N

3.31 0.06 10

8.8 0.7 10

2.03 0.07 10

102 0 10

4.2 0.1 10

140 1 10

31 1 10

0.94 0.03 10

Testitem 100mg/kg

Mean SEM N % P

3.04 0.16 9 -8 NS

8.7 0.3 9 -1 NS

2.10 0.10 9 +3 NS

103 0 9 +1 NS

4.2 0.1 9 0 NS

140 0 9 0 NS

29 1 9 -6 NS

1.01 0.03 9 +7 NS

Testitem 300mg/kg

Mean SEM N % P

3.50 0.13 8 +6 NS

9.2 0.9 8 +5 NS

2.15 0.17 8 +6 NS

103 1 8 +1 NS

4.1 0.1 8 -2 NS

140 1 8 0 NS

31 2 8 0 NS

0.94 0.03 8 0 NS

Testitem 1000mg/kg

Mean SEM N % P

3.80 0.31 8 +15 NS

8.2 1.0 8 -7 NS

2.11 0.10 8 +4 NS

103 1 8 +1 NS

4.0 0.1 8 -5 NS

141 1 8 +1 NS

28 2 8 -10 NS

1.07 0.04 8 +14 ?

Threshold

0.62

2.7

0.38

2

0.3

2

5

0.12

Vehicle: Corn oil

NS:P>0.05, ?:P<0.05, ??:P<0.01, when compared with control group

Analysis of variance with Dunnett0s test if P <0.05

Table 7.5.1/8: clinical chemistry - female - day 92 (mean table)

Treatment

ALT

AST

ALP

TBIL

PROT

ALB

CREA

Vehicle

Mean SEM N

29 1 9

69 4 10

91 4 10

2.39 0.13 10

61 1 10

32 1 10

39 2 10

Testitem 100mg/kg

Mean SEM N % P

26 2 10 -10 NS

61 2 10 -12 NS

94 7 10 +3 NS

2.22 0.15 10 -7 NS

61 1 10 0 NS

33 1 10 +3 NS

38 1 10 -3 NS

Testitem 300mg/kg

Mean SEM N % P

27 1 10 -7 NS

60 1 10 -13 NS

78 6 10 -14 NS

2.26 0.13 10 -5 NS

62 2 10 +2 NS

33 1 10 +3 NS

38 1 10 -3 NS

Testitem 1000mg/kg

Mean SEM N % P

42 5 9 +45 ??

79 9 9 +14 NS

107 11 9 +18 NS

2.13 0.09 9 -11 NS

61 1 9 0 NS

32 1 9 0 NS

39 3 9 0 NS

Threshold

9

16

25

0.46

5

2

6

Treatment

UREA

GLU

CHOL

Cl

K

Na

GLOB

AG

Vehicle

Mean SEM N

4.56 0.25 10

7.0 0.2 10

2.06 0.07 10

103 0 10

3.9 0.1 10

141 1 10

29 1 10

1.12 0.03 10

Testitem 100mg/kg

Mean SEM N % P

4.50 0.24 10 -1 NS

6.8 0.3 10 -3 NS

1.81 0.08 10 -12 NS

104 0 10 +1 NS

3.7 0.0 10 -5 NS

142 0 10 +1 NS

28 1 10 -3 NS

1.18 0.03 10 +5 NS

Testitem 300mg/kg

Mean SEM N % P

4.11 0.16 10 -10 NS

7.4 0.3 10 +6 NS

2.01 0.10 10 -2 NS

104 0 10 +1 NS

3.7 0.1 10 -5 NS

141 1 10 0 NS

29 1 10 0 NS

1.15 0.03 10 +3 NS

Testitem 1000mg/kg

Mean SEM N % P

4.63 0.30 9 +2 NS

6.9 0.4 9 -1 NS

1.90 0.15 9 -8 NS

103 1 9 0 NS

3.9 0.1 9 0 NS

141 1 9 0 NS

29 1 9 0 NS

1.13 0.03 9 +1 NS

Threshold

0.83

1.0

0.35

2

0.3

2

3

0.11

Vehicle: Corn oil

NS:P>0.05, ??:P<0.01, when compared with control group

Analysis of variance with Dunnett0s test if P <0.05

Table 7.5.1/9: urinalysis - male - day 92 (mean table)

Treatment		CREAU	KU	NaU	VOLU
Vehicle	Mean SEM N	7398 529 10	70 5 10	13 3 10	12 1 9
Testitem 100mg/kg	Mean SEM N % P	9090 1658 9 +23 NS	66 5 9 -6 NS	17 5 8 +31 NS	10 1 9 -17 NS
Testitem 300mg/kg	Mean SEM N % P	7032 678 8 -5 NS	51 3 8 -27 NS	13 3 7 0 NS	10 1 8 -17 NS
Testitem 1000mg/kg	Mean SEM N % P	6464 445 8 -13 NS	70 6 8 0 NS	27 7 8 +108 NS	11 1 8 -8 NS
	Threshold	3283	17	16	4

Vehicle: Corn oil

NS:P>0.05, when compared with control group

Analysis of variance with Dunnett's test if P <0.05

Table 7.5.1/10: urinalysis - female - day 92 (mean table)

Treatment		CREAU	KU	NaU	VOLU
Vehicle	Mean SEM N	8233 1746 9	70 11 9	20 5 9	5 1 9
Testitem 100mg/kg	Mean SEM N % P	8855 1098 10 +8 NS	71 12 10 +1 NS	17 4 9 -15 NS	5 1 10 0 NS
Testitem 300mg/kg	Mean SEM N % P	7965 1302 10 -3 NS	59 10 10 -16 NS	21 5 10 +5 NS	6 1 10 +20 NS
Testitem 1000mg/kg	Mean SEM N % P	7845 1159 9 -5 NS	63 12 9 -10 NS	23 5 8 +15 NS	6 1 9 +20 NS
	Threshold	4653	39	17	3

Vehicle: Corn oil

NS:P>0.05, when compared with control group

Analysis of variance with Dunnett's test if P < 0.05

Table 7.5.1/11: organ weight (absolute weight) - male - day 92 (mean table)

Treatment		BodyWeight	Liver	Spleen	Kidneys	Adrenals
Vehicle	Mean SEM N	506 12 10	13.7 0.5 10	0.747 0.035 10	2.66 0.15 10	0.055 0.003 10
Testitem 100mg/kg	Mean SEM N % P	455 11 10 -10 ?	12.1 0.5 10 -12 NS	0.666 0.023 10 -11 NS	2.69 0.10 10 +1 NS	0.058 0.003 10 +5 NS
Testitem 300mg/kg	Mean SEM N % P	485 23 8 -4 NS	13.0 0.9 8 -5 NS	0.680 0.024 8 -9 NS	2.62 0.11 8 -2 NS	0.062 0.001 8 +13 NS
Testitem 1000mg/kg	Mean SEM N % P	407 15 8 -20 ??	12.7 0.7 8 -7 NS	0.577 0.025 8 -23 ??	2.39 0.10 8 -10 NS	0.066 0.002 8 +20 ?
	Threshold	53	2.1	0.098	0.42	0.010

%: variation expressed in percentage in relation to control values

Vehicle: Corn oil

NS:P>0.05, ?:P<0.05, ??:P<0.01, when compared with control group

Analysis of variance with Dunnett's test if P <0.05

Table 7.5.1/12: organ weight (absolute weight) - female - day 92 (mean table)

Treatment		Body Weight	Liver	Spleen	Kidneys	Adrenal	sOvaries	Uterusand oviducts	Thymus	Heart	Brain
Vehicle	Mean SEM N	266 5 10	7.20 0.35 10	0.497 0.024 10	1.64 0.04 10	0.0661 0.0021 10	0.0855 0.0041 10	0.614 0.072 10	0.288 0.022 10	0.976 0.033 10	2.02 0.01 10
Test item 100 mg/kg	Mean SEM N % P	260   9   10 -2 NS	6.68   0.26   10 -7 NS	0.458   0.020   10 -8 NS	1.56   0.04   10 -5 NS	0.0642   0.0027   10 -3 NS	0.0840   0.0044   10 -2 NS	0.766   0.076   10 +25 NS	0.281   0.026   10 -2 NS	0.944   0.029   10 -3 NS	2.05   0.02   10 +1 NS
Test item 300 mg/kg	Mean SEM N % P	264   6   10 -1 NS	7.22   0.24   10 0 NS	0.486   0.019   10 -2 NS	1.66   0.06   10 +1 NS	0.0704   0.0022   10 +7 NS	0.0862   0.0042   10 +1 NS	0.612   0.021   10 0 NS	0.244   0.015   10 -15 NS	0.987   0.033   10 +1 NS	2.07   0.03   10 +2 NS
Test item 1000 mg/kg	Mean SEM N % P	273   6   9 +3 NS	8.24   0.25   9 +14 ?	0.506   0.028   9 +2 NS	1.77   0.06   9 +8 NS	0.0739   0.0038   9 +12 NS	0.0879   0.0058   9 +3 NS	0.568   0.045   9 -7 NS	0.263   0.017   9 -9 NS	0.951   0.055   9 -3 NS	2.05   0.02   9 +1 NS
	Threshol	d            23	0.98	0.077	0.18	0.0095	0.0162	0.201	0.071	0.129	0.07

%:variation expressed in percentage in relation to control values

Vehicle:Corn oil

NS:P>0.05,?:P≤0.05,??:P≤0.01,when compared with control group

Analysis of variance with Dunnett's test if P≤0.05

Table 7.5.1/13: Macroscopic observations - Group incidences - Male (mean table)

Organs	Observations	Vehicle	Testitem100 mg/kg	Testitem300 mg/kg	Testitem1000 mg/kg
Liver	Multiplepunctateor punctate Totalanimalsinvolved	1/10   1	0/9   0	1/8   1	0/8   0
Heart	Punctateorpoint Totalanimalsinvolved	0/10 0	0/9 0	1/8 1	0/8 0
Lung	Enlarged Punctateorpoint Pale Area Totalanimalsinvolved	0/10 0/10 1/10 1/10 1	0/9 0/9 1/9 1/9 1	0/8 2/8 0/8 0/8 2	1/8 1/8 1/8 1/8 1
Thymus	Dark Punctateorpoint Area Totalanimalsinvolved	0/10 1/10 0/10 1	0/9 2/9 1/9 3	0/8 4/8 2/8 6	1/8 1/8 0/8 2
Mesentericlymph nodes	Dark   Totalanimalsinvolved	0/10   0	0/9   0	0/8   0	1/8   1
Spleen	Enlarged Totalanimalsinvolved	1/10 1	0/9 0	0/8 0	0/8 0
Sub-maxillarylymph node	Dark   Punctateorpoint Totalanimalsinvolved	0/10   4/10 4	4/9   2/9 4	0/8   1/8 1	0/8   0/8 0
Adrenals	Enlarged Totalanimalsinvolved	1/10 1	0/9 0	0/8 0	0/8 0

Vehicle: Corn oil

Table 7.5.1/13: Macroscopic observations - Group incidences - female (mean table)

Organs	Observations	Vehicle	Testitem100 mg/kg	Testitem300 mg/kg	Testitem1000 mg/kg
Oesophagus	Mass Total animals involved	1/10 1	0/10 0	0/10 0	0/9 0
Heart	Dark Firm area Total animals involved	0/10 0/10 0	0/10 0/10 0	1/10 1/10 1	0/9 0/9 0
Lung	Mottled Punctate or point Pale AreaTotal animals involved	0/10 0/10 1/10 0/10 1	1/10 0/10 0/10 0/10 1	0/10 0/10 0/10 0/10 0	1/9 1/9 0/9 1/9 2
Uterus	Dilatation Total animals involved	1/10 1	1/10 1	0/10 0	1/9 1
Thymus	Enlarged Dark Punctate or point Area Total animals involved	0/10 0/10 1/10 1/10 2	1/10 0/10 0/10 1/10 2	0/10 1/10 0/10 0/10 1	0/9 0/9 0/9 0/9 0
Sub-maxyllarylymph node	Dark Punctate or point Total animals involved	0/10   0/10 0	1/10   2/10 2	0/10   0/10 0	1/9   1/9 1

Vehicle: Corn oil

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions, N-(2-hydroxypropyl) Oleamide Batch No. 160602013073 w/o Solvent, administered by the oral route for 13 weeks at 1000 mg/kg induced mortality and clinical signs, hepatic changes including higher hepatic plasma enzyme activity, higher liver weight and periacinar
hepatocytic hypertrophy. There were also bone marrow microscopic changes.
At 300 mg/kg, there was mortality in male and slight clinical signs. Higher hepatic plasma enzyme activity was not accompanied by microscopic changes.
At 100 mg/kg, one male was killed early on D77. No lesions at histopathology examination suggested the cause of death; however similar effects as those seen at the highest dose was noted such as higher hepatic enzymes activities or mild periacinar hepatocytic hypertrophy. In other animals treated at 100 mg/kg, there were no changes in hepatic plasma enzyme and no microscopic changes.
Whatever the tested doses, N-(2-hydroxypropyl) Oleamide induced mortality, a lower food consumption and lower body weight gain in males. The male gender seems to more sensitive than female. Taken into consideration the mortality and similar effects as those seen at the highest dose in a single male at 100 mg/kg, the No Observed Adverse Effect Level (NOAEL) was not be determined in males. In females, the NOAEL corresponds to 300 mg/kg.

Executive summary:

In a GLP-compliant Repeated Dose 90-Day Oral Toxicity Study in Rodents performed according to OECD guideline 408, N-(2-hydroxypropyl) Oleamide diluted in corn oil was administered by gavage to groups of male and female Sprague-Dawley rats (10/sex/dose) at the dose levels of 0, 100, 300, 1000 mg /kg bw/ day for 13 weeks (at constant administration volume of 5 mL/kg bw).

Morbidity/mortality checks were performed twice daily.

Clinical observations were performed before the first dosing and daily. A full clinical examination was performed once a week. Functional and neurobehavioural tests were performed before the first dosing and in the last week.

Body weight was recorded at predose and once a week.

Food consumption was measured weekly.

Ophthalmological examination was performed before the first dosing and during the last week.

Blood samples for haematology parameters and clinical chemistry analysis and urine were collected on D92.

All animals were killed on D92 (or D91 for one female). Selected organs were weighed, fixed and preserved at necropsy and examined histopathologically. Epidydimides were collected on D92 for sperm analysis.

5 males treated with test item (2 males at 300 mg/kg and 2 males at 1000 mg/kg) were found dead between D59 and D88 and one male treated with test item at 100 mg/kg was killed early on D77. One female treated with test item at 1000 mg/kg was found dead on D91.

On the days before death, there were no particular clinical signs but on the day of the death, there was mainly bradypnoea or polypnoea or absence or decrease in spontaneous locomotor activity

There was dose related statistically significant higher ALT, AST and ALP activities in males treated with test item at 300 and 1000 mg/kg and statistically significant higher ALT activity in females treated at 1000 mg/kg.

There were statistically significant changes at 1000 mg/kg:

• higher liver weight in males and females

• higher adrenals weight in males

• lower thymus weight in males

• higher testes and epididymides weight in males

Under the experimental conditions, N-(2-hydroxypropyl) Oleamide Batch No. 160602013073 w/o Solvent, administered by the oral route for 13 weeks at 1000 mg/kg induced mortality and clinical signs, hepatic changes including higher hepatic plasma enzyme activity, higher liver weight and periacinar hepatocytic hypertrophy. There were also bone marrow microscopic changes. At 300 mg/kg, there was mortality in male and slight clinical signs. Higher hepatic plasma enzyme activity was not accompanied by microscopic changes. At 100 mg/kg, one male was killed early on D77. No lesions at histopathology examination suggested the cause of death; however similar effects as those seen at the highest dose was noted such as higher hepatic enzymes activities or mild periacinar hepatocytic hypertrophy. In other animals treated at 100 mg/kg, there were no changes in hepatic plasma enzyme and no microscopic changes.

Whatever the tested doses, N-(2-hydroxypropyl) Oleamide induced mortality, a lower food consumption and lower body weight gain in males. The male gender seems to more sensitive than female. Taken into consideration the mortality and similar effects as those seen at the highest dose in a single male at 100 mg/kg, the No Observed Adverse Effect Level (NOAEL) was not be determined in males. In females, the NOAEL corresponds to 300 mg/kg.