[No public or meaningful name is available]

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:

2017-10-18 to 2018-02-04

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:

2017

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)

Version / remarks:

adopted 03 October 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Directive 96/54/EC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

{2-[(2-aminoethyl)amino]ethyl}(benzyl)amine

Cas Number:

39549-34-3

Molecular formula:

C11H19N3

IUPAC Name:

{2-[(2-aminoethyl)amino]ethyl}(benzyl)amine

Constituent 2

Reference substance name:

benzyl(2-{[2-(benzylamino)ethyl]amino}ethyl)amine

Cas Number:

17327-80-9

Molecular formula:

C18H25N3

IUPAC Name:

benzyl(2-{[2-(benzylamino)ethyl]amino}ethyl)amine

Constituent 3

Reference substance name:

Piperazine-1,4-diethylamine

EC Number:

229-428-8

EC Name:

Piperazine-1,4-diethylamine

Cas Number:

6531-38-0

Molecular formula:

C8H20N4

IUPAC Name:

2,2'-piperazine-1,4-diyldiethanamine

Constituent 4

Reference substance name:

2-piperazin-1-ylethylamine

EC Number:

205-411-0

EC Name:

2-piperazin-1-ylethylamine

Cas Number:

140-31-8

Molecular formula:

C6H15N3

IUPAC Name:

2-piperazin-1-ylethanamine

Constituent 5

Reference substance name:

N-(2-aminoethyl)-N'-[2-(benzylamino)ethyl]ethylenediamine

EC Number:

244-734-1

EC Name:

N-(2-aminoethyl)-N'-[2-(benzylamino)ethyl]ethylenediamine

Cas Number:

22029-44-3

Molecular formula:

C13H24N4

IUPAC Name:

N-(2-aminoethyl)-N'-[2-(benzylamino)ethyl]ethane-1,2-diamine

Constituent 6

Reference substance name:

2-{4-[(benzylamino)methyl]piperazin-1-yl}ethan-1-amine

Molecular formula:

C14H24N4

IUPAC Name:

2-{4-[(benzylamino)methyl]piperazin-1-yl}ethan-1-amine

Constituent 7

Reference substance name:

[2-(benzylamino)ethyl][2-(piperazin-1-yl)ethyl]amine

Molecular formula:

C15H26N4

IUPAC Name:

[2-(benzylamino)ethyl][2-(piperazin-1-yl)ethyl]amine

Constituent 8

Reference substance name:

1,12-diphenyl-2,5,8,11-tetraazadodecane

Cas Number:

140840-03-5

Molecular formula:

C20H30N

IUPAC Name:

1,12-diphenyl-2,5,8,11-tetraazadodecane

Constituent 9

Reference substance name:

benzyl(2-{4-[2-(benzylamino)ethyl]piperazin-1-yl}ethyl)amine

Cas Number:

176906-02-8

Molecular formula:

C22H32N4

IUPAC Name:

benzyl(2-{4-[2-(benzylamino)ethyl]piperazin-1-yl}ethyl)amine

Test material form:

liquid: viscous

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were
obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were
examined for signs of ill-health or injury. The animals were acclimatized for eight days
during which time their health status was assessed. A total of forty animals (twenty males
and twenty females) were accepted into the study. At the start of treatment the males
weighed 175 to 216g, the females weighed 136 to 165g, and were approximately six weeks
old.
Animal Care and Husbandry
The animals were housed in groups of five by sex in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The
animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad
Global Certified Diet, Envigo RMS (UK) Limited., Oxon, UK) was used. A certificate of
analysis of the batch of diet used is given in Annex 5. Mains drinking water was supplied
from polycarbonate bottles attached to the cage. Environmental enrichment was provided in
the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
The diet, drinking water, bedding and environmental enrichment were considered not to
contain any contaminant at a level that might have affected the purpose or integrity of the
study.
The animals were housed in a single air-conditioned room within the Envigo Research
Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at
least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to
give twelve hours continuous light and twelve hours darkness. Environmental conditions
were continuously monitored by a computerized system, and print-outs of hourly
temperatures and humidities are included in the study records. The Study Plan target ranges
for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term
deviations from these targets were considered not to have affected the purpose or integrity of
the study; see deviations from Study Plan.
The animals were randomized and allocated to dose groups on arrival. Subsequent group
mean body weights were checked on Day–2 and animals reallocated where necessary to
ensure similarity between the groups. The cage distribution within the holding rack was also
randomized. The animals were uniquely identified within the study by an ear punching
system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered daily, for up to twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of Polyethylene glycol 400.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Dose levels were based on available toxicity data, including the results of the Seven Day Repeated Dose Oral (Gavage) Range-Finding Study in the Rat (Envigo Study Number LR59HW). In this preliminary study it was concluded that a dosage of 250 mg/kg bw/day was too high for more long term investigation of toxicity, such as this twenty-eight day toxicity study. Dosages of 0 (control), 30, 75 and 150 mg/kg bw/day, using a treatment volume of 5 mL/kg body weight, have been chosen, in collaboration with the Sponsor, for investigation in this study.

Vehicle:

polyethylene glycol

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item was prepared at the appropriate concentrations as a
solution in Polyethylene glycol 400. The stability and homogeneity of the test item
formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical
Services. Results show the formulations to be stable for at least five hours when stored at
ambient temperature in the light. Formulations were therefore prepared daily during the
treatment period.
Samples of the test item formulations were taken and analyzed on two occasions for
concentration of Benzylated polyamine at Envigo Research Limited, Shardlow, UK,
Analytical Services. The method used for analysis was GC.
The results indicate that the prepared formulations were within 78 to 105% of the nominal concentration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

75 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 male and 5 female

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were allocated to treatment groups as follows:
Treatment Group Dose Level (mg/kg bw/day) Treatment Volume (mL/kg) Concentration (mg/mL) Animal Numbers
Male Female
Control 0 5 0 5 (1-5) 5 (6-10)
Low 30 5 6 5 (11-15) 5 (16-20)
Intermediate 75 5 15 5 (21-25) 5 (26-30)
High 150 5 30 5 (31-35) 5 (36-40)

The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All
observations were recorded.
Body Weight
Individual body weights were recorded on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to terminal kill.
Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.
Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes except during Week 3 where water intake was measured gravimetrically.
Functional Observations
Prior to the start of treatment and on Days 7, 14, 21 and 26, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on
all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.
Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for
Behavioral Assessments and Sensory Reactivity Tests.
Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were
randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory
conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during
the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979). Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each
animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn
along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive
trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).
Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin
(1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach
Normal range data for Functional Performance Assessments are given
In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all surviving animals from each test and control group at the end of the study (Day 28). Blood samples were
obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.
Hematology
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).
Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++) Triglycerides (Trigs)

Sacrifice and pathology:

Necropsy
On completion of the dosing period all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at lower than -60 °C. No treatment-related effects on the
pituitary-thyroid axis were identified, therefore these samples were discarded.
Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed
before fixation:
Adrenals Liver
Brain Ovaries
Epididymides Spleen
Heart Testes
Kidneys Thymus
Pituitary (weighed after partial fixation) Thyroid/Parathyroid (weighed after partial fixation)
Prostate and Seminal Vesicles Uterus with Cervix (and oviducts)
(with coagulating glands and fluids)
Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals Ovaries
Aorta (thoracic) Pancreas
Bone & bone marrow (femur including stifle joint) Pituitary
Bone & bone marrow (sternum) Prostate
Brain (including cerebrum, cerebellum and Rectum
pons) Salivary glands (submaxillary)
Cecum Sciatic nerve
Colon Seminal vesicles (with coagulating
Duodenum glands and fluids)
Epididymides ♦ Skin
Esophagus Spinal cord (cervical, mid thoracic
Eyes * and lumbar)
Gross lesions Spleen
Heart Stomach
Ileum Testes ♦
Jejunum Thymus
Kidneys Thyroid/Parathyroid
Liver Trachea
Lungs (with bronchi)# Urinary bladder
Lymph nodes (mandibular and mesenteric) Uterus & Cervix (and oviducts)
Mammary gland Vagina
Muscle (skeletal)
All tissues were dispatched to the histology processing Test Site (Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, UK) for processing (Principal Investigator:
N Fower). The tissues shown in bold from all control and 150 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and
stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. In addition, sections of testes from all
Control and 150 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.
Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the histopathology results for the study was conducted by Vasanthi Mowat at
Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS. A histology and histopathology examination phase report is presented in Annex 1 and
represents the consensus view of both pathologists.

Other examinations:

Necropsy
On completion of the dosing period all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the serum
from each animal was stored frozen at lower than -60 °C. No treatment-related effects on the
pituitary-thyroid axis were identified, therefore these samples were discarded.
Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed
before fixation:
Adrenals Liver
Brain Ovaries
Epididymides Spleen
Heart Testes
Kidneys Thymus
Pituitary (weighed after partial fixation) Thyroid/Parathyroid (weighed after partial fixation)
Prostate and Seminal Vesicles Uterus with Cervix (and oviducts)
(with coagulating glands and fluids)
Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered
10% formalin, except where stated:
Adrenals Ovaries
Aorta (thoracic) Pancreas
Bone & bone marrow (femur including stifle joint) Pituitary
Bone & bone marrow (sternum) Prostate
Brain (including cerebrum, cerebellum and Rectum
pons) Salivary glands (submaxillary)
Cecum Sciatic nerve
Colon Seminal vesicles (with coagulating
Duodenum glands and fluids)
Epididymides ♦ Skin
Esophagus Spinal cord (cervical, mid thoracic
Eyes * and lumbar)
Gross lesions Spleen
Heart Stomach
Ileum Testes ♦
Jejunum Thymus
Kidneys Thyroid/Parathyroid
Liver Trachea
Lungs (with bronchi)# Urinary bladder
Lymph nodes (mandibular and mesenteric) Uterus & Cervix (and oviducts)
Mammary gland Vagina
Muscle (skeletal)
All tissues were dispatched to the histology processing Test Site (Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, UK) for processing (Principal Investigator:
N Fower). The tissues shown in bold from all control and 150 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and
stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. In addition, sections of testes from all
Control and 150 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.
Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the histopathology results for the study was conducted by Vasanthi Mowat at
Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS. A histology and histopathology examination phase report is presented in Annex 1 and
represents the consensus view of both pathologists.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect
the significance of intergroup differences from control; statistical significance was achieved
at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry,
Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module
as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method.
The homogeneity of variance from mean values was analyzed using Bartlett’s test.
Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with
appropriate covariates. Any transformed data were analyzed to find the lowest treatment
level that showed a significant effect using the Williams Test for parametric data or the
Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity
of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel
(non-parametric) test to determine significant difference from the control group. Where the
data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test
(parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Increased salivation was seen in 3/5 males and 2/5 females dosed with 150 mg/kg bw/day; two males and one female showed this observation on a single isolated occasion, one male
showed it on two occasions (Days 25 and 28), and one female showed it on five occasions
(Days 10, 14, 16, 27 and 28). One female dosed at 75 mg/kg bw/day showed increased salivation on an isolated occasion. At the level observed this probably represents difficulty in
dosing isolated animals on a few occasions. 3/5 males and 4/5 females treated with 150 mg/kg bw/day showed noisy respiration on a number of occasions throughout the study. 3/5
males and 4/5 females treated with 75 mg/kg bw/day showed noisy respiration on a number of occasions. One female treated with 30 mg/kg bw/day showed noisy respiration on a single
occasion. One male dosed at 150 mg/kg bw/day was observed to have a decreased respiratory rate on Day 17, though only for a transient period. These findings may represent
accidental aspiration of the test item during the dosing procedure and are considered not to be indicative of systemic toxicity.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female dosed with 75 mg/kg bw/day was found dead and cannibalised on Day 9 relative to the start of dosing (after having received eight consecutive daily doses). No clinical signs
had been apparent for this animal prior to this event. Necropsy findings revealed a tear in the esophagus and therefore this death was considered to be due to trauma during the dosing
procedure, and not related to the administration of test item. Pale discoloration of the heart and small discolored (red) lungs were also apparent at necropsy, but these findings were
considered to be incidental.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment on bodyweight or body weight gains for either sex at 30, 75 or 150 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment on food consumption or food conversion efficiency for either sex at 30, 75 or 150 mg/kg bw/day.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

There was no effect of treatment on water consumption for either sex at 30, 75 or 150 mg/kg bw/day; daily visual inspection of water bottles, and gravimetric measurement of water consumption during Week 3 revealed no overt intergroup differences.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of hematology parameters did not indicate any obvious effect of treatment for either sex at 30, 75 or 150 mg/kg bw/day.
A statistically significant increase in reticulocyte count was identified in all treated animals, although no dosage relationship was present. All values for treated animals were within historical control ranges, and in isolation, without any effects on other erythrocyte parameters or supporting histopathological changes, the finding can be considered to be incidental and unrelated to treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Assessment of blood chemistry parameters did not indicate any adverse effect of treatment for either sex at 30, 75 or 150 mg/kg bw/day.
Mean glucose levels for all treated animals were statistically significantly lower than controls. However 4/5 control male individual values exceeded the historical control normal
range, whereas all treated male individual values were within the normal range, with the exception of one male dosed at 75 mg/kg bw/day. For female individual values only 1/5
controls exceeded the normal historical control range and all values for treated females were within the normal historical control range. This finding is likely to represent unusually high
control values, rather than any true effect of treatment.
Mean calcium levels were statistically significantly increased for females treated with 150 mg/kg bw/day compared to controls. However all individual female control values and all
individual values for females treated with 150 mg/kg bw/day were below normal historical ranges. The significance of this increase is therefore equivocal and considered to be of notoxicological significance.
Alanine aminotransferase (ALAT) levels were statistically significantly decreased in females dosed with 30 and 75 mg/kg bw/day. This enzyme is typically released into the bloodstream as a result of damage
affecting the liver, thus a decrease in alanine aminotransferase is not an adverse effect. Furthermore, in the absence of any histopathological liver findings, or similar findings in females treated with 150 mg/kg
bw/day this finding is considered to be of no toxicological significance. Aspartate aminotransferase (ASAT) and ALAT levels were significantly higher in males dosed with
150 mg/kg bw/day. Similar to ALAT, ASAT is released when cells lyse, although it is found in high concentrations within several highly metabolic tissues such as the heart, liver, skeletal
muscle, kidneys and pancreas. In absence of any histopathological changes in these organs, these findings in isolation cannot be considered adverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behavioral Assessments did not indicate any effect of treatment for either sex at 30, 75 or 150 mg/kg bw/day. There were no clinical signs apparent during behavioural assessments for either sex at 30 or 75 mg/kg bw/day. One male dosed at 150 mg/kg bw/day showed noisy respiration on Day 26, this same male had shown instances of noisy respiration previously during the study. There were no clinical signs during behavioural assessments for females at 150 mg/kg bw/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Assessment of organ weights did not indicate any adverse effect of treatment for either sex at 30, 75 or 150 mg/kg bw/day.
At the end of the treatment period, statistically significant increases in absolute and body weight relative liver weights were identified in females treated with 150 mg/kg bw/day in
comparison with controls. However, for both absolute and bodyweight relative values, 4/5 individual values were within the historical control range. Furthermore there was no
consistent dosage relationship for either absolute or relative liver weights, and in the absence of any supporting histopathological change, this finding was considered to be incidental and
of no toxicological significance.
Males treated with 75 mg/kg bw/day showed a statistically significant increase in absolute and body weight relative thyroid weights. In the absence of a similar effect at 150 mg/kg bw/day or any
histopathological correlates, the intergroup difference was considered of no toxicological significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The incidence, type and distribution of macroscopic findings observed at terminal necropsy of surviving animals did not indicate any effect of treatment at dosages of 30, 75 or 150 mg/kg bw/day.
Incidental findings included one control male with a small thymus, one male treated at 30 mg/kg bw/day with a pale yellow fluid filled mass on both the left and right epididymides, one male treated
at 75 mg/kg bw/day with small epididymides and testes, and one male reated with 150 mg/kg bw/day with a small prostate. In isolation, these findings were considered to be of no toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No microscopic findings which could be attributed to administration of the test item were noted; all findings noted were considered to be incidental and not related to the
administration of the test item.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No microscopic findings which could be attributed to administration of the test item were noted; all findings noted were considered to be incidental and not related to the
administration of the test item.

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Oral administration of Benzylated polyamine to male and female Wistar Han™:RccHan™:WIST strain rats for up to twenty-eight consecutive days at dose levels of 30, 75 and 150 mg/kg bw/day resulted in no adverse treatment related changes. The No Observed Adverse Effect Level (NOAEL) was considered to be 150 mg/kg bw/day.

Executive summary:

The test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for up to twenty-eight consecutive days, at dose levels of 30, 75 and 150 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Polyethylene glycol 400). Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

One female dosed with 75 mg/kg bw/day was found dead and cannibalized on Day 9 relative to the start of dosing. No clinical signs had been apparent for this animal prior to this event. Necropsy findings revealed a tear in the esophagus and therefore this death was considered to be due to trauma during the dosing procedure, and not related to the administration of test item.

 There were no clinical signs observed that indicated any systemic effect of treatment at dosages of 30, 75 and 150 mg/kg bw/day. Behavioral Assessments did not indicate any effect of treatment for either sex at 30, 75 or 150 mg/kg bw/day. Functional performance tests did not indicate any effect of treatment for either sex at 30, 75 or 150 mg/kg bw/day. Intergroup differences observed in the scores for sensory reactivity did not indicate any effect of treatment for either sex at 30, 75 or 150 mg/kg bw/day.

There was no effect of treatment on bodyweight or body weight gain, no effect on food consumption or food conversion efficiency and no effect on water consumption for either sex at 30, 75 or 150 mg/kg bw/day. The assessment of hematology parameters did not indicate any effect of treatment for either sex at 30, 75 or 150 mg/kg bw/day.

The incidence, type and distribution of macroscopic findings observed at terminal necropsy of surviving animals did not indicate any effect of treatment at dosages of 30, 75 or 150 mg/kg bw/day. Assessment of organ weights did not indicate any adverse effect of treatment for either sex at 30, 75 or 150 mg/kg bw/day. No microscopic findings which could be attributed to administration of the test item were noted.

Oral administration of Benzylated polyamine to male and female Wistar Han™:RccHan™:WIST strain rats for up to twenty-eight consecutive days at dose levels of 30, 75 and 150 mg/kg bw/day resulted in no adverse treatment related changes. The No Observed Adverse Effect Level (NOAEL) was considered to be 150 mg/kg bw/day.