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Description of key information

In a preliminary 7 day- DRF study with Wistar rats dose levels were 0, 125, 250 and 500 mg/kg bw/d. The aim of this study was to determine suitable dose levels for the oral 28 day repeated dose toxicity study. The results indicated severe toxicological effects in the highest dose groups such as increased mortality and severe clinical effects (prostration, piloerection, lethargy, hypothermia, hunched posture, loss of righting reflex and laboured respiration) and decreased body weight. In macroscopic examinations a mottled appearance of the liver for all animals, with the liver for two animals also appearing dark, gaseous distension of the stomach with the non-glandular region appearing thin in all animals and reddened appearance of the lungs for two animals were found. In the 250 mg/kg dose group also severe clinical effects occurred mainly in male animals such as lethargy, hunched posture, increased respiration rate and staining around snout and further notable body weight loss between Days 5-7. Based on these effects one male animal was subsequently killed on Day 7.

Female rats were found to be less sensitive to the test material.

Based on the results of this study, the high dosage for more long term investigation of toxicity in male and female rats should not lie much greater than 125 mg/kg bw/day (Envigo, 2018).

In the subsequently performed 28d oral repeated dose toxicity with male and female Wistar Han™:RccHan™:WIST strain rats 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.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
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
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
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.
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
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
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
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)
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
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
Critical effects observed:
no
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.

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Dose range finding study for 28 day RDT study (Envigo 2018)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
05 June 2017 - 05 July 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Short term toxicity study as dose range finding study
Justification for type of information:
The short term study is used as dose range finding study for the repeated dose toxicity study (28 days).
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study was conducted in accordance with the UK Home Office Guidance document on regulatory Toxicology and Safety Evaluation Studies and the OECD guidance document on recognition, assessment and use of clinical signs as humane endpoints for experimental animals used in safety evaluation.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wister Han: RccHan:WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: males between 244 - 282 g, Females: 175 - 199 g
- Acclimation period: 21 days
- Housing: in groups of three by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet: ad libitum, Pellet diet (at first (Rodent 2018C Teklad Global Certified Diet then for main study Rodent 2014C Teklad Global Certified Diet)
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
Animals were housed in a single air-conditioned room within Envigo research Limited, shardlow, UK barrier Maintained Rodent Facility.
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): at least 15
- Photoperiod: 12 hrs dark and 12 hrs light

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily, for seven 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.
Vehicle:
polyethylene glycol
Remarks:
Polyethylene glycol 400
Details on oral exposure:
The animals were allocated to dose groups using a randomization procedure based on stratified body weights and the group mean body weights were then determined to ensure
similarity between the dose groups. The animals were uniquely identified within the study, by an ear punch system routinely used in these laboratories.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
For the purpose of this study the test item was prepared at the appropriate concentrations in Polyethylene glycol 400.
The test item was administered within approximately two hours of it being formulated. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation.
Duration of treatment / exposure:
Seven days
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Concentration: 0 mg/ml
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
Concentration: 25 mg/ml
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Concentration: 50 mg/ml
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Concentration: 100 mg/ml
No. of animals per sex per dose:
Three animals per sex and dose group
Control animals:
yes, concurrent vehicle
Details on study design:
In the absence of any toxicity data, preliminary toxicity work was undertaken, treating one male and one female rat at 75 mg/kg bw/day for three days, then increasing to 150 mg/kg
bw/day for the following three days, then dosed at 300 mg/kg bw/day over a further three days. Further preliminary toxicity work was carried out on two further pairs of animals. One
pair of animals were dosed at 600 mg/kg bw/day. A second pair of animals were dosed at 300 mg/kg bw/day for three days, then the dose level was increased to 1000 mg/kg bw/day.
Animals treated with 1000 mg/kg bw/day were found dead on Day 3, prior to dosing.
Observations and examinations performed and frequency:
All animals were examined for overt signs of toxicity, ill health or behavioral change immediately before dosing, up to thirty minutes after dosing and one hour after dosing.
Additional observations were also made four hours following dosing (not at weekends or on public holidays). All observations were recorded.
Individual body weights were recorded on Days 1, 3, 5 and 8.
Food consumption was recorded for each cage group for Days 1 to 3, 3 to 5 and 5 to 8. Food conversion efficiency was calculated retrospectively.
Water intake was measured and recorded daily for each cage group.
Sacrifice and pathology:
On completion of the dosing period, all surviving animals were killed by carbon dioxide asphyxiation followed by exsanguination and subjected to an internal and external
macroscopic examination. Any tissues showing macroscopic abnormalities were preserved in buffered 10% formalin.
Statistics:
Data were processed to give summary incidence or group mean and standard deviation values where appropriate. All data were summarized in tabular form.
The following computerized systems were used on the study:
Provantis
Delta BMS
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs for the surviving male animals on Day 6 of HD rats before termination for animal welfare included prostration, piloerection, lethargy, hypothermia (cold to touch), hunched posture, loss of righting reflex and laboured respiration. The females showed no clinical signs until day 6, however, they were terminated on day 6.
In the intermediate dose group one male showed adverse clinical signs: lethargy, hunched posture, increased respiration rate and staining arround snout.
Mortality:
mortality observed, treatment-related
Description (incidence):
In the HD group one male was found dead on day 6 and the two other males showed adverse clinical effects and were therefore terminated.
One male of the intermediate group was killed on day 7 due to adverse clinical signs.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male animals in HD showed notable weight loss between days 3-5 and days 5-6. Food consumption was lower than control during days 1-3 and to a gretaer extent days 3-5.
The females showed slight body weight loss between days 3-5 and also slight decreased food consumption.
Males of the intermediate group showed loss of body weight between day 5-8 and also food consumption was lower than control throughout the study. For the females body weight gain was slightly lower than controls from day 3 of the study. Food intake was also lower during day 5-8.
In the low dose group there was only a slightly lower food intake and subsequently also only a slightly lower body weight gain during the first two days of the study of the male rats.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In the HD group macroscopic necropsy examination of the males revealed a mottled appearance of the liver for all animals, with the liver for two animals (including the decedent) appearing dark, gaseous distension of the stomach with the nonglandular region appearing thin in all animals and reddened appearance of the lungs for two animals (including the decedent). Two females showed gaseous distension of the stomach.
In the intermediate group, necropsy revealed a thin non-glandular region of the stomach in all three male animals, with a thickened glandular region for two animals and gaseous distension of the stomach for the remaining animal.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
500 mg/kg bw/day
Treatment of males at 500 mg/kg bw/day was associated with notable mean body weight losses between Days 3-5 and Days 5-6, with all individual animals being affected. Food consumption was lower than control during Days 1-3 and, to a greater extent, Days 3-5. Water consumption was lower than control during Days 3-4 and 4-5 but, to a certain extent, these values may have been influenced by the lower food consumption. On Day 6, one male was found dead and the two surviving males showed adverse clinical signs and were therefore terminated. Clinical signs for the surviving male animals on Day 6 included prostration, piloerection, lethargy, hypothermia (cold to touch), hunched posture, loss of righting reflex and laboured respiration. Macroscopic necropsy examination of the males revealed a mottled appearance of the liver for all animals, with the liver for two animals (including the decedent) appearing dark, gaseous distension of the stomach with the nonglandular region appearing thin in all animals and reddened appearance of the lungs for two animals (including the decedent). Although no clinical signs had been apparent for females at 500 mg/kg bw/day, they were terminated on Day 6 of the study as this dosage had clearly been demonstrated to be unsuitable in the male animals. Females at this dosage did show a slight mean body weight loss between Days 3-5 (two females affected) and food consumption was lower than control during Days 1-3 and 3-5. Water consumption was higher than control throughout their treatment period. Macroscopic necropsy examination revealed gaseous distension of the stomach for two females.
250 mg/kg bw/day
Treatment of males at 250 mg/kg bw/day was associated with slight mean body weight loss between Days 3-5. One male was subsequently killed on Day 7 after showing lethargy, hunched posture, increased respiration rate and staining around snout and a more notable body weight loss between Days 5-7. The two surviving males showed body weight loss between Day 5 and Day 8 (termination). Food consumption was lower than control throughout the study, with differences being more marked as the study progressed. Water consumption was also lower than control from Day 6 but, to a certain extent, values may have been influenced by lower food consumption. Necropsy revealed a thin non-glandular region of the stomach in all three animals, with a thickened glandular region for two animals and gaseous distension of the stomach for the remaining animal. Report Envigo Study Number: LR59HW Page 18 For females at this dosage, body weight gain was slightly lower than control from Day 3 of the study. Food consumption was lower than control between Days 3-5 but there was no obvious effect on water consumption. There were no clinical signs apparent during the study and no macroscopic necropsy findings were apparent at termination.
125 mg/kg bw/day
For males at 125 mg/kg bw/day, body weight gain and food consumption were slightly lower than control during the first two days of treatment. Thereafter body weight gain was similar to control to termination, although slightly lower food intake was again apparent during Days 5-8. There were no obvious effects on body weight gain or food consumption for females at this dosage. No clinical signs were apparent for either sex during the study and water consumption for both sexes was similar to control. No macroscopic necropsy findings were apparent for either sex at termination.
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
gross pathology
mortality
Conclusions:
Based on the results of this study, the high dosage for more long term investigation of toxicity in the rat should not lie much greater than 125 mg/kg bw/day and a dosage sequence
of 0 (Control), 30, 75 and 150 mg/kg bw/day may be appropriate.
Executive summary:

The test item was administered by gavage to three groups, each of three male and three female Wistar Han™:RccHan™:WIST strain rats, for up to seven consecutive days, at dose levels of 125, 250 and 500 mg/kg bw/day. A control group of three males and three females was dosed with vehicle alone (Polyethylene glycol 400) over the same treatment period. Clinical signs, body weight change, dietary intake and water consumption were monitored during the study.All animals were subjected to gross necropsy examination.

Treatment of males at 500 mg/kg bw/day was associated with body weight losses between Days 3-5 and Days 5-6, for all animals. Food consumption was lower than control during Days 1-3 and, to a greater extent, Days 3-5. Water consumption was lower than control during Days 3-4 and 4-5. On Day 6, one male was found dead and the two surviving males were terminated due to adverse clinical signs including prostration, piloerection, lethargy, hypothermia (cold to touch), hunched posture, loss of righting reflex and laboured respiration. Macroscopic necropsy revealed a mottled appearance of the liver for all animals, with the liver for two animals also appearing dark, gaseous distension of the stomach with the non-glandular region appearing thin in all animals and reddened appearance of the lungs for two animals. Females at 500 mg/kg bw/day were terminated on Day 6 of the study as this dosage was clearly unsuitable for the male animals. Females showed a slight mean body weight loss between Days 3-5 (with two females affected) and lower food consumption during Days 1-3 and 3-5 compared to control. Water consumption was higher than control throughout their treatment period. No clinical signs were apparent for females at this dosage. Macroscopic necropsy revealed gaseous distension of the stomach for two females.

Treatment of males at 250 mg/kg bw/day was associated with slight mean body weight loss between Days 3-5. One male was subsequently killed on Day 7 after showing lethargy, hunched posture, increased respiration rate and staining around snout and further notable body weight loss between Days 5-7. The two surviving males also showed body weight loss between Day 5 and Day 8 (termination). Food consumption was lower than control throughout the study, with differences being more marked as the study progressed and water consumption was lower than control from Day 6. Necropsy revealed a thin non-glandular region for the stomach in all three animals, with a thickened glandular region for two animals and gaseous distension of the stomach for the remaining animal. For females at 250 mg/kg bw/day, body weight gain was slightly lower than control from Day 3 of the study. Food consumption was lower than control between Days 3-5 but there was no effect on water consumption. No clinical signs or macroscopic necropsy findings were apparent for females at this dosage.

For males at 125 mg/kg bw/day, body weight gain and food consumption were slightly lower than control during the first two days of treatment; slightly lower food intake was also apparent during Days 5-8. There were no effects on body weight gain or food consumption for females at this dosage. Water consumption for both sexes was similar to control and no clinical signs or macroscopic necropsy findings were apparent for either sex at 125 mg/kg bw/day during the study.

Based on the results of this study, the high dosage for more long term investigation of toxicity in the rat should not lie much greater than 125 mg/kg bw/day and a dosage sequence of 0 (Control), 30, 75 and 150 mg/kg bw/day may be appropriate.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study planned
Justification for type of information:
There are no GLP, non-GLP studies nor any historical human data for the registered substance available. Application of grouping, read-across and (Q)SAR is difficult due to the complex composition of the material (UVCB). Furthermore, the registrant is not aware of any In vitro method which can fulfil data requirements for this endpoint. There are no studies which can be used in combination to fullfil data requirements using weight of evidence approach. Substance-tailored exposure driven testing is not applicable for the registered substance and relevant uses.
As a consequence we have to propose a 90 day oral repeated dose toxicity study to fulfill the endpoint repeated-dose toxicity. Because of the fact that the compound is classified to be corrosive to the skin and the eyes (Skin Corr. cat. 1B), dose concentrations have to be selected below the corrosive concentration. To get a more detailed picture of the likely systemic effects caused by the test material, the oral administration is selected.
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In the available oral 28-day study which was performed as a dose range finding study, no adverse treatment related effects occured up to the highest tested dose so that no LOAEL could be derived. As a consequence, a testing proposal for an oral subchronic (90-day) repeated dose study is included in the dossier. Because of the fact that the substance is classified to be corrosive to the skin and the eyes the selected dose levels of the 90 day study have to be below the corrosive concentration to be able to gain further information on the systemic effects of the test substance.

Justification for classification or non-classification

Based on the results of an oral 28 day repeated dose toxicity study, the test material is not classified according to CLP regulation 1272/2008.