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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2020-02-20 to 2021-03-03
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
No. 440/2008 and subsequent
revisions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 25 June 2018
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N-bis[3-(dimethylamino)propyl]-N',N'-dimethylpropane-1,3-diamine
EC Number:
251-459-0
EC Name:
N,N-bis[3-(dimethylamino)propyl]-N',N'-dimethylpropane-1,3-diamine
Cas Number:
33329-35-0
Molecular formula:
C15H36N4
IUPAC Name:
N,N-bis[3-(dimethylamino)propyl]-N',N'-dimethylpropane-1,3-diamine
Constituent 2
Reference substance name:
1,3-Propanediamine, N,N-bis[3-(dimethylamino)propyl]-N',N'-dimethyl
IUPAC Name:
1,3-Propanediamine, N,N-bis[3-(dimethylamino)propyl]-N',N'-dimethyl
Constituent 3
Reference substance name:
Polycat 9
IUPAC Name:
Polycat 9
Constituent 4
Reference substance name:
Tris(3-Dimethylamino)Propylamine
IUPAC Name:
Tris(3-Dimethylamino)Propylamine
Test material form:
other: pale yellow clear liquid
Details on test material:
Appearance: pale yellow clear liquid
Density (25 oC): 0.8487
Viscosity (25 oC): 10 cps
Flash point: 102 oC
Boiling point: 285 oC
Solubility in water (25 oC): . 0.5 g/mL
Solubility in methanol (25 oC): . 0.5 g/mL
Solubility in acetone (25 oC): . 0.5 g/mL
Solubility in chloroform (25 oC): . 0.5 g/mL
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Evonik Operations GmbH, UW19417870
- Expiration date of the lot/batch: 2022-02-16
- Purity test date: 2022-02-16
-Purity: 97.9%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light and oxidants (under
nitrogen)
- Stability under storage conditions: stable
- Stability under test conditions: In an analytical study conducted from test laboratory, a 28-hour stability at room temperature and an 8-day stability at 2-8°C were verified in the range from 1 to 50mg/mL. According to test laboratory SOPs, solutions are considered to be stable if concentration, after the defined period of storage, is still acceptable (90%-110%).

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley SD rat was the species and strain of choice because it is accepted by
many regulatory authorities and there are ample experience and background data on this
species and strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females nulliparous and non-pregnant: [yes]
- Age at study initiation: 27-29 days old
- Weight at study initiation: 75-100 g (range of 103.5-119.9 g for males and 89.7-101.4 g for females)
- Fasting period before study: no
- Housing: The animals were housed up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages. Nesting material was provided inside suitable bedding bags and changed at least twice a week.
- Diet (e.g. ad libitum): ad libitum except when blood samples (for thyroid hormone determination) were taken under light isoflurane anesthesia and condition of food deprivation.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 18 days before start of the treatment

DETAILS OF FOOD AND WATER QUALITY: Drinking water was supplied ad libitum to each cage via water bottles. A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4,
20019, Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
There was no information available to indicate that any non-nutrient substance likely to
influence the effect of the test item was present in the drinking water or the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C±2°C
- Humidity (%): 55%±15%
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2020-02-20 To: 2020-06-08

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as it is one of the possible routes of unintentional and exaggerated
exposure of the test item in man.
Dose levels were selected based on information from preliminary studies.
Vehicle:
water
Remarks:
The vehicle used was softened water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was dissolved in the vehicle. The preparations were
made daily or weekly (concentrations of 3 and 10mg/mL for the entire treatment period,
30mg/mL for Days 1 to 3, and 20mg/mL from Day 4 up to the end of the study), according
to stability data. The pH value on each preparation day was checked and recorded.
Concentrations were calculated and expressed in terms of test item as supplied.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in the range from 1 to 50 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.98; accuracy 90-110%; precision CV < 5%).
With regards to the test item a 28-hour stability at room temperature and an 8-day stability at 2-8°C were verified in the range from 1 to 50mg/mL.
The proposed preparation procedure for the test item was checked in the range from 1 to
50 mg/mL by chemical analysis (concentration) during the pre-treatment period to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in the laboratory SOPs for concentration (90-110%).
Samples of the preparations prepared on Weeks 1 and 13 were analyzed to check the concentration.
Results of the analyses were within the acceptability limits stated in test laboratory SOPs
for concentration (90-110%).

Duration of treatment / exposure:
13 weeks plus two weeks recovery limited to the control and high-dose groups.
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Due to unexpected severe clinical signs and mortality, the high dose level was reduced fromm 300 mg/kg/day to 200 mg/kg/day starting formDay 4 of the study.
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 for all groups in the main study and 5 in the recovery groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on information from
preliminary studies. The test item was administered orally by gavage at a dose volume of 10mL/kg b.w./day.
Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry: yes, at the last week of treatment

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during the study, twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to necropsy.


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


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Both eyes of all animals were examined prior to the commencement of treatment.
- Dose groups that were examined: The eyes of all animals from high dose
and control groups were re-examined duringWeek 13 of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: last week of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all aniamals in the main phase groups
- Parameters were examined as follows: Haematocrit
– Haemoglobin
– Red blood cell count
– Reticulocyte count
– Mean red blood cell volume
– Mean corpuscular haemoglobin
– Mean corpuscular haemoglobin concentration
– White blood cell count
– Differential leucocyte count
· Neutrophils
· Lymphocytes
· Eosinophils
· Basophils
· Monocytes
· Large unstained cells
– Platelets
--Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: same as above
- Animals fasted: Yes
- How many animals: same as above
- Parameters were examined as follows: – Alkaline phosphatase
– Alanine aminotransferase
– Aspartate aminotransferase
– Gamma-glutamyltransferase
– Urea
– BUN (blood urea nitrogen)
– Creatinine
– Glucose
– Triglycerides
– Inorganic phosphorus
– Total bilirubin
– Total cholesterol
– HDL
– LDL
– Total protein
– Albumin
– Globulin
– A/G Ratio
– Sodium
– Potassium
– Calcium
– Chloride

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The motor activity (MA) of all animals was measured once during Week 12 of treatment and once during Week 2 of the recovery.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No


OTHER: Oestrous cycle
At the end of the treatment and recovery periods, just prior to necropsy, vaginal smears
were taken from all animals

Sacrifice and pathology:
GROSS PATHOLOGY: Yes: Adrenal glands; Aorta; Bone marrow (from sternum); Brain (cerebrum, cerebellum, medulla/pons); Caecum; Coagulating glands; Colon; Duodenum; Epididymides; Eyes; Femur with joint; Heart; Ileum O O
Jejunum (including Peyer’s patches); Kidneys; Liver; Lungs (including mainstem bronchi); Lymph nodes – cervical; Lymph nodes – mesenteric; Mammary area; Nasal cavity; Oesophagus; Ovaries; Oviducts; Pancreas; Parathyroid glands; Pituitary gland; Prostate gland; Rectum; Salivary glands Sciatic nerve; Seminal vesicles; Skeletal muscle; Skin; Spinal column; Spinal cord; Spleen; Stomach; Testes; Thymus (where present); Thyroid gland; Trachea; Urinary bladder; Uterus – cervix; Vagina.


HISTOPATHOLOGY: Yes. Adrenal glands; Aorta; Bone marrow (from sternum); Brain (cerebrum, cerebellum, medulla/pons); Caecum; Coagulating glands; Colon; Duodenum; Epididymides; Eyes; Femur with joint; Heart; Ileum O O
Jejunum (including Peyer’s patches); Kidneys; Liver; Lungs (including mainstem bronchi); Lymph nodes – cervical; Lymph nodes – mesenteric; Mammary area; Nasal cavity; Oesophagus; Ovaries; Oviducts; Pancreas; Parathyroid glands; Pituitary gland; Prostate gland; Rectum; Salivary glands Sciatic nerve; Seminal vesicles; Skeletal muscle; Skin; Spinal cord; Spleen; Stomach; Testes; Thymus (where present); Thyroid gland; Trachea; Urinary bladder; Uterus – cervix; Vagina.
Other examinations:
Spermanalysis
During the necropsy procedure, one cauda from one epididymis of each animal completing
the scheduled test period in the high dose and the control groups was taken for spermcount
ad evaluation of motility and morphology. The animals of the mid- and low dose groups
were dosed until the evaluation of the control and high dose animals was performed. Since no treatment-related effects were seen between control and high dose males, no
assessment was carried out in males from the mid- and low dose groups as well as from
recovery groups.
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables
the significance of the differences amongst groups was assessed by analysis of variance.
Differences between each treated group and the control group were assessed by Dunnett’s
test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s
test before Dunnett’s test. If the data were found to be inhomogeneous aModified t test
(Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the
actual values in the computer without rounding off. Statistical analysis of histopathological
finding was carried out by means of a non-parametric Kolmogorov-Smirnov test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
DAILY CLINICAL SIGNS
During the treatment period, dyspnoea, rales, piloerection and hunched posture were the most relevant and treatment-related clinical signs observed in animals of Group 4 (300/200mg/kg/day). These signs were mainly recorded during the first two/three weeks of dosage and, as a results of the reduction of the high dose level from 300 to 200mg/kg/day, their incidence was significantly reduced. Rales and dyspnoea, however, were still observed in some males treated at 200mg/kg/day, up to the day of sacrifice. These signs were no longer observed during the recovery period, with the exception of one male of Group 4, still showing rales during the whole recovery period. Other signs, such as missing teeth, damaged ear and hairloss, were occasionally recorded during the study in single animals, with no correlation with the dose and were, thus, considered as minor clinical signs and not related to the administration with the test item.
Palpable masses were seen in a single female animal of the control group, starting from Day 43 of the treatment period.

WEEKLY DETAILED CLINAL SIGNS (REMOVAL FROM CAGE AND OPEN FIELD MEASUREMENTS)
No treatment-related changes were found at the weekly clinical examination during treatment and recovery periods, which included an evaluation of neurotoxicity. Statistically significant fluctuations (mostly increases without any correlation with the dosing period) in rearing number were occasionally recorded in males and some females at 100 and 300/200mg/kg/day (from +5% to 26% in the male, from -8% to +26% in the females). No toxicological significance was attributed to this finding, since it was occasional and the values obtained were comparable to the other values recorded during the study. It was, therefore, considered to be incidental.

For further details see Attached background materia
Mortality:
mortality observed, treatment-related
Description (incidence):
Seven cases of premature death, two males and five females from the high dose group
Occurred during the study.
In these animals, dyspnoea, hunched posture, rales, piloerection, decreased activity and
swollen abdomen were the most relevant and treatment-related clinical signs observed,
before their death.
The factors contributory to the ill status of the high dose animals sacrificed for humane
reasons could be attributed to gastrointestinal and /or respiratory findings due to an accidental
aspiration of irritant effect of the initial high dose formulation (300mg/kg/day) as a result of reflux after gavage dosing (enhanced by the presence of alveolar macrophages in the alveoli associated with chronic inflammation) or procedure-related. The pathological picture of the three high dose females found dead during the study did not allow to establish the cause of death.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
When compared to control animals, no changes were noted in mean body weights and
body weight gain in both genders, during treatment and recovery periods. For further details see Attached background materia
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
During the study, statistically significant increases and/or decreases were occasionally
recorded in males and females of all treated groups, when compared to controls. These
variations in food consumption can be mainly ascribed to fluctuations in food intake seen
in control animals, being the food consumption of treated animals almost constant during
the study.
No alterations were recorded during the recovery period, in both genders. Attached background materia
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Before the start of treatment, all animals showed no ocular abnormality at the ophthalmoscopic
examination.
Both eyes of animals from groups 1 and 4 were re-examined duringWeek 13 of treatment
(on Study Day 85) and no ocular findings were detected
Haematological findings:
no effects observed
Description (incidence and severity):
DOSING PHASE: Statistically significant differences between control and treated animals were recorded, such as: haematocrit in males from all treated groups (-4% in all groups), neutrophils
and haemoglobin in males dosed at 30mg/kg/day (-23% and -5%, respectively), neutrophils,
mean corpuscular haemoglobin concentration and eosinophils in those receiving 300/200mg/kg/day (+53%, +2% and +50%, respectively), basophils in females dosed at
30mg/kg/day (-38%) and mean corpuscular volume in females dosed at 300/200mg/kg/day
(-3%).
Due to the absence of dose-relation, the consistency between sexes and other related
findings, the above changes were considered to be incidental.

RECOVERY PHASE
All findings recorded atWeek 13 showed a complete reversibility after 2 weeks of Recovery.
The statistically significant differences recorded (reticulocytes and lymphocytes in females)
were not observed during the Dosing Phase, therefore they were considered to be unrelated
to treatment

COAGULATION
Dosing Phase
Prothrombin time was statistically significantly higher than controls in females dosed at
300/200mg/kg/day (5%) and lower than controls in those receiving 30 and 100mg/kg/day
(5%, both).
Due to the absence of dose-relation, these findings were considered to be unrelated to
treatment.

Recovery Phase
No changes we recorded, confirming reversibility.

For further Deatils see Attached background material
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
DOSING PHASE
No changes that could be considered to be adverse were observed. Compared with controls, statistical significant fluctuactions of some biochemical parameter were recorded in treated animals. Relevant data are reported in Table 6 in Any other information on results incl. tables.
Most of the differences showed in Table 6 were not dose-related and/or not consistent between sexes, thus considered to be incidental. The other findings recorded which could be possibly related to administration of the test item (decreases of alkaline phosphatase and protein) were of minimal severity and no other related changes were observed, therefore the were considered to be not adverse.

RECOVERY PHASE
Alkaline phosphatase and calcium still showed statistically significant differences with controls in treated females (-27% and + 4%, respectively). In addition, chloride was still lower than control in treated males (-2%).
As for the dosing phase, these findings were minimal and not associated with other related changes, thus they were considered to be not adverse.
The ther changes recorded at dosing phase showed reversibility.
The statistically significant difference of gluscose recorded between control and treated males was not observed at the end of dosing, thuis it was considered to be unrelated to treatement. For further details see Attached background material
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
FINAL SACRIFICE
Terminal body weight of treated animals was comparable to the control group No treatment related changes were observed in absolute and relative (% to body weight) organ weights in all the treatments groups for both sexes, when compared to control data.
RECOVERY SACRFICE
After 2 weeks of recovery, no changes in terminal body weight, nor treatment-related organ weight changes were reported in animals of both sexes previously treated with high dose, when compared with controls.
For further details see Attached background materia
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Final sacrifice:
The only relevant change observed following gross pathology examination was a single
dark area in the azygos lobe of one high dose treated female corresponding microscopically
to alveolar macrophage aggregation, peribronchial chronic inflammation and subpleural
haemorrhage.

Recovery sacrifice:
No treatment-related changes were noted, following gross pathology examination
Neuropathological findings:
no effects observed
Description (incidence and severity):
Several functional tests, such as hind limb landing foor splay, sensory reactivity to stimuli, incliding grip strendth and motor activity, were performed during Week 12 of treatment and at the end of the recovery period, for neurotoxicity assessment. Increased landing foot splay measurements were recorded in male animals dosed at 30 mg/kg/ day (+ 18%) and 100 mg/kg day (+30%), when compared to control animals. Differences
were, anyway, not dose-related, since animals dosed at 300/200 mg/kg/day had
comparable values with those of control animals, hence no toxicological significance was
attributed to these values. These alterations were not observed at the end of the recovery
period, in animals receiving 200 mg/kg/day.
Motor activity measurements and sensory reaction to stimuli performed at the end of the
treatment and recovery periods did not show any toxicologically significant differences
between treated animals and controls.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Final sacrifice
Treatment-related lesions were noted only in a few cases in lungs of high dose treated
animals of both sexes consisting in interstitial chronic inflammation and fibrosis, in general
surrounding interstitial haemorrhage of lungs associated with the presence of alveolar
macrophages. Anyway it could be speculated that these lung lesions occurred in a few
subjects and may be the result of gastroesophageal reflux induced in the first three days of
administration.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle
and to the integrity of the various cell types within the different stages; regular layering in
the germinal epithelium was noted in all control and treated males.
Normal physiology of the oestrous cycle (oestrous, metestrous, diestrous and proestrous)
was noted in control and treated females associated with no morphological changes of
each “oestrous phase” in the ovaries, uterus/cervix and vagina.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Panel Analyses
The determination of T3, T4 and TSH was performed by the Test Site on samples from all
animals of all groups.
TSH was higher than controls in 3 males dosed at 300/200mg/kg/day and, compared
with mean control data, the mean group value was 70% above controls. However, since
no relevant changes of the other hormones were recorded and no thyroid findings were
observed, the above change was considered to be not adverse.

Spermanalysis:
Samples for evaluation of sperm motility, morphology and concentration were obtained from the epididymal cauda at necropsy from all males of the control and high dose groups, killed at the end of the treatment period. No differences that can be ascribed to treatment were seen in motility, morphology and concentration expressed as million sperm/gram caudal epididymal tissue.
Since no treatment-related effects were seen between control and high dose males, no assessment was performed in males of the mid- and low dose groups as well as on recovery groups.


Oestrous cycle:
No treatment-related anomalies were noted in the oestrous cycle of treated females, when
compared to controls.
Details on results:
For further details on Results see Attached background material

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related systemic effect which could be considered to be adverse was observed following dosing at 200mg/kg for at least 87 days or at 100 and 30mg/kg/day. Thus, the high dose level of 200mg/kg/day may be considered the NOAEL for this study.
Remarks on result:
other:
Remarks:
some treatment-related effects (including mortality) were observed at 300/200mg/kg/day. However, these effects were ascribed to a strong local effect of the test item due to its chemical nature (strong base) to the gastrointestinal and respiratory tracts, following initial 4-day dosing at 300mg/kg/day

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Overall, on the basis of the above results, some treatment-realted effects (including mortality) were observed at 300/200 mg/kg/day. However, these effects were ascribed to a strong local effect on the test item due to its chemical nature (strong base) to the gastrointestinal and respiratory tracts, following initial 4-day dosing at 300 mg/kg/day. No treatment-related systemic effect which could be considered to be adverse was observed following dosing at 200 mg/kg bw/day for at least 87 days or at 100 and 30 mg/kg/day. Thus, it can be concluded that the high dose level of 200 mg/kg/day may be considered as no Observed Adverse Effect Level (NOAEL) for this study, under the reported experimental conditions.