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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
ong
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N-bis(3-aminopropyl)methylamine
EC Number:
203-336-8
EC Name:
N,N-bis(3-aminopropyl)methylamine
Cas Number:
105-83-9
Molecular formula:
C7H19N3
IUPAC Name:
bis(3-aminopropyl)(methyl)amine
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): N,N-Bis(3-aminopropyl)methylamine
- Physical state: liquid/colorless, clear
- Analytical purity: 99.6 area-%
- Lot/batch No.: O 2903

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-11 weeks (Males/females)
- Weight at study initiation:
- Fasting period before study: no
- Housing: individually (during study period) Makrolon type M II cages
Exceptions: During overnight matings, male and female mating partners were housed together in Makrolon type M III cages. Pregnant animals and their litters were housed together until PND 4 (end of lactation). For motor activity (MA) measurements the animals were housed individually in polycarbonate cages.
- Diet (e.g. ad libitum): ad libitum (Kliba maintenance diet mouse-rat “GLP”, meal, Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water (e.g. ad libitum): ad libitum (drinking water)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): fully air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
N,N-Bis(3-aminopropyl)methylamine was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, deionized water was filled up to the desired volume, subsequently released with a magnetic stirrer. The test substance preparations were produced at least once a week.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in deionized water over a period of 7 days at room temperature was proven before the start of the study. Concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start of the administration period.
Duration of treatment / exposure:
The duration of treatment covered a 2 week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and 4 days of lactation period in females.
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
25, 100, 500 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal.
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size.

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter and without positive evidence of sperm in the vaginal smear or waiting for necropsy were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION: Yes
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0 - 7, 7 - 14 and 14 - 20.
- Food consumption of F0 females, which gave birth to a litter was determined for PND 1 - 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

WATER CONSUMPTION: Yes
Generally, water consumption was determined once a week (over a period of max. 4 days) for male and female parental animals, with the following exceptions:
- Water consumption was not determined during the mating period (male and female F0 animals)
- Water consumption of the F0 females with evidence of sperm was determined on GD 6 - 7, 13 - 14 and 19 - 20. - Water consumption of F0 females, which gave birth to a litter was determined for PND 3 - 4.
Water consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning towards the end of the administrative period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Parameters checked: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET), Prothrombin time (Hepato Quick’s test) (HQT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In the morning towards the end of the administrative period
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Parameters checked: Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase; EC 2.6.1.2.), Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.), Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; EC 3.1.3.1.), Gamma-Glutamyltransferase (GGT) (Gamma -glutamyl) peptide: aminoacid-gamma-glutamyl-transferase; EC 2.3.2.2.), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)

URINALYSIS: Yes
- Time schedule for collection of urine: overnight towards the end of the administrative period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, Turbidity, Volume.

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery (FOB) was performed in the first five surviving male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period.
Home cage observations:
Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Impairment of gait, other findings.
Open field observations: behavior on removal from the cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupilsize, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, gait abnormalities, activity/arousal level, feces excreted within 2 minutes (number/appearance/consistency), urine excreted within 2 minutes (amount/color), rearing within 2 minutes
Sensory motor tests/ reflexes: Reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), other findings, grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test.
Motor activity (MA) was also measured on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
All male and female animals of test group 3 (500 mg/kg bw/d) were sacrificed moribund with exception of animals No. 35 and 133 which died intercurrently. All of them were necropsied and assessed by gross pathology as soon as possible after their death.
Organ weights:
- The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Epididymides, Testes
- The following weights were determined in 5 animals/sex and test group (females withlitters, same animals as used for clinical pathology examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

HISTOPATHOLOGY: Yes
The following organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde or in modified Davidson’s solution:
Adrenal glands, All gross lesions, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve, Esophagus, Extraorbital lacrimal gland, Epididymides (modified Davidson’s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands, (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Target organs,Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.

The testes, epididymides and ovaries of animals that died/were sacrificed intercurrently were fixed in 4% neutral-buffered formaldehyde solution.
Statistics:
DUNNETT test (two-sided): Food consumption (parental animals), Water consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days
FISHER'S EXACT test (one-sided): Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups
WILCOXON test (one-sided+) with BONFERRONI-HOLM: Mating days until day 0 pc, % postimplantation loss, pups tillborn, % perinatal loss
WILCOXON test (one-sided+) with BONFERRONI-HOLM: Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index
WILCOXON test (two-sided): % live male day x, %live female day x
KRUSKALWALLIS test (two-sided) or WILCOXON test (twosided): Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
KRUSKAL-WALLIS test or WILCOXON-test (twosided): Blood parameters
WILCOXON-test (one-sided): Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity
KRUSKAL-WALLIS test or WILCOXON-test: Urine pH, volume, specific gravity, color and turbidity
KRUSKAL-WALLIS test (two-sided) or WILCOXON-test (two-sided): Weight parameters

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
Parental animals:
One female animal of test group 3 (500 mg/kg bw/d) was found dead on study day one and one male of test group 3 (500 mg(kg bw/d) was found dead on study day three. Due to their reduced general state of health and the severe observations all remaining animals of test group 3 (500 mg/kg bw/d) were sacrificed in a moribund state during the first five days of the administration period.
Several males and females of test group 3 (500 mg/kg bw/d) showed e.g. labored respiration, piloerection, unsteady gait, hypothermia, semiclosed eyelids and abdominal position during study days one to four. Therefore they were sacrificed in a moribund state between study day 1 and 4 of the premating period.
One female (No. 120) animal of test group 1 (25 mg/kg bw/d) showed severe salivation, moderately reduced nutritional condition, paleness and piloerection on GD 23.
One female animal of test group 1 (25 mg/kg bw/d; animal No. 120) showed severe salivation, slightly to moderately reduced nutritional condition, paleness and piloerection intermittent during lactation. Furthermore, this female did not nurse its pups properly on lactation days 1 to 3. No comparable observation was made in any other animal of the study. Therefore, this finding was assessed as being incidental.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight changes were not statistically significantly affected in male and female animals in test group 1 and 2 (25 and 100 mg/kg bw/d, respectively). In test group 2 (100 mg/kg bw/d) all females showed slight body weight loss during lactation. Nobody weight was determined in test group 3 (500 mg/kg bw/d) after exposure because the animals were found dead or were sacrificed moribund before the first scheduled body
weight measurement on study day 7. Therefore, it could not be excluded that the slight body weight loss in test group 2 was treatment-related during lactation.

FOOD CONSUMPTION
No significant deviations from control in male and female animals in test group 1 (25 mg/kg bw/d) and test group 2 (100 mg/kg bw/d) were observed during the study period. No food consumption was determined in test group 3 (500 mg/kg bw/d) because the animals were found dead or were sacrificed moribund before the first scheduled food measurement on study day 7.
One female animal of test group 1 (25 mg/kg bw/d; animal No. 120) showed a low food consumption during lactation. No comparable observation was made in any other animal of the study. Therefore, this finding was assessed as being incidental.

WATER CONSUMPTION
During gestation the water consumption in test group 1 (25 mg/kg bw/d) was slightly lower in comparison to control but did not reached a significant level. The water consumption was significantly lower in females of test group 2 (100 mg/kg bw/d) on gestation day 20 (-19%). No water consumption was determined in test group 3 (500 mg/kg bw/d) because the animals were found dead or were sacrificed moribund before the first scheduled water measurement on study day 7. Therefore, it could not be excluded that the significantly reduced water consumption was treatment-related during gestation. One female animal of test group 1 (25 mg/kg bw/d; animal No. 120) showed a low water consumption during lactation. This observation was an isolated finding in this dose group and was not considered as treatment-related. No other significant deviations in male and female animals were observed during the study.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.

URINALYSIS
No treatment-related changes among urinalysis parameters were observed.

NEUROBEHAVIOUR
Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative Parameters: No test substance-related effects were observed.
Motor activity measurement: There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals of all test groups in comparison to the concurrent control group. Regarding single intervals in males and females no significant deviations were detected.

ORGAN WEIGHTS
When compared to control group 0 (set to 100%), the mean absolute weights of the heart of males in test group 1 (25 mg/kg bw/d) was significantly decreased (90%*) without a dose-dependent relationship. Therefore, this change was regarded as incidental and not related to treatment.
When compared to control group 0 (set to 100%), the mean relative weights of the testes were significantly increased (108%*) in males of test group 2 (100 mg/kg bw/d). This change had no histopathological correlate and was therefore regarded as incidental.
All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.

GROSS PATHOLOGY
One female animal of test group 2 (100 mg/kg bw/d) showed a yellow discoloration in the glandular stomach that correlated with an inflammation of the serosa which was regarded as incidental. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Deceased/sacrificed animals: With the exception of female No. 136, all male and female animals of test group 3 (500 mg/kg bw/d) that died or were sacrificed moribund showed one or more of the findings red discolorations either in the forestomach or glandular stomach and / or duodenum, which were regarded as treatment-related.
Fertility: The female animals (Nos. 101, 123, 125 and 129), which were not pregnant as well as the male mating partners (Nos. 1, 23, 25 and 29) did not show gross lesions that could affect fertility.

HISTOPATHOLOGY
No treatment-related findings were observed in male and females of test groups 2 (100 mg/kg bw/d). All findings occurred either individually or were biologically equally distributed over control and treatment groups. One female animal of test group 2 (100 mg/kg bw/d) showed in the serosa of the glandular stomach, a mixed-cell inflammation with granulation tissue. The same findings were noted in the adjacent adipose tissue. In addition, the papillary process of the liver in this animal was hemorrhagic and necrotic and was surrounded by granulation tissue. This finding most likely corresponded to a liver lobular torsion. All of these findings correlated with macroscopic changes and were regarded as incidental. All other histopathological changes in males and females of test group 2 (100 mg/kg bw/d) were considered to be incidental or spontaneous in origin and without any relation to treatment.
Deceased/sacrificed animals: In the forestomach and glandular stomach, extensive areas of erosion and/or ulceration were seen in males (minimal to severe) and females (severe to massive). Hemorrhagic inflammation was observed in the mucosa of the duodenum in males (minimal to mild) and in the duodenum and jejunum of females (mild to moderate). The hemorrhagic inflammation was mostly accompanied by fusion and blunting of the duodenal/jejunal villi, most likely representing a reaction to previous erosion. The findings in the gastrointestinal tract correlated with red discoloration at gross pathology. In the liver, single cell necrosis/apoptosis was seen in 1 of 5 males (slight) and 3 of 5 females (minimal to moderate). In the kidneys, tubular necrosis of the cortex was seen in 4 of 5 males (minimal to severe) and all females (minimal to massive). In males, a concomitant tubular
regeneration (basophilic tubules) was also observed. In the thymus, apoptotic necrosis in the cortex and/or medulla was observed in 2 of 5 males (slight and moderate) and all females (minimal to slight), whereas in the spleen, minimal apoptotic necrosis was seen in the periarteriolar lymphoid sheath (PALS) and/or follicles of single male and females. The mesenteric and axillary lymph nodes were minimally to slightly affected by reduced lymphoid cellularity in the paracortex in only 1 of 5 males and all females. The reduced size of the prostate, seminal vesicles and coagulating glands in males was attributed to the lower age of these animals on the day of death compared to the controls.
Fertility: The female animals (Nos. 101, 123, 125 and 129), which were not pregnant as well as the male mating partners (Nos. 1, 23, 25 and 29) did not show relevant histopathological findings. Male animal (Nos. 13, 14, 17, 19) and female animals (Nos. 113, 114, 117, 119) were not investigated histopathologically.






Effect levels

Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The stability of the test substance in deionized water was demonstrated over a period of 7 days at room temperature.

Given that the test substance was completely miscible with deionized water, solutions were considered to be homogenous without further analysis.

The concentrations of N,N-Bis(3-aminopropyl)methylamine in deionized water were found to be in the range of 90 % – 110 % of the nominal concentration. These results demonstrated the correctness of the concentrations of N,N-Bis(3-aminopropyl)methylamine in deionized water.

Applicant's summary and conclusion