Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an OECD 422 study a NOAEL (parental systemic toxicity) of 100 mg/kg bw/day was obtained with test substance N,N-Bis(3-aminopropyl)methylamine. The NOEL for fertility and reproduction was 100 mg/kg bw/d and the NOEL for developmental toxicity in the F1 was found to be 100 mg/kg bw/d.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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
Route of administration:
oral: gavage
Vehicle:
water
Details on 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.
Details on mating procedure:
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group.
The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".
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
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
Parental animals: Observations and examinations:
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.
Oestrous cyclicity (parental animals):
no
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

BODY WEIGHT
The pups were weighed on the day after birth (PND 1) and on PND 4.

CLINICAL OBSERVATION
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

NECROPSY OBSERVATION
All pups with scheduled sacrifice on PND 4. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
Postmortem examinations (parental animals):
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.
Postmortem examinations (offspring):
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2.
All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-bycase basis, depending on the type of finding noted.

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
Reproductive indices:
Male reproduction data:
The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:
Male mating index (%) = (number of males with vomfirmed mating* / number of males placed with females) x 100
*defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = (number of males proving their fertility* / number of males placed with females) x 100
* defined by a female with implants in utero

Female reproduction and delivery data:
The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females. For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:
Female mating index (%) = (number of females mated* / number of females placed with males) x 100
* defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = (number of females pregnant* / number of females mated**) x 100 * defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant*) x 100
* defined as the number of females with implants in utero

Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100

Post implantation loss (%) = ((number of implantations - number of pups deliverd) / number of implantations) x 100
Offspring viability indices:
Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated according to the following formula:

Viability index (%) = ( number of live pups on day 4 after birth / number of live pups on the day of birth) x 100

Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.
The sex ratio was calculated at day 0 and day 4 after birth according to the following formula:

Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x 100

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.
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
Dose descriptor:
NOEL
Remarks:
(reproductive toxicity)
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
VIABILITY (OFFSPRING)
The viability index indicating pup mortality during lactation (PND 0 - 4) was 100% (test group 0), 83.3% (test group 1) and 96.0% (test group 2) basedon three pups of test group 2 (100 mg/kg bw/d) and 4 pups of test group 1 (25 mg/kg bw/d) were found dead. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
One pup of test group 2 (100 mg/kg bw/d) and 3 pups of test group 1 (25 mg/kg bw/d) were found stillborn. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

SEX RATIO
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature

CLINICAL SIGNS (OFFSPRING)
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.

BODY WEIGHT (OFFSPRING)
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group.
One male and one female runt were seen in test groups 0 and 1 (25 mg/kg bw/d), respectively.

GROSS PATHOLOGY (OFFSPRING)
In test group 2 (100 mg/kg bw/d) two male pups showed post mortem autolysis. Four pups (one male and three female pups of test group 1, 25 mg/kg bw/d) showed also post mortem autolysis. These findings were assessed as being spontaneous in nature and without toxicological relevance.
Dose descriptor:
NOEL
Remarks:
(developmental toxicity)
Generation:
F1
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Reproductive effects observed:
not specified

The stability of the test substance in deionized water was demonstrated over a period of 7 days at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed.

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
Guideline study conducted in accordance with GLP.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In an OECD 422 study conducted with N,N-Bis(3-aminopropyl)methylamine, the test substance was given daily as a solution to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 25, 100 and 500 mg/kg body weight/day. Control animals were dosed daily with the vehicle only (deionized water).

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 up to one day prior to the day of schedule sacrifice of the animals.

F0 animals were mated 13 days after the beginning of treatment to produce a litter (F1 generation pups). A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation (days 0 - 7, 7 - 14, 14 - 20) and lactation (days 1 - 4). Water consumption of the F0 parents was determined once weekly during premating. In dams water consumption was determined for gestation days 6 - 7, 13 – 14, 19 – 20 and lactation days 3 – 4. In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0 and 4. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4 and their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO2 and examined macroscopically for external and visceral findings at necropsy.

At the test group of 500 mg/kg bw/d several males developed a moribund state showing e.g. piloerection, hypothermia, labored respiration, semiclosed eyelids, abdominal position, unsteady gait and respiratory sounds during study days one to four.Moribund animals were sacrificed. One male was found dead on study day three and on study day four the remaining four males were sacrificed moribund. One female was found dead on study day one. Several females developed a moribund status showing e.g. piloerection, labored respiration, semiclosed eyelids, hypothermia, unsteady gait, and abdominal position on study day one. On study day one the remaining nine females were sacrificed in a moribund state. The parental animals of test group 100 mg/kg bw/d showed a decreased water consumption at the end of gestation and in female animals a slight body weight loss during lactation. Regarding pathology, there were neither treatment-related organ weight changes, nor gross lesions in males and females of test group 2 (100 mg/kg bw/d). The moribund state as well as the death of males and females in test group 3 (500 mg/kg bw/d) was attributed to the extensive erosive and ulcerative lesions in the gastrointestinal tract together with the renal cortical necrosis. 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. At 25 mg/kg bw/d there were no test substance-related adverse findings observed.

In conclusion, under the conditions of the present Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats the NOEL (no observed effect level) for general, systemic toxicity was 25 mg/kg bw/d for

the parental rats. 100 mg/kg bw/d was the LOEL (lowest observed effect level) based on the decreased water consumption at the end of gestation and a higher incidence of slight body weight loss observed during lactation. The corresponding NOAEL (no observed adverse effect level) was 100 mg/kg bw/d for the parental rats because of no adverse findings in clinic, in the clinical pathology and in the pathology. The NOEL for fertility and reproductive performance was 100 mg/kg bw/d. The NOEL for developmental toxicity in the F1 progeny of the test substance-treated group was found to be 100 mg/kg bw/d.

Effects on developmental toxicity

Description of key information
In an OECD 422 study a NOAEL (parental systemic toxicity) of 100 mg/kg bw/day was obtained with test substance N,N-Bis(3-aminopropyl)methylamine. The NOEL for fertility and reproduction was 100 mg/kg bw/d and the NOEL for developmental toxicity in the F1 was found to be 100 mg/kg bw/d.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In an OECD 422 study conducted with N,N-Bis(3-aminopropyl)methylamine, the test substance was given daily as a solution to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 25, 100 and 500 mg/kg body weight/day. Control animals were dosed daily with the vehicle only (deionized water).

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 up to one day prior to the day of schedule sacrifice of the animals.

F0 animals were mated 13 days after the beginning of treatment to produce a litter (F1 generation pups). A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation (days 0 - 7, 7 - 14, 14 - 20) and lactation (days 1 - 4). Water consumption of the F0 parents was determined once weekly during premating. In dams water consumption was determined for gestation days 6 - 7, 13 – 14, 19 – 20 and lactation days 3 – 4. In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0 and 4. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4 and their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO2 and examined macroscopically for external and visceral findings at necropsy.

At the test group of 500 mg/kg bw/d several males developed a moribund state showing e.g. piloerection, hypothermia, labored respiration, semiclosed eyelids, abdominal position, unsteady gait and respiratory sounds during study days one to four.Moribund animals were sacrificed. One male was found dead on study day three and on study day four the remaining four males were sacrificed moribund. One female was found dead on study day one. Several females developed a moribund status showing e.g. piloerection, labored respiration, semiclosed eyelids, hypothermia, unsteady gait, and abdominal position on study day one. On study day one the remaining nine females were sacrificed in a moribund state. The parental animals of test group 100 mg/kg bw/d showed a decreased water consumption at the end of gestation and in female animals a slight body weight loss during lactation. Regarding pathology, there were neither treatment-related organ weight changes, nor gross lesions in males and females of test group 2 (100 mg/kg bw/d). The moribund state as well as the death of males and females in test group 3 (500 mg/kg bw/d) was attributed to the extensive erosive and ulcerative lesions in the gastrointestinal tract together with the renal cortical necrosis. 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. At 25 mg/kg bw/d there were no test substance-related adverse findings observed.

In conclusion, under the conditions of the present Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats the NOEL (no observed effect level) for general, systemic toxicity was 25 mg/kg bw/d for the parental rats. 100 mg/kg bw/d was the LOEL (lowest observed effect level) based on the decreased water consumption at the end of gestation and a higher incidence of slight body weight loss observed during lactation. The corresponding NOAEL (no observed adverse effect level) was 100 mg/kg bw/d for the parental rats because of no adverse findings in clinic, in the clinical pathology and in the pathology. The NOEL for fertility and reproductive performance was 100 mg/kg bw/d. The NOEL for developmental toxicity in the F1 progeny of the test substance-treated group was found to be 100 mg/kg bw/d.

Justification for classification or non-classification

Based on the available data, the classification criteria according to Regulation (EC) No 1272/2008 (CLP) are not met.