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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2020-04-30 to 2020-07-23
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
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:
- Storage condition of test material: Room temperature, protected from light and oxidants (under
nitrogen)
- Stability under storage conditions: stable
- Stability under test conditions: The required amount of PU-2019-871 was dissolved/suspended in the vehicle. The formulation was prepared daily or weekly
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The stability of the preparations at 1-50mg/mL was assessed at:
• up to 28 hours at room temperature
• after 8 days at 2-8°C

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 9 weeks old (virgin females), 11 weeks old (males)
- Weight at study initiation: 203-223 g (virgin females); 340 g (males)

- Housing: Before and after the pairing period, the animals were housed no more than 5 of one sex in polysulfone solid bottomed cages, measuring 59.5×38×20cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week. In addition, suitable nesting material was provided as necessary. Nesting material was changed at least 2 times a week. During the pairing period, the rats were housed on the basis of 1 male to 1 female in clear polysulfone cages measuring 42.5×26.6×18.5cm with a stainless-steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week before the start of the treatment

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

IN-LIFE DATES: From: 2020-05-22 To: 2020-06-19

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved/suspended in the vehicle. The formulation was prepared daily or weekly according to stability data obtained from test laboratory study (concentrations of 3.75, 7.5 and 15mg/mL), unless specified otherwise.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Preparation of the test item:
The required amount of the test item was dissolved/suspended in the vehicle. The formulation was prepared daily or weekly according to stability data obtained from a separate study (concentrations of 3.75, 7.5 and 15mg/mL), unless specified otherwise.

Analysis of test item preparations:
Analysis was performed in a separate study in order to validate the analytical method and the preparation procedure and to verify the stability of the preparations. The stability of the preparations at 1-50mg/mL was assessed at:
– up to 28 hours at room temperature
– after 8 days at 2-8°C
Samples of the preparations prepared during the first and last week of treatment of the
current study were analyzed to check the concentration. Results of the analyses were within the acceptability limits stated in test laboratory SOPs for concentration of solutions (90-110%).
The validated software used for this activity was Analyst 1.6.2 (ABSciex).
Details on mating procedure:
Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: yes. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of mating was made.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear or vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
All animals were dosed once a day from Day 3 through Day 19 post coitum.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
37.5 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels of 37.5, 75 and 150 mg/kg/day were selected by the based on information from a preliminary non-GLP compliant study (ERBC Study no. Y0470). In thepreliminary study, the dose levels of 100, 200 and 400 mg/kg/day were used. The test item induced slight to moderate signs of maternal toxicity at all dose levels, in terms of clinical
signs recorded and decreased body weight gain. These effects were more evident at the two
highest dose levels in which unscheduled death and decrease in absolute weight gain were
also noted.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.


DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # GD 20
- Organs examined: Thyroid gland

OTHER: Abnormalities
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes /
- Number of early resorptions: Yes: only placental remnants visible
- Number of late resorptions: Yes: placental and foetal remnants visible.
Fetal examinations:
- External examinations: Yes: half per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-
Wallis test and intergroup differences between the control and treated groups assessed by a
non-parametric version of the Williams test.
Indices:
-Pre, post- and totoal implantation loss were calculated as a percentage.
- Sex ratios of the foetuses were calculated as the percentage of males per litter.
- Anogenital distance (AGD): The AGD of each live foetus was measured on Day 20 post
coitum. The AGD was normalized to the cube root of body weight collected on Day 20 post
coitum. Individual and mean data were reported.
-

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
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:
no effects observed
Description (incidence and severity):
Terminal body weight, uterus weight and mean absolute weight gains (body weight at
necropsy minus gravid uterus weight, minus body weight at Day 0 of pregnancy) of treated
females were comparable to the control group
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Description (incidence and severity):
No differences between control and treated animals were recorded.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
not examined
Other effects:
no effects observed

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
changes in number of pregnant
changes in pregnancy duration
dead fetuses
early or late resorptions
effects on pregnancy duration
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
Key result
Dose descriptor:
NOAEL
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved/suspended in the vehicle. The formulation was prepared daily or weekly according to stability data obtained from test laboratory study (concentrations of 3.75, 7.5 and 15mg/mL), unless specified otherwise.
The test item was administered, by oral gavage, to mated female rats (25 females per dose group) from Days 3 to 19 post coitum. The dose levels were 0, 37.5, 75 and 150 mg/kg/day. On Day 20 post coitum the dams were killed and macroscopically examined.
Gravid uterus and thyroid were weighed, and fetuses were examined after caesarian section. External, visceral and skeletal examinations were performed on fetuses of all groups. No mortalities were observed. One female receiving 75 mg/kg/day was found not pregnant at necropsy.
Further, daily clinical observations during the gestation period did not reveal any remarkable findings in the animals’ appearance, general condition or behavior amongst the dosing and control groups.
No significant differences in mean body weights, body weight gain and food consumption were observed amongst the control and treated groups.
There were no statistically significant differences between the control and treated groups in the number of corpora lutea, number of implantations, live fetuses and early and late resorptions, nor in the pre- and post-implantation loss or in the sex ratio of the fetuses.
Furthermore, no significant differences in the litter and mean fetal weights were observed between control and treated groups.
No remarkable differences in gravid uterus weight, terminal body weight or absolute weight gain were observed between the control group and the groups receiving test item.
Fetal external observations, visceral and skeletal examination did not reveal any remarkable findings which could be related to treatment.
Based on the results obtained in the study, the NOAEL for maternal and developmental toxicity could be set at 150 mg/kg/day.