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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)
Remarks:
High Level (Main Group 4)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
Medium Level (Main Group 3)
Dose / conc.:
37.5 mg/kg bw/day (actual dose received)
Remarks:
Low Level (Main Group 2)
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
Description (incidence and severity):
The clinical signs (scabs or hairloss) recorded during the the study were not considered
treatment related since they were equally observed among the treated and control groups.
Piloerection was seen in one female receiving 150 mg/kg/day on Day 4 post coitum only.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study. One Female receving 75 mg/kg/day was found not pregnat at necropsy. The remaining females sacrifices on Day 20 post-coitim were pregnant.
Pregnancy status: There were 25/25, 25/25, 24/25 and 25/25 pregnant females in control,
37.5, 75 and 150 mg/kg/day. All pregnant females had viable foetuses.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight and mean body weight gain did not give any indication of test -Item related effect.
Food efficiency:
no effects observed
Description (incidence and severity):
No statistical significant differences in food consumption were observed amongst the control and the groups receiving the test item at the dose levels of 37.5, 75 and 150 mg/kg/day.
Water consumption and compound intake (if drinking water study):
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
no effects observed
Description (incidence and severity):
With regard Thyroid horme determination -Delegated phase
No deifferences between control and treated animals were recorded.
For more information see Results on Thyroid hormone measurments and maternal developmental toxicity attached in under in the "Attached background material" under "Overall Remarks"
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. No treatment-related was seen in thyroid weight if treated females compared to the control. The decrease of relavtive (% to body weight) (approximately 18%) seen in females receiving 150 mg/kg/day, compared to controlm was not considered test item.related since no concurrent histopathological findings were noted.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Females that completed the treatment period did not show relevant macroscopic changes
that could be considered treatment-related.
No treatment-related changed were noted in control and treated females, following gross pathological examination. All observed changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups and /or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
Neuropathological findings:
not examined
Description (incidence and severity):
No treatment-related finsing were noted in the microscopic evaluation of the thyroid gland of treaed females, when compared to their controls.
Histopathological findings: non-neoplastic:
no effects observed
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
Description (incidence and severity):
There were no statistically significant differences between the control and treated groups
in the number of corpora lutea, number of implantations, live foetuses and early and late
resorptions, nor in the pre- and post implantation loss or in the sex ratio of the foetuses.
Furthermore, no significant differences in the litter and mean foetal weights were observed
between control and treated groups
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Post-implantation losses, as percentage of implantation sites, were 2.5%, 1.8%, 2.2% and 1.9% in control and in females treated at 37.5, 75 and 150 mg/kg/day, respectively. Number of live fetuses corrrespond to 97.4%, 98.4%, 97.7% and 97.8% of implantation sites in control and in females treated at 37.5, 75 and 150 mg/kg/day, respectively.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Litter mean of treated groups were similar to the control and therefore were not considered to be affected by the treatment with the test item.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No test item-related effects on the sex ratio of the fetuses were noted at any dose level. The
proportion of male foetuses was 52%, 50%, 50% and 52% in order of ascending dose levels.

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
Description (incidence and severity):
Mean fetal weights (combined or by sex) of treated groups were similar to the control and therefore were not considered affected by the treatment with the test item .
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
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
No changes were seen in AGD (anogenital distance) between control and treated groups. No differences were noted in the mean values of the AGD of the fetuses of both sexes, maternally exposed at all dose levels compared to the control group.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
During the external examination of fetuses, abnormal findings were noted in the three fetuses of control groups, in one fetus at 37.5 mg/kg/day, in seven fetusus at 75 mg/kg/day and three fetuses at 150 mg/kg/day. Abnormal findings such as small fetusus (weight < 2.7 g), acaudia, imperforate anus and malrotated forelimbs were considered to be incidental due the the presence also in control group (acaudia and imperforate anus) and / or because they occured without dose-dependancy
Skeletal malformations:
no effects observed
Description (incidence and severity):
Abnormalities and variations noted during the skeletal examination were observed in control and treated groups wihtout of dose-dependency, thus they were not considered to be attributed to the treatment with the test item. During skeletal examination of the fetuses, findings were noted in:

- 90% examined fetuses (in 100% litters) in control group:;
- 88% examined fetuses (in 100% litters) at 37.5 mg/ kg bw/day:
- 90% examined fetuses (in 100% litters) at 75 mg/ kg bw/ day;
- 81% examined fetuses (in 100% litters) at 150 mg/kg bw/ day.

The distribution of these alteration among the groups, control and treated, did not indicate any test item related effects.
Major abnormality (pubis bone no ossification) were noted in one control fetus ( in 1 litter), 1 fetus maternally exposed at 37. 5 mg/ kg bw/ day (out of 1 litter) , in 6 fetuses (out of 2 litters) exposed at 75 mg/kg bw/ day and in 3 fetuses (out of 2 litter) exposed at 150 mg/kg bw/ day. Two additional fetuses, one maternaly exposed at 37.5 mg/kg bw / day and one at 150 mg/ bw/ day showed cleft palate and polydactyly, respectively. The distribution of these alterations among the groups, control and treated, did not indicate any test-tem related effects.
Visceral malformations:
no effects observed
Description (incidence and severity):
There was no visceral malformation associated with treatment with the test item.
Oveerall, fetal variations observed during the microdissection were commonly seen in rat foetuses in this age. Thus, these findings could be incidental and not attributed to the treatment with the test item . Consequently, under the conditions described for the present study, the test item did not reveal teratorgenic potential up to and including the dose level of 150 mg/ kg bw/ day
Other effects:
not examined

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

Any other information on results incl. tables

Results in Tabular form about effects level on Maternal animals and fetuses  are available under in the "Attached background material" under "Overall Remarks"

Applicant's summary and conclusion

Conclusions:
The test item was administered, by oral gavage, to mated female Sprague Dawley rats (25 females per dose group) from Days 3 to 19 post coitum.
The vehicle was softened water (by reverse osmosis) and the dose volume used was 10 mL/kg.
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 behaviour 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 the 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.