Amines, N-(3-aminopropyl)-N'-(C16-18 and C18-unsatd. alkyl)trimethylenedi-

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21-Apr-2021 to 08-Dec-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

Official Journal of the European Union No. L142, May 2008.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(3-aminopropyl)-N'-C16-18 (evennumbered), C18 unsaturated alkyl -propane-1,3-diamine

Cas Number:

1219458-14-6

IUPAC Name:

N-(3-aminopropyl)-N'-C16-18 (evennumbered), C18 unsaturated alkyl -propane-1,3-diamine

Test material form:

not specified

Details on test material:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, container flushed with nitrogen.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

OTHER SPECIFICS
- physical description: white paste
- pH: 10.2 - 10.6 (at concentration of 1%)

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Arrival: Untreated females will be mated at the Supplier and will be at Day 0 or 2 post-coitum on
arrival at the Test Facility (Day 0 post-coitum is the day of successful mating). The actual age
of animals received will be listed in the Final Report.
- Age at study initiation: 17 – 20 weeks old
- Weight at study initiation: Approximately 3000 to 4300 g
- Fasting period before study: No fasting, See Diet
- Housing: Cages with perforated floors (Ebeco, Germany, dimensions
67 x 62 x 55 cm) equipped with water bottles.Animals individually housed. Cages will be arranged on the racks according to a Latin-square model.
- Diet (e.g. ad libitum): KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from
Granovit AG, Kaiseraugst, Switzerland. Pellets. Restricted access. On arrival animals will receive approximately 25 grams pelleted diet and on subsequent days 140-160 grams will be supplied.
- Water (e.g. ad libitum): Municipal tap water. Freely available to each animal via water bottles. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The actual daily mean temperature during the study period was 18 to 19°C
- Humidity (%): actual daily mean relative humidity of 48 to 61%
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hours light and 12-hours dark (may be interrupted for designated procedures)

IN-LIFE DATES: From: To: 19April2021 - 21May2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Frequency: Once daily
Duration: Day 7 to Day 28 post-coitum.
Formulations were heated to a maximum temperature of maximally 60°C for a maximum of 25 minutes to obtain visual homogeneity. Formulations were released for dosing when they have obtained a temperature of 40°C or lower.
Formulations (w/w) were prepared at least weekly, homogenized to visually acceptable levels and stored at room temperature (15-25°C) under nitrogen until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.
Method: The dose volume for each animal were based on the most recent body weight measurement. The doses were given using a plastic catheter attached to a plastic disposable syringe. Dose pot identification via Provantis was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

VEHICLE
- Concentration in vehicle: 0, 4, 8 and 16 mg/mL for vehicle, 5, 10 and 20 mg/kg bw/day groups.
- Amount of vehicle: 1.25 mL/kg bw
- Justification for use and choice of vehicle (if other than water): Based on trial formulations conducted by study facility and approved by study sponsor.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During week 1 of treatment dose formulation samples were collected for analysis.

For concentration analysis, samples from the middle part of the dosing container of all dose groups were taken (2 x approximately 500 mg). Samples were stored at a temperature set to maintain 18-22°C. For concentration, all mean sample concentration results within or equal to ± 10% (solutions) ± 15% (suspensions) of theoretical concentration were considered acceptable.

For homogeneity analysis, samples from the top, middle and bottom parts of the dosing container of groups 2 and 4 (low- and high-dose groups) were taken (2 x approximately 500 mg). Samples were stored at a temperature set to maintain 18-22°C. For homogeneity, a relative standard deviation (RSD) of concentrations of ≤10% for each group was considered acceptable.

Stability analyses performed previously in conjunction with the method development and validation study (ABL Study No. 501610) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Details on mating procedure:

- M/F ratio per cage: 1/1 (one female was cohabitated with one stock male)
- Age at start of mating of the females in the study: Approximately 17-20 weeks
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually.
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

Day 7 to day 28 post-coitum.

Frequency of treatment:

once daily 7d/wk

Duration of test:

from Day 0 to Day 29 (scheduled euthanasia)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Remarks:

Low-dose group

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

Mid-dose group

Doses / concentrations 4

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Remarks:

High-dose group

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of the dose range finding study (Project 20279148)
- Rationale for animal assignment: Upon detection of mating (Day 0 post-coitum), the females were distributed in a random sequence over the test groups. Females which were mated on the same day were classified in the same subgroup.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: mortality was checked at least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency were at least
once daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstance of any death was recorded in detail.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: At least once daily; starting on Day 7 post-coitum up to the day prior to necropsy.
Observations will be made postdose. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

BODY WEIGHT
- Time schedule for examinations: On Days 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

FOOD CONSUMPTION
- Daily from Day 3 post-coitum onwards.

WATER CONSUMPTION
Regular basis throughout the study. Water consumption will be monitored by visual inspection of the water bottles. If inter group differences are noted, consumption may be assessed by weight.

GENERAL REPRODUCTION DATA
- Mating date and confirmation of pregnancy was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.

Ovaries and uterine content:

Each ovary and uterine horn of all animals was dissected and examined as quickly as
possible to determine the following as part of the necropsy procedure:
• The number of corpora lutea.
• The weight of the uterus (not for animals found dead, sacrificed before planned
necropsy or that started to deliver).
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of early and late resorptions

Fetal examinations:

External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

All statistical tests were be conducted at the 5% significance level. All pairwise comparisons
were conducted using two sided tests and are reported at the 1% and 5% levels, unless
otherwise noted.

- Parametric/Non-Parametric: Levene’s test will be used to assess the homogeneity of group variances. The groups will be compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis
test is found to be significant, then pairwise comparisons will be conducted using Dunnett’s
or Dunn’s test, respectively.

- Non-Parametric: The groups will be compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test is found to be significant, then the above pairwise comparisons will be conducted using Dunn’s test.

- ANCOVA: The data corresponding to a response variable of interest and to a related covariate will be submitted to an analysis of covariance (ANCOVA), including only groups with at least three
non-missing paired values and if found to be significant, then pairwise comparisons will be conducted using Dunnett’s test.

- Incidence: A Fisher’s exact test will be used to conduct pairwise group comparisons of interest.

Indices:

For each litter the following calculations were performed:
Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) / number of corpora lutea x 100
Post-implantation loss (%) = (number of implantation sites - number of live fetuses) / number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where: Viable fetuses affected / litter (%) = number of viable fetuses affected / litter x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In total, 19/22 females treated at 20 mg/kg/day survived until scheduled necropsy of which
14/19 females were dosed for the entire treatment period.
Due to overall clinical condition of five females treated 20 mg/kg/day, it was decided to allow
a dosing holiday (for 2 to 4 days) for these females. This temporary dosing intermission was
initiated for Female Nos. 67, 76, 79 and 84 from 12 May 2021 onwards (Days 21 to 24
post-coitum, depending on mating date) until recovery in body weight and/or food
consumption was observed again. Female No. 78 was not dosed on Day 24 post-coitum, due
to its clinical signs.
Considering that this pause in dosing occurred after completion of the organogenesis,
generally considered the period between Days 6-18 post-coitum, and based on clinical signs
the animals have been dosed up to their respective maximum tolerability also after this
period, it is considered that the results from the evaluation of the litters of these animals
remain valid for the evaluation for developmental toxicity of the substance up to maximum
tolerable dose levels.
Clinical signs observed for females treated at 20 mg/kg/day were considered test item-related
unless stated otherwise.
- Pregnant females surviving until scheduled necropsy -
At 20 mg/kg/day, a thin appearance was observed for Female No. 68 on Days 26 to 27.
Reduced feces production was observed in 11/20, 8/20, 16/20 and 9/10 pregnant females in
the control, 5, 10 and 20 mg/kg/day groups, respectively. As this occurred in all groups,
including control, at comparable incidences and based on the time of onset and duration this
was considered unrelated to treatment with the test item.
Any other clinical signs noted during the treatment period occurred within the range of
background findings to be expected for rabbits of this age and strain which are housed and
treated under the conditions in this study and did not show any apparent dose-related trend. At
the incidence observed, these were considered to be unrelated to treatment with the test item.
These clinical signs included fur loss, small skin lesions on mouth or muzzle, scabs on lip,
muzzle or nose, swollen muzzle, and broken teeth.
- Non-Pregnant females surviving until scheduled necropsy -
Erected fur was observed for Female No. 72 on Day 26 post-coitum.
Reduced feces production was observed 2/2, 1/1 and 3/4 non-pregnant females in the control,
10 and 20 mg/kg/day groups, respectively. As this occurred in groups, including control, at
comparable incidences and based on the time of onset and duration this was considered
unrelated to treatment with the test item.
- Females treated at 20 mg/kg/day with a dosing holiday up to four days -
Erected fur was observed for 3/4 pregnant females (up to seven consecutive days). One
pregnant female was additionally observed with a thin appearance (Days 25 to 27 postcoitum) and pale feces (Days 25 to 28 post-coitum), whereas another pregnant female was
observed with hunched posture (Days 24 and 25 post-coitum).
Female No. 78 was apathic on Day 24 post-coitum and was therefore not dosed as fixation for
dosing was unsuccessful (recorded in study daybook).
Female No. 79 (non-pregnant) was observed with abnormal breathing sounds and erected fur.
Other clinical signs included reduced and/or absent feces production for all females with a
dosing holiday and scabs, which were considered unrelated to treatment with the test item.
Females treated at 20 mg/kg/day sacrificed in extremis
Erected fur was observed in 2/3 females (up to five consecutive days). Female No. 83 was
observed with wheezing and labored breathing on Day 13 post-coitum.
Other clinical signs included reduced feces production for 2/3 females and an injured right
eye, including a superficial ulcer on the cornea and conjunctivitis for one female. These
clinical signs were considered unrelated to treatment with the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

Six females were sacrificed during the study period, of which three (high dose) were
considered test item-related, and three (low or mid dose) were considered unrelated to
treatment with the test item.
At the moment of early termination, all females, except Female No. 83, were gravid.
Test item-related mortality at 20 mg/kg/day:
Female No. 83 (20 mg/kg/day) was sacrificed in extremis on Day 13 post-coitum, based on
clinical signs.
Female Nos. 74 and 82 were sacrificed in extremis on Days 22 and 19 post-coitum,
respectively, due to effects on food consumption and body weight.
As findings were in line with the those of surviving animals, clinical signs, body weight, food
consumption and macroscopic findings of these moribund animals are mentioned in the
sections below).
Mortality unrelated to treatment with the test item:
Female No. 42 (5 mg/kg/day) was sacrificed on Day 8 post-coitum because of poor
acclimatization after arrival. Food consumption was minimal to absent (ranging between
0-6 grams feed per day) during the acclimatization period (Days 3-6 post-coitum) and after
initiation of treatment (Day 7 post-coitum), and body weight loss (2% compared to Day 7
post-coitum; 10% compared to Day 0 post-coitum, non-GLP) was observed. Clinical signs
included decreased fecal production on Days 7 and 8 post-coitum. No macroscopic findings
were observed.
Female No. 30 (5 mg/kg/day) was sacrificed in extremis on Day 9 post-coitum as a result of
the oral gavage procedure. Directly after dosing, the animal showed labored breathing and the
animal was necropsied after consulting the veterinarian. At necropsy, brown-gritty fluid was
found in the thoracic cavity, red fluid in the trachea and the lungs were dark-red discolored.
Body weight and food consumption were considered normal.
Female No. 64 (10 mg/kg/day) was sacrificed on Day 27 post-coitum for animal welfare
reasons, due to a necrotic lesion with discharge (40 x 30 mm) in the cervical ventral region
observed from Day 25 post-coitum onwards. Other clinical signs included scabs and
decreased fecal production. Food consumption was reduced from Day 22 post-coitum
onwards but was considered normal in the periods before. Normal body weight gain was
observed from Day 7 to 24 post-coitum, with a slight body weight loss (2%) on Day 27
post-coitum.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Pregnant females surviving until scheduled necropsy -
At 20 mg/kg/day, mean body weight gain over the total study period (Days 7-29 post-coitum)
was within the same range as the control group. However, a mean lower body weight gain
(not statically significant) was observed compared to control between Days 7-9 post-coitum.
At 5 and 10 mg/kg/day, mean body weight gain was comparable to concurrent control.
The higher body weight gain observed over Day 7-9 post-coitum in females treated at
5 mg/kg/day was considered to be unrelated to treatment with the test item as no trend was
apparent regarding dose and duration of treatment.
Mean body weights of the control group were higher (not statistically significant) compared
to the 5, 10 and 20 mg/kg/day groups (up to 6%) upon randomization and at the initiation of
dosing, which resulted in mean body weights that were statistically significantly lower on
Days 12, 27 and 29 post-coitum at 10 mg/kg/day and from Day 9 post-coitum onwards at
20 mg/kg/day.
Body weight gain adjusted for the gravid uterus was considered unaffected up to
20 mg/kg/day.
- Females treated at 20 mg/kg/day with a dosing holiday up to four days -
For 2/5 females, body weight loss up to 9% compared to their maximum body weight was
observed, resulting in a dosing holiday for several days. After this dosing holiday body
weight gain recovered to normal.
For 2/5 females, a stable body weight was observed during the treatment period, with one of
these females losing 4% of its weight between Days 27 and 29 post-coitum.
For 1/5 females, normal body weight gain was observed.
- Females treated at 20 mg/kg/day sacrificed in extremis -
2/3 females were observed with body weight loss up to 7 to 9% compared to the initiation of
treatment. 1/3 females had a stable body weight prior to sacrifice.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Pregnant females surviving until scheduled necropsy -
Mean food consumption was considered unaffected upon treatment up to 20 mg/kg/day.
- Females treated at 20 mg/kg/day with a dosing holiday up to four days -
Overall, food consumption was lower compared to normal values (between 140-160 gram per
day) for all 5 females, with various periods showing a severely reduced food consumption
(≤20 grams per day) for 4/5 females. In 2/5 females, food consumption recovered to normal
values after the dosing holiday was initiated.
- Females treated at 20 mg/kg/day sacrificed in extremis -
Food consumption was reduced in 3/3 females during of the treatment period. Two females
were observed with a reduced food consumption from the initiation of treatment, developing
in an absent food consumption (0-13 gram per day) from Day 12 or 14 post-coitum onwards.
For the other female, food consumption was considered to be within normal ranges during the
first days of treatment, but a reduced food consumption prior to necropsy was observed.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Pregnant and non-pregnant females surviving until scheduled necropsy -
At 10 mg/kg/day, one pregnant female was observed with mucoid white content on the
fundus wall of the stomach and dark-black foci on the gallbladder wall. As the mucoid white
content on the stomach wall was also observed in one non-pregnant female treated at
20 mg/kg/day (as well as dark-black foci on the stomach), a test item-relation could not be
excluded.
Macroscopic observations at necropsy did not reveal any alterations that were considered to
have arisen as a result of treatment with the test item in the remaining animals.
Findings among test item-treated animals included dark-black foci on the lungs, prominent
lobular architecture of the liver and/or gray and hard nodule in the liver. In the absence of a
dose response and/or occurrence of the finding in the control group as well, they were
considered changes of no toxicological significance.
- Females treated at 20 mg/kg/day with a dosing holiday up to four days -
No macroscopic abnormalities at necropsy were observed.
- Females treated at 20 mg/kg/day sacrificed in extremis -
For 1/3 females, dark-black foci on the glandular stomach and mucoid white content on the
fundus wall were observed, which was considered test item-related.
The eye-injury of 1/3 females was confirmed at necropsy, which was considered unrelated to
the test item. For 1/3 females, no macroscopic findings were observed.

Details on results:

In total, 19/22 females treated at 20 mg/kg bw/day survived until scheduled necropsy of which 14/19 females were dosed for the entire treatment period.
Due to overall clinical condition of five females treated 20 mg/kg bw/day, it was decided to allow a dosing holiday (for 2 to 4 days) for these females. This temporary dosing intermission was initiated for 4 females until recovery in body weight and/or food consumption was observed again. One female was not dosed on Day 24 post-coitum, due to its clinical signs.
Considering that this pause in dosing occurred after completion of the organogenesis, generally considered the period between Days 6-18 post-coitum, and based on clinical signs the animals have been dosed up to their respective maximum tolerability also after this period, it is considered that the results from the evaluation of the litters of these animals remain valid for the evaluation for developmental toxicity of the substance up to maximum tolerable dose levels.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean numbers of corpora lutea and implantation sites in the control, 5 and 10 mg/kg bw/day groups were comparable and in the range of normal biological variation.

At 20 mg/kg bw/day pre- and post-implantation loss was comparable to that observed for the control group and in the range of normal biological variation.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

The mean numbers of pre- and post-implantation loss in the control, 5 and 10 mg/kg bw/day groups were comparable and in the range of normal biological variation.

At 20 mg/kg bw/day two females scored as non-pregnant were observed with corpora lutea (one which had a dosing holiday and one which was sacrificed in extremis). In rabbits it is hard to distinguish between non-pregnancy and early resorption of the litter that occurred during the first days after implantation, as they are both presenting as an empty uterus. As the total number of females with litters was reduced at the dose level on which severe maternal toxicity was observed, it cannot be excluded that possible implantation points were resorbed in a very early stage of the pregnancy and therefore were not visible during necropsy.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All females, including those that were necropsied preterm, were found to be pregnant, except for two females of the control group, two of the 5 mg/kg bw/day, one female of the 10 mg/kg bw/day and five females of the 20 mg/kg bw/day. Of these females, three were observed with corpora lutea (one control female: 2 corpora lutea, and two females of the 20 mg/kg bw/day having 3 or 13 corpora lutea).
Excluding non-pregnant females and females that did not survive until scheduled necropsy, there were 20 females with viable litters in the control group, and 18, 20 and 15 females at 5, 10 and 20 mg/kg bw/day, respectively. Of these high dose group females, four had a temporary dosing intermission of up to four days during the study between Days 21 and 28 post-coitum, depending on mating date.
At 20 mg/kg bw/day, the number of pregnant females at necropsy was remarkably lower compared to concurrent control and historical control data (Historical Control Data, Albino, New Zealand White Rabbit (2016-2020): No. of Animals Examined at Laparohysterectomy = 729; No. Non-gravid = 61).

Details on maternal toxic effects:

All females, including those that were necropsied preterm (see Section 7.2.1), were found to
be pregnant, except for Female Nos. 09 and 16 (control group), Nos 27 and 41 (5 mg/kg/day),
No. 48 (10 mg/kg/day) and Nos. 72, 73, 79, 83 and 86 (20 mg/kg/day). Of these females,
three were observed with corpora lutea (control Female No. 9; 2 corpora lutea, No. 79;
3 corpora lutea and No. 83; 13 corpora lutea, both treated at 20 mg/kg/day).
Excluding non-pregnant females and females that did not survive until scheduled necropsy,
there were 20 females with viable litters in the control group, and 18, 20 and 15 females at 5,
10 and 20 mg/kg/day, respectively. Of these high dose group females, Nos. 67, 76, 78 and 84
had a temporary dosing intermission of up to four days during the study between Days 21 and
28 post-coitum, depending on mating date.
The developmental data from the 20 mg/kg/day group was not used for any conclusions and
was reported separately to avoid misinterpretation as surviving females did not meet the minimum number of 16 surviving to termination with implantations. Below an overview is given of
the females of which the data could not (fully) be used for a rough, overall description of the
developmental data at 20 mg/kg/day. Breakdown of Females in the 20 mg/kg/day group (Group 4) of which Developmental Data were not used for any Conclusions.
Female No. Reason for Interpretation of the Data with Caution/Exclusion:
F67 Temporarily dosing intermission Not dosed on Days 24, 25, 27 and 28 post-coitum; F72 Not pregnant – Data excluded; F73 Not pregnant – Data excluded; F74 Euthanized in extremis on Day 22 post-coitum – Data excluded; F76 Temporarily dosing intermission Not dosed on Days 23 and 26 post-coitum; F78 Temporarily dosing intermission Not dosed on Day 24 post-coitum; F79
Temporarily dosing intermission – Data excluded Not dosed on Days 22, 23 and 25 post-coitum
Not pregnant; F82 Euthanized in extremis on Day 19 post-coitum – Data excluded; F83 Euthanized in extremis on Day 13 post-coitum – Data excluded Not pregnant; F84 Temporarily dosing intermission Not dosed on Days 21, 22, 24 and 25 post-coitum; F86 Not pregnant – Data excluded.
The mean numbers of corpora lutea and implantation sites, and pre- and post-implantation
loss in the control, 5 and 10 mg/kg/day groups were comparable and in the range of normal
biological variation.
At 20 mg/kg/day, the number of pregnant females at necropsy was remarkably lower
compared to concurrent control and historical control.
Two females scored as non-pregnant were observed with corpora lutea (No. 79 which had a dosing holiday and No. 83 which was sacrificed in extremis). In rabbits it is hard to distinguish between non-pregnancy and early resorption of the litter that occurred during the first days after
implantation, as they are both presenting as an empty uterus. As the total number of females
with litters was reduced at the dose level on which severe maternal toxicity was observed, it
cannot be excluded that possible implantation points were resorbed in a very early stage of
the pregnancy and therefore were not visible during necropsy.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Remarks on result:

other:

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related relevant effects on fetal body weights (both sexes) noted by
treatment up to 10 mg/kg/day. Note: At 20 mg/kg/day, the mean litter size was considered within normal values.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 10 mg/kg/day.
Note: At 20 mg/kg/day, the male:female ratio was considered normal

Changes in litter size and weights:

not specified

Description (incidence and severity):

There were no test item-related relevant effects on fetal body weights (both sexes) noted by
treatment up to 10 mg/kg/day.
At 20 mg/kg/day, mean fetal body weights (both sexes) were lower compared to control,
reaching statistical significance for male weights only.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No external variations or malformations that were considered to be test item-related were
observed up to at least 10 mg/kg/day.
One control fetus, No. 22-L9, was observed with an inwards malrotated right hind-paw and
one fetus at 5 mg/kg/day (No. 34-R8) which was dead, had a distended abdomen. In addition,
one late resorption of the control group (No. 15-R7) had gastroschisis. Based on the single
occurrence and/or the occurrence in the control group only, these were considered to be
unrelated to treatment with the test item. The dead fetus at 5 mg/kg/day (No. 34-R8) that had a distended abdomen was also observed with the external variation: “localized subcutis, edema”. Its single incidence rules out a test item-effect.
At 20 mg/kg/day, no external variations or malformations were observed

Skeletal malformations:

not specified

Description (incidence and severity):

No skeletal variations or malformations that were considered to be test item-related were
observed up to at least 10 mg/kg/day.
Skeletal malformations were discovered in the ribs and vertebra in 3 (3), 10 (8) and
2(2) fetuses (litters) of the control, 5 and 10 mg/kg/day groups, respectively. These were
observed at low incidences and/or not in a dose dependent manner; consequently, all were
ruled out as test-item related.
All skeletal variations observed up to 10 mg/kg/day, occurred in the absence of a dose-related
incidence trend and/or infrequently or occurred in conjunction with a malformation.
Therefore, they were considered not test item-related.
At 20 mg/kg/day, skeletal malformations were discovered in the ribs and vertebra in 4 (3)
fetuses (litters).
Noteworthy were the skeletal variations “unossified hind-paw phalanges” and “misaligned
sternabra”, that had a statistical significant increased incidence compared to concurrent
control.
For the variation “un-ossification of hind-paw phalanges”, this increase was caused by three
fetuses out of three litters (Nos. 67-L1, 77-R10 and 88-L3). Two of these fetuses, which were
observed with low body weights as well (17.7 and 21.7 grams), showed additional signs of
retarded ossification for the talus and sternebrae. These findings are in line with the slightly
lower group mean fetal weights at this dose level.
The increase for “misaligned sternebra” was not accompanied by an increased misalignment
of other bones i.e. ilium, costal cartilage and vertebra.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No visceral variations or malformations that were considered to be test item-related were
observed up to at least 10 mg/kg/day.
Visceral malformations were observed in 3 (2), 2 (2) and 2 (2) fetuses (litters) of the control,
5 and 10 mg/kg/day groups, respectively. As the malformations occurred in single fetuses
and/or in control fetuses only, these were considered spontaneous events and not test
item-related.
Visceral variations occurred across a variety of structures, occurring at low incidences and/or
in the control group only, ruling out any test item-relationships. The statistically significant
increase in the incidence of “retrocaval right ureters” observed at 5 mg/kg/day was considered
not related to treatment with the test item in the absence of a dose dependency.
At 20 mg/kg/day, visceral malformations were observed in 2(1) fetuses (litters).

Details on embryotoxic / teratogenic effects:

Note: As according to the guidelines, a minimum of 16 females with implantations at
termination is required in each group for an adequate evaluation, the 15 such animals
available at 20 mg/kg/day were considered not sufficient for a robust and valid evaluation of
the developmental data. As such, the developmental data from the 20 mg/kg/day group cannot
be used for any conclusions. However, despite formally one litter short, it is important to note
that even at 20 mg/kg bw/day, which is possibly just above the maximally tolerated level, still
the only developmental effects observed involved a slight increase of skeletal variations.
These involved “unossified hind-paw phalanges” and “misaligned sternabra” and are in
general in line with the also observed slightly lower group mean fetal weights at this dose
level.No test item-related changes were noted up to 10 mg/kg/day in any of the developmental
parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external,
visceral and skeletal malformations and developmental variations).

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

> 10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other:

Remarks on result:

other: Possible reduction observed at 20 mg/kg bw/day. Though based off guidance dose group does not qualify for conclusions.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

DOSE FORMULATION ANALYSES

Table 1 - Accuracy and Homogeneity Test

Group	Sample Position	Concentration^1 [mg/mL]		Accuracy [%]		Homogeneity [%]
		Target	Analyzed	Individual	Mean
1	50% height	0 0	nd nd	na na	na	na
2	90% height   50%height   10% height	4 4 4 4 4 4	4.24 4.19 4.39 4.25 4.25 4.23	106 105 110 106 106 106	106	1.6
3	50% height	8 8	9.19 8.64	115 108	111	na
4	90% height   50%height   10% height	16 16 16 16 16 16	17.7 17.3 17.9 17.5 16.6 16.9	111 108 112 109 104 106	108	2.8

¹ Due to issues with the analytical system, sample extracts prepared on 26 Apr 2021 were re-diluted and analyzed on 30 Apr 2021. The sample extracts were stored in normal laboratory conditions at room temperature from 26 Apr 2021 until analysis on 30 Apr 2021.

n.d. Not detected.

n.a. Not applicable.

Accuracy
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The determined mean values ranged from 106% to 111%. No test item was detected in the Group 1 (vehicle) formulation.

Homogeneity
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%). The determined mean values ranged from 1.6% to 2.8%.

 

 

Table 2 - Summary of Body Weights: Gestation (mean values, grams)

	Day(s) Relative to Mating
	0	7	9	12	15	18	21	24	27	29
0 mg/kg bw/day	3764.0	3717.4	3743.0	3807.1	3893.9	3947.7	3978.2	4025.1	4074.3	4116.1
5 mg/kg bw/day	3615.4	3561.4	3639.9	3665.2	3757.4	3808.1	3856.7	3915.0	3974.6	4033.8
10 mg/kg bw/day	3490.6	3491.5	3524.9	3574.8*	3675.9	3728.3	3778.6	3810.6	3819.7*	3864.4*
20 mg/kg bw/day	3498.8	3486.1	3485.7*	3513.1**	3586.6**	3593.8**	3621.0**	3722.1*	3771.9**	3822.0*

Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

 

Table 3 - Summary of Food Consumption: Gestation (mean values, g/animal/day)

	Day(s) Relative to Mating
	7 to 9 [G]	9 to 12 [G1]	12 to 15 [G1]	15 to 18 [G]	18 to 21 [G]	21 to 24 [G1]	24 to 27 [G1]	27 to 29 [G]	7 to 29 [G1]
0 mg/kg bw/day	133.88	129.38	102.62	116.30	130.73	116.10	98.32	104.28	116.21
5 mg/kg bw/day	143.29	138.15	109.39	129.33	131.72	120.85	103.98	113.14	123.31
10 mg/kg bw/day	140.69	129.86	111.32	129.46	137.46	111.78	72.21*	90.28	114.85
20 mg/kg bw/day	122.26	104.84*	83.45	93.08	97.25	102.64	89.82	95.80	102.41

[G] - Kruskal-Wallis & Dunn
[G1] - Anova & Dunnett: * = p ≤ 0.05

 

Table 4 - Summary of Maternal Performance and Mortality

		0 mg/kg bw/day	5 mg/kg bw/day	10 mg/kg bw/day	20 mg/kg bw/day
Group size – females		22	22	22	22
Number of Females Pregnant	N %	20 90.9	20 90.9	21 95.5	17 77.3
Female with Live Fetuses	N %	20 100.0	20 100.0	21 100.0	17 100.0
Total Resorptions	N %	0 0.0	0 0.0	0 0.0	0 0.0
Female with all Nonviable	N %	0 0.0	0 0.0	0 0.0	0 0.0
Terminal Euthanasia	N %	22 100.0	20 90.9	21 95.5	19 86.4
Unscheduled Death/Euthanasia	N %	0 0.0	2 9.1	1 4.5	3 13.6
Unscheduled Euthanasia	N %	0 0.0	2 9.1	1 4.5	3 13.6

 

Table 5 - Summary of Ovarian and Uterine Examinations and Litter Observations

		0 mg/kg bw/day	5 mg/kg bw/day	10 mg/kg bw/day	20 mg/kg bw/day
Female with Live Fetuses	N %	20 100.0	18 100.0	20 100.0	15 100.0
Number of Corpora Lutea [k]	Mean %Diff	11.4 -	11.3 -0.1	10.9 -4.4	11.1 -1.9
Number of Implantations [k]	Mean %Diff	10.5 -	10.6 1.1	9.6 -8.6	10.6 1.0
Pre-implantation Loss (%) [k]	Mean %Diff	7.21 -	6.33 -12.13	11.98 66.16	4.86 -32.61
Total Number of Fetuses [k]	Mean %Diff	9.9 -	9.9 0.4	8.9 -10.6	10.1 1.7
Number of Live Fetuses [k]	Mean %Diff	9.9 -	9.9 -0.1	8.9 -10.6	10.1 1.7
Number of Dead Fetuses [k]	Mean %Diff	0.0 -	0.1 -	0.0 -	0.0 -
Number of Early Resorptions [k]	Mean %Diff	0.5 -	0.4 -11.1	0.6 20.0	0.2 -60.0
Number of Late Resorptions [k]	Mean %Diff	0.1 -	0.2 122.2	0.2 50.0	0.3 233.3
Total Number of Resoprtions [k]	Mean %Diff	0.6 -	0.7 11.1	0.8 25.0	0.5 -11.1
Post-implantation Loss (%) [k]	Mean %Diff	5.40 -	6.77 25.23	7.98 47.70	4.45 -17.62
Number of Live Male Fetuses [k]	Mean %Diff	4.5 -	5.3 18.5	4.6 1.1	5.0 11.1
Number of Live Feale Fetuses [k]	Mean %Diff	5.4 -	4.6 -15.6	4.3 -20.4	5.1 -6.2
Live Male Fetus/Litter (%) [k]	Mean %Diff	46.80 -	53.23 13.74	50.17 7.20	49.92 6.67
Live Female Fetuses/Litter (%)	Mean %Diff	53.20 -	46.15 -13.25	49.83 -6.33	50.08 -5.86
Mean Fetal Weight Males (g) [G]	Mean %Diff	40.71 -	41.07 0.89	41.19 1.18	35.94* -11.72
Mean Fetal Weight Females (g) [G]	Mean %Diff	38.83 -	39.16 0.86	39.75 2.36	36.12 -6.98
Mean Fetal Weight all (g) [G]	Mean %Diff	39.61 -	40.36 1.89	40.46 2.15	35.95 -9.24

[k] - Kruskal-Wallis & Dunn

[G] - Anova & Dunnett: * = p ≤ 0.05

Applicant's summary and conclusion

Conclusions:

The prenatal developmental toxicity study with C16-18, C18-unsaturated-alkyl dipropylene triamine in time-mated female New Zealand White rabbits resulted to a maternal NOAEL of 10 mg/kg/day, based on mortality and effects on body weights and food consumption at 20 mg/kg/day. The developmental NOAEL was 10 mg/kg/day being the highest dose available for evaluation.
The developmental data from the 20 mg/kg/day group was not used for any conclusions as with 15 evaluable litters the minimum required number of 16 litters for a robust and valid evaluation of the developmental data was not reached. However, the available limited results from this dose level do not indicate developmental effects.

Executive summary:

This study was conducted according to GLP in line with TG 414. Time-mated female New Zealand White rabbits were treated with C 16-18, C18-unsaturated-alkyl dipropylene triamine from Day 7 to 28 post-coitum, inclusive by daily oral gavage at dose levels of 5, 10 and 20 mg/kg/day. The rabbits of the control group received the vehicle, propylene glycol, alone.
Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

In total, 19/22 females treated at 20 mg/kg/day survived until scheduled necropsy. Of these, five females required a dosing pause for one to up to four days between Days 21 and 28 post-coitum due to overall very poor clinical condition. Considering that this pause in dosing occurred after completion of the organogenesis, generally considered the period between Days 6-18 post-coitum, and based on clinical signs the animals have been dosed up to their respective maximum tolerability also after this period, it is considered that the results from the evaluation of the litters of these animals remain valid for the evaluation for developmental toxicity of the substance up to maximum tolerable dose levels.

At 20 mg/kg/day, three females were sacrificed in extremis based on body weight loss, reduced food consumption and/or clinical signs. For one of these females, the observed macroscopic findings (dark-black foci on the glandular stomach and mucoid white content on the fundus wall) were considered test item-related. Comparable clinical signs and effects on body weight and/or food consumption were observed for approximately 25% of the remaining females, and therefore resulting in a dosing holiday for these females to prevent early termination. As these findings were in line with the females that were early terminated, these effects were considered to be adverse.

At 5 and 10 mg/kg/day, no test item-related clinical signs or effects on body weight and food consumption were observed. Macroscopic findings observed in a single female treated at 10 mg/kg/day were considered non-adverse due to its single occurrence and in the absence of other clinical symptoms for this animal.

No test item-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e. corpora lutea, implantation sites and pre- and post-implantation loss). This thus resulted in the conclusion of a maternal NOAEL of 10 mg/kg bw/day.

Excluding non-pregnant females and females that did not survive until scheduled necropsy, there were 20 females with viable litters in the control group, and 18, 20 and 15 females at 5, 10 and 20 mg/kg/day, respectively. As according to the guidelines, a minimum of 16 females with implantations at termination is required in each group for an adequate evaluation, the 15 such animals available at 20 mg/kg/day were considered not sufficient for a robust and valid evaluation of the developmental data. As such, the developmental data from the 20 mg/kg/day group cannot be used for any conclusions. However, despite formally one litter short, it is important to note that even at 20 mg/kg bw/day, which is possibly just above the maximally tolerated level, still the only developmental effects observed involved a slight increase of skeletal variations (79.85%, 81.10%, 82.66% and 84.80% in control, 5, 10 and 20 mg/kg respectively). These involved “unossified hind-paw phalanges” and “misaligned sternabra” and are in general in line with the also observed lower group mean fetal weights at this dose level. At 20 mg/kg/day, mean fetal body weights (both sexes) were lower compared to control, reaching statistical significance for male weights only. No other notable effects were observed in dose groups 5 and 10 mg/kg/day that were attributed to dosing. This thus results in an effective developmental NOAEL of 10mg/kg/day.