3-aminopropyldimethylamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Sep 2020 - 16 Oct 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-aminopropyldiethylamine

EC Number:

203-236-4

EC Name:

3-aminopropyldiethylamine

Cas Number:

104-78-9

Molecular formula:

C7H18N2

IUPAC Name:

N,N-diethylpropane-1,3-diamine

Test material form:

liquid

Details on test material:

Appearance: colorless liquid
Storage condition: At room temperature in transparent glass flasks in an opaque plastic flask

Specific details on test material used for the study:

- Source: Arkema, La Chambre, France
- Appearance: colorless liquid
- Storage condition of test material: At room temperature in transparent glass flasks in an opaque plastic flask
- Specific requirements (handling conditions): See Safety Data sheet (Diethylaminopropylamine is very toxic, sensitizing and irritant agent and must be handled with care)

Test animals

Species:

rabbit

Strain:

other: KBL New Zealand White, Specific Pathogen Free (KBL NZW SPF)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France (Châtillon-sur-Chalaronne, France)
- Age at study initiation: approximately 18-20 weeks
- Weight at study initiation: mean body weight 3629 g (range: 2995 g to 4310 g)
- Females: Sexually mature and primigravid
- Fasting period before study: No
- Housing: individually, in noryl cages (Tecniplast, floor area: 4200 cm²/height: 46 cm). Each cage contained dumbbell, music and hay for environmental enrichment.
- Diet: breeding pelleted diet "type 110C", (3409 CRL, KLIBA, Kaiseraugst, Switzerland), ad libitum. In addition, carrots were distributed to one animal in the control group on Days 12 and 13 p.c., one animal in the mid-dose group on Day 22 p.c., and one animal in the high-dose group on Days 12 and 13 p.c., because of drastically reduced food consumption
- Water: tap water (filtered with a 0.22 µm filter), ad libitum
- Contaminant analyses: The batches of diet were analyzed regularly by the Supplier for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet or drinking water at levels which could be expected to interfere with, or prejudice, the outcome of the study.
- Acclimation period: 4 or 5 days before the beginning of the treatment period. For some of the receptions of animals over the last years, this period was shortened to 4 up to 3 days (although according to OECD414 test guidance, animals should be acclimated to the environmental conditions of a study for at least 5 days before dosing).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C (set to maintain)
- Humidity (%): 50 ± 20% (set to maintain)
- Air changes (per hr): about 5 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 8h dark/16h light

IN-LIFE DATES: From: 10 Sep 2020 - 16 Oct 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Drinking water treated by reverse osmosis ELIX 5 (Millipore SA)

Details on exposure:

- Type of test item formulation (visual observation): Solution in the vehicle
- Preparation procedure: According to Study No. 42758 AHS (Tarlet, 2016) (homogeneity and stability testing) describing the preparation procedure for a range of concentrations of 2 to 200 mg/mL. All formulations were adjusted to pH to 8.0 (± 0.5). Dose formulations ranging from 2 mg/mL to 200 mg/mL were considered to be suitable for routine administration in GLP Toxicological studies, for up to 4 days after preparation and those ranging from 10 to 150 mg/mL for up to 10 days after preparation.
- Frequency of preparation: Test item dose formulations: based on test item dose formulation stability (Study No. 42758 AHS (Tarlet, 2016)) and vehicle expiry; Control dose formulations: according to the frequency of test item dose formulation preparation
- Storage and delivery conditions (control and test item dose formulations): At room temperature and protected from light

ADMINISTRATION
The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time. The quantity of the dose formulation administered to each animal was adjusted according to the most recently recorded body weight. A constant dosage volume of 2 mL/kg bw/day was used. Control animals (group 1) received the vehicle only. The dose formulations were maintained under delivery conditions (at room temperature and protected from light) throughout the administration procedure. The control and test item dose formulations were stirred just before administration and were maintained under continuous magnetic stirring throughout the administration procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analytical technique: Gas chromatography with flame ionization detection (GC FID)
- Principle and validation of the method: Analytical method developed and validated at Charles River Laboratories Evreux Study No. 42757 VAA (Bezard/Garapon, 2016/2021) prior to dose formulation analysis. Checked parameters, acceptance criteria and obtained results are detailed in the validation report.
- Determination of test item concentrations in dose formulations: Twice in the study (Days 6 and 28 p.c.). A sample was taken from control and test item dose formulations and analyzed using the validated method.
- Acceptance criterion: Measured concentration = nominal concentration ± 10%.

Details on mating procedure:

Females were mated at the breeder's facilities. The day of confirmed mating (visual assessment) was designated as Day 0 p.c.

Duration of treatment / exposure:

Day 6 to Day 28 post coitum (p.c.) inclusive; i.e. from implantation to the day prior to the scheduled hysterectomy

Frequency of treatment:

Once daily

Duration of test:

Day from arrival of animals to the test facility (Day 1 or 2 p.c.) till scheduled euthanasia and hysterectomy (Day 29 p.c.)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Low-dose group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Mid-dose group

Doses / concentrations 3

Dose / conc.:

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

Remarks:

High-dose group

No. of animals per sex per dose:

24 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

ALLOCATION TO GROUPS
During the acclimation period, the animals were allocated to the groups, according to a stratified procedure base on body weight recorded on Day 1 or 2 p.c., to ensure comparatively similar mean body weight of the groups.

ADMINISTRATION ROUTE RATIONALE
The oral route was selected since it is one of the routes of administration recommended by the regulatory authorities.

DOSE SELECTION RATIONALE
The dose levels were selected in agreement with the Sponsor, on the basis of the results of a preliminary prenatal development study [Study No. 47473 RSL (Papineau, 2020)] in which New Zealand White female rabbits (8 mated females per group) received the test item at 0, 100, 300 or 750 mg/kg bw/day, from Day 6 to Day 28 p.c., inclusive.
At 300 and 750 mg/kg bw/day, 4/8 and 8/8 females were prematurely euthanized for ethical reasons between Days 15 and 23 p.c. and between Days 10 and 12 p.c., respectively. Prior to euthanasia, signs of poor clinical condition were observed (i.e. soft feces or absence of feces, soiled urogenital area, emaciated appearance, cold to the touch, ventral decubitus, hypotonia, body weight loss and/or almost no food intake). The cause of these signs was probably related to gastrointestinal changes. At hysterectomy, on Day 29 p.c., all surviving females were pregnant (with live fetuses at termination). No test item-related clinical signs were reported in the 4/8 surviving females at 300 mg/kg bw/day or in females at 100 mg/kg bw/day.
At 300 mg/kg bw/day and when compared with controls, there was a severe reduction in food consumption (down to -82%) between Days 6 and 15 p.c. (p<0.01 or p<0.001) which tend to be less pronounced in surviving females until the end of the treatment period (down to -54%; not statistically significant). This finding correlated with body weight loss and lower body weight gain over the whole treatment period (+70 g vs. +405 g in controls; p<0.01). The body weight was slightly affected from Day 12 p.c. (down to -10% vs. controls).
At 100 mg/kg bw/day, no effects on body weight or food consumption were noted although a low body weight gain was observed over the whole treatment period (+298 g vs. +405 g in controls). Test item-related macroscopic observations were noted at = 100 mg/kg bw/day in the stomach, at = 300 mg/kg bw/day in the kidneys and at 750 mg/kg bw/day in the intestines and liver. At 300 and 100 mg/kg bw/day, low net body weight change (-393.3 g and -269.2 g vs. -187.2 g in controls, respectively) was noted. At 300 mg/kg bw/day, lower gravid uterus weight (-22% vs. controls) and lower fetal weight ( 11% vs. controls) were observed. There were no effects on hysterectomy data at 100 or 300 mg/kg bw/day and no effects on fetal body weight at 100 mg/kg bw/day. There were no external variations or malformations at fetal examination at 300 or 100 mg/kg bw/day.
Therefore, 130 mg/kg bw/day was selected as the high-dose level. The low-dose and mid-dose were selected using a ratio representing approximately a 3-fold interval (i.e. 15 and 50 mg/kg bw/day).

Examinations

Maternal examinations:

MORBIDITY AND MORTALITY: Yes
- Time schedule: Each animal, once a day before the treatment period and at least twice a day during the treatment period, including weekends.

CLINICAL SIGNS: Yes
- Time schedule: From arrival, each animal once a day as part of routine examinations. From the start of the treatment period, each animal once a day
- Observations made: clinical signs (including evidence of abortion)

BODY WEIGHT: Yes
- Time schedule: each animal, on Days 2, 4, 5, 6, 9, 12, 15, 19, 24 and 29 p.c.

FOOD CONSUMPTION: Yes
- Time intervals: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-19, 19-24 and 24-29 p.c. Any obvious spillage of food was documented.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: By an intravenous injection of sodium pentobarbital (prematurely and scheduled (29 p.c.) euthanized females).
- Macroscopic post-mortem examinations: principal thoracic and abdominal organs (prematurely and scheduled euthanized females).
- Preservation of tissues: Macroscopic lesions and stomach (prematurely and scheduled euthanized females). Since remarkable macroscopic lesions were observed in test item-treated group animals, the corresponding tissues of 5 control animals were sampled and preserved.

MICROSCOPIC EXAMINATIONS: Yes
- Dose groups examined: control and low-, mid- and high-dose, all animals
- Tissue examined: Stomach (and macroscopic lesions)
- Preservation: 10% buffered formalin
- Preparation of histological slides: embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with haematoxylin-eosin.

Ovaries and uterine content:

EXAMINATION OF OVARIES AND UTERINE CONTENT: Yes, after termination (29 p.c.)
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number and distribution of dead and live fetuses: Yes
- Number and distribution of early and late resorptions: Yes
- Number and distribution of uterine scars: Yes
- Number and distribution of implantation sites: Yes
A gross evaluation of placentas was also undertaken.

CLASSIFICATION USED TO RECORD
- uterine scar: uterine implantation without implant
- dead fetus: dead fetus with no degenerative changes
- early resorption: evidence of implant without recognizable embryo
- late resorption: dead embryo or fetus with degenerative changes

Blood sampling:

Not performed

Fetal examinations:

POST-MORTEM EXAMINATIONS
- Sacrifice: By intraperitoneal injection of sodium pentobarbital.
- Body weight and sex: Yes, of each fetus. Sex was determined by internal examination of the sexual organs.
- External examinations: Yes, each fetus, which included the observation of all visible structures, surfaces and orifices.
- Body examinations: Yes, as soon as possible after euthanasia, which included observation of all the organs and structures of the neck, thorax and abdomen and indication of incomplete testicular descent/cryptorchidism in male fetuses. The fetuses were then eviscerated.
- Head and brain examinations: Yes, one half of the fetuses were decapitated and the head was fixed in Harrison’s fluid with decalcification. Serial sectioning was performed for evaluation of brain, nasal passages and tongue (and other structures, where appropriate). In the other half of the fetuses, the brain of each fetus was sampled and fixed in Harrison’s fluid. Serial section was made for examination of the organ.
- Skeletal examinations: Yes, the carcasses were fixed with ethyl alcohol, detailed examination were performed of skeleton (bones + cartilage) after staining with alizarin red S and alcian blue. This examination included observation of all the bone and cartilage structures of the skull (50% of fetuses), spine, rib cage, pelvis and limbs (100% of fetuses).
- Anogenital distance: No

CLASSIFICATION USED TO RECORD
- Malformation: A permanent structural change that is likely to adversely affect the survival or health.
- Variation: A change that occurs within the normal population under investigation and is unlikely to adversely affect the survival or health (this might include a delay in growth or morphogenesis that otherwise followed a normal pattern of development).

Statistics:

Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).

Indices:

Data are recorded and calculated using computerized validated software. Data of non-pregnant females are not included in group mean calculations (body weight, body weight change, food consumption and litter data).

The following parameters were calculated:
For each pregnant female:
• body weight change for different intervals,
• net body weight (presented as carcass weight): Body weight on Day 29 p.c. - gravid uterine weight
• net body weight change: Body weight on Day 29 p.c. - body weight on Day 6 p.c. - gravid uterine weight
For each litter:
• total number of resorptions: Sum of uterine scars + early resorptions + late resorptions
• total number of dead fetuses: Sum of dead fetuses
• % of dead fetuses per litter: (Total number of dead fetuses / Number of implantation sites) x 100
• total number of live fetuses: Sum live male + live female fetuses
• % of live fetuses per litter: (Total number of live fetuses / Number of implantation sites) x 100
• % of pre-implantation loss: ((Number of corpora lutea - Number of implantation sites) / Number of corpora lutea) x 100
• % of post-implantation loss: ((Number of implantation sites - Number of live fetuses) / Number of implantation sites) x100
• average fetal body weight: Sum of individual fetal weights / Number of live fetuses
• average placental weight: Sum of individual placental weights / Number of placenta weighed

Parameters calculated for each group are given in section "Any other information on materials and methods incl. tables".

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs were observed during the study at any dose level. All clinical signs recorded in surviving females were considered to be unrelated to the test item treatment as they were present both in control and test item-treated animals and/or were observed in isolated animals.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

At 50 mg/kg bw/day, two females were prematurely euthanized as follows:
- One female was prematurely euthanized on Day 22 p.c. due to abortion. Blood and three placentae were found in the bedding. At maternal necropsy, no macroscopic post-mortem lesions were noted. This isolated abortion was considered to be incidental.
- One female was prematurely euthanized on Day 25 p.c. due to poor clinical condition: emaciated appearance along with soft feces or absence of feces from Day 21 p.c., associated with body weight loss on Days 12-25 p.c. (-26%) and almost no food intake from Day 12 p.c.. At necropsy, equivocal reddish colored areas were noted in the stomach along with thickened and gelatinous mucosa. A relationship to the test item treatment was considered to be unlikely as there were no unscheduled deaths or signs of marked toxicity in females at the higher dose level.
No unscheduled deaths occurred in females at 0, 15 or 130 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights and mean body weight changes recorded in control and test item treated animals are summarized in Table 1 of section "Any other information on results incl. tables".
At 130 and 50 mg/kg bw/day, when compared with controls, slightly lower mean body weight gain was recorded between Days 6 and 29 p.c. (+293 g and +318 g vs. +363 g in controls, respectively; not statistically significant). These differences were especially due to continuous slightly lower mean body weight gain at 50 mg/kg bw/day or resulted from transiently lower mean body weight gain (statistically significant on Days 6-9 p.c. and 24-29 p.c.) at 130 mg/kg bw/day. While a relationship with the test item treatment could not be excluded, the differences in the overall mean body weight changes were not statistically significant and did not impact the Day 29 p.c. body weight. They were therefore considered to be non-adverse.
At 15 mg/kg bw/day, no effects on mean body weight or mean body weight change were noted.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption values recorded during the study are summarized in Table 2 of section "Any other information on results incl. tables".
At 130 mg/kg bw/day and when compared with controls, slightly lower mean food consumption was noted between Days 6 and 9 p.c. (-19%; p<0.05) and between Days 24 and 29 p.c. (-34%; not statistically significant). As these differences were of slight magnitude and did not affect the mean body weight, they were considered to be non-adverse. At 50 and 15 mg/kg bw/day, no effects on mean food consumption were noted.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine 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):

No effects were noted on mean gravid uterus weight, carcass weight or net body weight changes.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related macroscopic findings. All gross observations were considered to be consistent with spontaneous findings encountered in the rabbits of these strain and age. This included the reddish colored areas (red discolorations) in the stomach from a few females treated at = 15 mg/kg bw/day that correlated with microscopic lesions (congestion and hemorrhage) seen also in controls.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic findings. The few minimal to slight congestions and hemorrhages were seen with similar incidence and severity in both controls and test item-treated animals and thus were considered to be part of the spontaneous background and/or to be related to the euthanasia/necropsy procedure.

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One female in the mid-dose group lost the fetuses due to abortion on Day 22 p.c.. Blood and three placentae were found in the bedding. At maternal necropsy, no macroscopic post mortem lesions were noted. This isolated abortion was considered to be incidental.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Hysterectomy data are presented in Table 3 of section "Any other information on results incl. tables".
When compared with controls, there were no significant effects on hysterectomy parameters (mean number of corpora lutea, implantation sites, pre-implantation loss, live fetuses and post implantation loss) at any dose level. Although a slight increase in the mean post implantation loss was recorded from 50 mg/kg bw/day, these differences were not statistically significant, remained within the range of the HCD and were therefore not considered to be adverse.
Some statistically significant differences (i.e. total number of live fetuses, total number of resorptions + scars and early resorptions) were observed. They were not considered to be of toxicological importance as they were poorly dose-related and/or as the mean number of live fetuses per female and the mean number of early resorptions were not statistically significantly different from controls.

Total litter losses by resorption:

not specified

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Some statistically significant differences in total number of resorptions were observed. They were not considered to be of toxicological importance as the mean number of early resorptions were not statistically significantly different from controls (see Table 3 in section "Any other information on results incl. tables").

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead fetuses in all dose groups (see Table 3 in section "Any other information on results incl. tables").

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

At hysterectomy on Day 29 p.c., 23/24, 21/24, 22/24 and 23/24 females were pregnant with live fetuses in the groups treated at 0, 15, 50 and 130 mg/kg bw/day, respectively (see Table 3 in section "Any other information on results incl. tables").

Other effects:

not examined

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal body are presented in Table 4 of section "Any other information on results incl. tables".
A lower mean fetal body weight was noted at 130 mg/kg bw/day (-7% vs. controls), that correlated with delayed ossification noted at skeletal examination at this dose level. As this difference was of minimal magnitude and not statistically significant, it was not considered to be adverse. No relevant differences from controls were observed for the mean fetal body weight at the other dose levels.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was not affected by the test item treatment at any dose level.

Changes in litter size and weights:

not examined

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

VARIATIONS
The distribution of the fetal and litter incidences of external variations is presented in Table 5 of section "Any other information on results incl. tables".
There was no test item-related increase of the frequency of external variations. At 130 mg/kg bw/day, one dam had two fetuses with abnormal color of amniotic fluid (that also showed omphalocele) and one dam had one fetus with blackish color of abdomen. At 50 mg/kg bw/day, one dam had one fetus with abnormal color of amniotic fluid along with polyhydramnios (that also showed proboscis, cyclopia and meningocele).
At 15 mg/kg bw/day, no external variations were observed. Although the above-mentioned findings were not reported among control animals, they were considered to be incidental as they were of isolated incidence, without a dose-relationship and/or with an incidence within the range of the HCD (historical control data).

MALFORMATIONS
The distribution of the fetal and litter incidences of external malformations are presented in Table 6 of section "Any other information on results incl. tables".
There was no test item-related increase in the frequency of external malformations.
At 130 mg/kg bw/day, one fetus from one litter showed omphalocele.
At 50 mg/kg bw/day, three fetuses from two litters showed external malformations:
• One fetus: proboscis, cyclopia and meningocele that correlated with visceral malformations,
• One fetus: open eye, ectrodactyly and ombilical hernia,
• One fetus: ombilical hernia.
As malformations observed at 130 and 50 mg/kg bw/day were noted without a dose relationship, were of isolated incidence and/or within the range of the HCD, they were considered to be unrelated to the test item.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

CARTILAGE
The distribution of significant differences in fetal and litter incidences of cartilage findings is presented in Table 9 of section "Any other information on results incl. tables".
When compared with controls, there were higher litter and/or fetal incidences of fetuses with metacarpal bone cartilage present from 50 mg/kg bw/day, median phalanx and/or pelvic girdle bone(s) cartilage present at 130 mg/kg bw/day. These findings were associated with a delayed ossification [i.e. unossification or incomplete ossification of 1st metacarpal, unossification or incomplete ossification of 5th forepaw median phalanx, and/or incomplete ossification of pubis] and were considered as test item treatment-related although they were within and/or very close to the range of the HCD, but they are not considered to be adverse. Other absence of cartilage [i.e. absence of cartilage of lumbar vertebra(e)] was of isolated occurrence and was within the range of the Historical Control Data. Therefore, a test item relationship was considered to be unlikely.

VARIATIONS
The distribution of significant differences in fetal and litter incidences of skeletal variations is presented in Table 10 of section "Any other information on results incl. tables".
At 130 mg/kg bw/day and when compared with controls, there were higher fetal and/or litter incidences of fetuses with unossification or incomplete ossification of 6th sternebra(e), unossification or incomplete ossification of 1st metacarpal, unossification or incomplete ossification of 5th forepaw median phalanx, and/or incomplete ossification of pubis. As these findings were associated with higher fetal and/or litter cartilage findings, they were considered as variations. Although incomplete ossification of 6th sternebra(e) and unossification of 1st metacarpal were with a dose-related occurrence, sometimes statistically significant as they were within the range of the HCD, they were therefore considered to be test item-related but non-adverse. At 50 mg/kg bw/day and when compared with controls, there were higher litter incidence of fetuses with incomplete ossification of 1st metacarpal. A relationship to the test item could not be ruled out for this dose-related variation which was also associated with higher litter cartilage findings, although their incidences were within the range of the Historical Control Data. The other variations [i.e. incomplete ossification of hyoid centrum, short supernumerary 13th rib, incomplete ossification of forepaw median phalanx and/or incomplete ossification of forepaw proximal phalanx and/or presence of 25 pre-sacral vertebra(e)] were of isolated occurrence, not dose-related, noted without a statistically significant occurrence and/or were within or close to the range of the Historical Control Data. Therefore, a test item-relationship was considered to be unlikely.

MALFORMATIONS
The distribution of significant differences in fetal and litter incidences of skeletal malformations is presented in Table 11 of section "Any other information on results incl. tables".
There was no test item-related increase in the frequency of skeletal malformations. Skeletal malformations that were observed at 15, 50 or 130 mg/kg bw/day were of isolated occurrence [absent lumbar vertebra(e)], not dose-related [nasal split, skullcap hole, branched rib, fused rib, fused sternebra(e)], observed in controls with a similar or low incidence [parietal split, absent thoracic hemivertebra(e), misaligned caudal vertebra(e)] and/or within the range of the HCD (all malformations). Therefore, a test item relationship was considered to be excluded.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

VARIATIONS
The distribution of the fetal and litter incidences of soft tissue variations is presented in Table 7 of section "Any other information on results incl. tables".
At 130 and 50 mg/kg bw/day and when compared with controls, there was a statistically significant higher fetal incidence of fetuses with absence of brachiocephalic trunk (p<0.001 and p<0.05, respectively). As this increase was dose-related and as incidences were slightly above the upper limit of the HCD at 130 mg/kg bw/day, this finding was attributed to the test item, but this variation is a minor abnormality as this change occurs within the normal population (i.e. 94.4% of litters and 36.0% of fetuses in HCD). This abnormality is a common branching variation (as described in Guidance of evaluation of reproductive toxicological data, Monograph No.31). It was therefore considered as non-adverse. Other soft tissue variations were observed without a dose relationship in the gall bladder, liver, kidneys, ovaries, innominate artery and thymus, with an isolated low incidence (i.e. fluid filled thoracic cavity) and/or with a litter incidence lower than controls (i.e. dilated renal pelvis). Therefore, they were considered to be incidental and unrelated to the test item treatment.

MALFORMATIONS
The distribution of the fetal and litter incidences of soft tissue malformations is presented in Table 8 of section "Any other information on results incl. tables".
There was no test item-related increase in the frequency of soft tissue malformations.
Soft tissue malformations observed at 130, 50 or 15 mg/kg bw/day were of isolated occurrence (i.e. small lungs and dilated pulmonary trunk), not dose-related [i.e. absent palatine, aglossia, absent upper and lower jaw, absence of cerebral ventricle(s), small cerebrum, misshapen cerebrum, small cerebral hemisphere and malpositioned cerebellum, hydropericardium, malpositioned kidney, narrowed pulmonary trunk], observed in controls with a similar or low incidence (i.e. ventricular septum defect, dilated aortic arch) and/or within the range of the HCD (in part). Therefore, a test item relationship was considered to be excluded.
At 130 mg/kg bw/day, two fetuses from two different litters showed malformations:
• one fetus with hydropericardium,
• one fetus with small lung and dilated pulmonary trunk.
At 50 mg/kg bw/day, one fetus from one litter showed polymalformations:
• one fetus with absent palatine, aglossia, absent upper and lower jaw, absence of cerebral ventricle(s), small cerebrum, misshapen cerebrum, small cerebral hemisphere and malpositioned cerebellum that corresponded with fetal external malformations.
At 15 mg/kg bw/day, three fetuses from three litters showed malformations:
• one fetus with ventricular septum defect, dilated aortic arch and narrowed pulmonary trunk,
• one fetus with hydropericardium,
• one fetus with malpositioned kidney.
At 0 mg/kg bw/day, one litter contained one malformed fetus with ventricular septum defect, dilated aortic arch and transposition of great vessels.

Other effects:

not examined

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

CHEMICAL ANALYSIS OF THE DOSE FORMULATION

The test item concentrations in the administered dose formulations analyzed on Days 6 and 28 p.c. remained within an acceptable range of variations (-4.5% to +5.2%) when compared with the nominal values (± 10% of the nominal concentrations).

No test item was detected in the control dose formulation.

 

BODY WEIGHTS AND BODY WEIGHT GAINS

Mean body weights and mean body weight changes recorded in control and test item-treated animals are summarized in Table 1.

Table 1: Mean Body Weights/Mean Body Weight Changes (g)

Dose level (mg/kg bw/day)	0	15	50	130
Body weight (g)
Day 6 p.c.	3625	3671	3659	3642
Day 9 p.c.	3689	3739	3718	3648
Day 12 p.c.	3740	3778	3755	3707
Day 15 p.c.	3802	3852	3795	3797
Day 19 p.c.	3838	3898	3814	3821
Day 24 p.c.	3880	3938	3861	3888
Day 29 p.c.	3988	4034	3981	3935
		(+1)	(0)	(-1)
Body weight change (g)
Days 6 - 9 p.c.	+65	+68	+59	+7*
Days 9 - 12 p.c.	+51	+39	+38	+59
Days 12 - 15 p.c.	+62	+74	+39	+90
Days 15 - 19 p.c.	+35	+46	+19	+24
Days 19 - 24 p.c.	+42	+40	+37	+67
Days 24 - 29 p.c.	+108	+96	+76	+47*
Days 6 - 29 p.c.	+363	+363	+318	+293
		(0)	(-12)	(-19)

Bold characters: test item-related; Statistically significant difference vs. controls: *: p<0.05; (): in brackets, percentage difference (%) vs. controls.

 

FOOD CONSUMPTION

Mean food consumption values recorded during the study are summarized in Table 2.

Table 2: Mean Food Consumption (g/animal/day)

Dose level (mg/kg bw/day)	0	15	50	130
Days 6 - 9 p.c.	159	172	161	128*
		(+8)	(+1)	(-19)
Days 9 - 12 p.c.	148	158	154	133
		(+7)	(+4)	(-10)
Days 12 - 15 p.c.	107	131	114	123
		(+22)	(+7)	(+15)
Days 15 - 19 p.c.	147	178	161	162
		(+21)	(+10)	(+10)
Days 19 - 24 p.c.	158	171	162	134
		(+8)	(+3)	(-15)
Days 24 - 29 p.c.	184	140	156	122
		(-24)	(-15)	(-34)

Bold characters: test item-related; Statistically significant difference vs. controls: *: p<0.05; (): in brackets, percentage difference (%) vs. controls.

 

HYSTERECTOMY DATA

Hysterectomy data are presented in Table 3.

Table 3: Hysterectomy Data on Day 29 p.c.

Dose level (mg/kg bw/day)	0	15	50	130	HCD CRL Evreux
Number of pregnant females at hysterectomy	23	21	22	23	103
Number of females with live fetuses at termination	23	21	22	23	100
Mean number of corpora lutea per animal	11.8	11.5	11.0	11.7	11.7-13.0
Mean number of implantation sites per animal	9.8	9.4	9.0	10.2	10.1-10.6
Mean percentage of pre-implantation loss (%)	16.7	17.8	19.3	11.6	9.4-21.7
Total number of live fetuses	219	189	182*	213**	949
Mean number of live fetuses per animal	9.5	9.0	8.3	9.3	8.9-9.9
Dead fetuses (%)	0.0	0.0	0.0	0.0	0.00-0.39
Total number of resorptions + scars	6	8	16*	22**	/
Mean number of resorptions + scars per female	0.3	0.4	0.7	1.0	/
Mean number of implantation scars per female	0.0	0.0	0.0	0.0	/
Total number of early resorptions	4	3	10	13*	/
Mean number of early resorptions per female	0.2	0.1	0.5	0.6	/
Total number of late resorptions	2	5	6	9	/
Mean number of late resorptions per female	0.1	0.2	0.3	0.4	/
Number of females with post-implantation loss	6	5	10	10	/
Mean percentage of post-implantation loss (%)	2.6	4.2	6.9	9.3	7.3-13.2

Statistically significant differences versus controls: *: p<0.05 and **: p<0.01. HCD Evreux: Historical Control Data in New Zealand White Rabbits (control data collected from five studies at Charles River Laboratories Evreux between 2019 and 2020), /: not recorded or calculated in HCD

 

FETAL BODY WEIGHT

Mean fetal body are presented in Table 4.

Table 4: Mean Fetal Body Weight (g)

Dose level (mg/kg bw/day)	0	15	50	130	HCD CRL Evreux
Mean fetal body weight	42.3	42.3 (0)	42.2 (0)	39.3 (-7)	31.5-60.9
Mean fetal body weight (males)	43.2	42.9 (-1)	42.9 (-1)	40.0 (-7)	28.7-60.9
Mean fetal body weight (females)	41.7	41.7 (0)	41.3 (-1)	38.5 (-8)	28.9-55.0

No statistically significant difference vs. controls; (): in brackets, percentage difference (%) vs. controls. HCD Evreux: Historical Control Data in New Zealand White Rabbits (control data collected from five studies at Charles River Laboratories Evreux between 2019 and 2020)

 

FETAL EXTERNAL VARIATIONS

The distribution of the fetal and litter incidences of external variations is presented in Table 5.

Table 5: Mean litter (L) and Fetal (F) Incidences of External Variations (%)

Dose level (mg/kg bw/day)	0	15	50	130	HCD CRL Evreux
Dams with live fetuses, n	23	21	22	23	100
Live fetuses, n	219	189	182	213	949
General
. abnormal color of amniotic fluid, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	4.3 (0.9)	5.0 (1.6)
. polyhydramnios, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	5.0 (0.5)
Trunk
. abdomen, blackish color, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.3 (0.5)	5.0 (0.5)
Litters affected, n (%)	0 (0)	0 (0)	1 (4.5)	2 (8.7)	3 (3.0)
Fetus affected, n (%)	0 (0)	0 (0)	1 (0.5)	3 (1.4)	5 (0.5)

n: number; No statistically significant differences vs. controls. HCD Evreux: Historical Control Data-maximum value of mean litter and fetal incidences in New Zealand White. Rabbits (control data collected from five studies at Charles River Laboratories Evreux between 2019 and 2020)

 

FETAL EXTERNAL MALFORMATIONS

The distribution of the fetal and litter incidences of external malformations are presented in Table 6.

Table 6: Mean litter (L) and Fetal (F) Incidences of Main Soft Tissue Malformations (%)

Dose level (mg/kg bw/day)	0	15	50	130	HCD CRL Evreux
Dams with live fetuses, n	23	21	22	23	100
Live fetuses, n	219	189	182	213	949
Nose, Nasal cavities
. proboscis, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	0.0 (0.0)
Eyes
. open eye, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	4.8 (0.5)
. cyclopia, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	0.0 (0.0)
Paw and digit
. ectrodactyly, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	0.0 (0.0)
Trunk
. ombilical hernia, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (1.1)	0.0 (0.0)	0.0 (0.0)
. omphalocele, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.3 (0.5)	5.6 (0.6)
Cranium
. meningocele, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	0.0 (0.0)
Litters affected, n (%)	0 (0.0)	0 (0.0)	2 (9.1)	1 (4.3)	7 (7.0)
Fetus affected, n (%)	0 (0.0)	0 (0.0)	3 (1.6)	1 (0.5)	8 (0.8)
Affected fetuses/Litter, Mean %	0.0	0.0	1.2	0.9

n: number; No statistically significant differences vs. controls. HCD Evreux: Historical Control Data-maximum value of mean litter and fetal incidences in New Zealand White. Rabbits (control data collected from five studies at Charles River Laboratories Evreux between 2019 and 2020)

 

FETAL SOFT TISSUE VARIATION

The distribution of the fetal and litter incidences of soft tissue variations is presented in Table 7.

Table 7: Mean Litter (L) and Fetal (F) Incidences of Soft Tissue Variations (%)

Dose level (mg/kg bw/day)	0	15	50	130	HCD CRL Evreux
Number of litters	23	21	22	23	100
Number of fetuses	219	189	182	213	949
Gall bladder
. enlarged, L (F)	8.7 (0.9)	4.8 (0.5)	13.6 (3.3)	4.3 (0.5)	20.0 (2.1)
. small, L (F)	8.7 (0.9)	0.0 (0.0)	0.0 (0.0)	8.7 (0.9)	18.2 (3.9)
. bilobed, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	4.8 (0.5)
Liver
. colored nodule, L (F)	0.0 (0.0)	0.0 (0.0)	9.1 (1.1)	0.0 (0.0)	9.5 (1.0)
Kidneys
. dilated renal pelvis, L (F)	21.7 (2.3)	19.0 (2.6)	13.6 (2.2)	13.0 (1.9)	22.2 (2.9)
Gonads
. hemorrhagic ovary, L (F)	0.0 (0.0)	4.8 (1.1)	4.5 (0.5)	4.3 (0.5)	16.7 (3.5)
Vessels
. absent innominate artery, L (F)	8.7 (1.8)	0.0 (0.0)	18.2 (2.2)	4.3 (0.5)	5.0 (1.6)
. absent brachiocephalic trunk, L (F)	87.0 (24.2)	81.0 (32.3)	77.3 (35.7*)	100.0 (39.9^#)	94.4 (36.0)
. short innominate artery, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	23.8 (3.8)
Thymus
. reddish color, L (F)	4.3 (0.9)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)
General
. fluid-filled thoracic cavity, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.3 (0.5)	4.8 (0.5)
Litters affected, n (%)	20 (87.0)	17 (81.0)	18 (81.8)	23 (100.0)	90 (90.0)
Fetus affected, n (%)	65 (29.7)	66 (34.9)	74* (40.7)	92** (43.2)	340 (35.8)
Affected fetuses/Litter, Mean %	31.3	32.7	44.1	44.1

n: number; Bold characters: test item-related. HCD Evreux: Historical Control Data-maximum value of mean litter and fetal incidences in New Zealand White. Rabbits (control data collected from five studies at Charles River Laboratories Evreux between 2019 and 2020); Statistical significance: *: p<0.05, **: p<0.01, ^#: p<0.001 for number of fetuses.

 

FETAL SOFT TISSUE MALFORMATION

The distribution of the fetal and litter incidences of soft tissue malformations is presented in Table 8.

Table 8: Mean Litter (L) and Fetal (F) Incidences of Soft Tissue Malformations (%)

Dose level (mg/kg bw/day)	0	15	50	130	HCD CRL Evreux
Number of litters	23	21	22	23	100
Number of fetuses	219	189	182	213	949
Mouth, Jaw, Palate
. absent palatine, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	0.0 (0.0)
. aglossia, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	0.0 (0.0)
. absent upper and lower jaw, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	0.0 (0.0)
Brain
. absence of cerebral ventricle(s), L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	0.0 (0.0)
. small cerebrum, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	4.8 (0.5)
. misshapen cerebrum, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	4.8 (0.5)
. small cerebral hemisphere, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	0.0 (0.0)
. malpositionned cerebellum, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	0.0 (0.0)
Heart
. ventricular septum defect, L (F)	4.3 (0.5)	4.8 (0.5)	0.0 (0.0)	0.0 (0.0)	5.3 (0.6)
. hydropericardium, L (F)	0.0 (0.0)	4.8 (0.5)	0.0 (0.0)	4.3 (0.5)	5.3 (0.6)
Lungs
. small lung, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.3 (0.5)	0.0 (0.0)
Kidneys
. malpositioned kidney, L (F)	0.0 (0.0)	4.8 (0.5)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)
Vessels
. dilated aortic arch, L (F)	4.3 (0.5)	4.8 (0.5)	0.0 (0.0)	0.0 (0.0)	5.6 (0.6)
. narrowed pulmonary trunk, L (F)	0.0 (0.0)	4.8 (0.5)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)
. dilated pulmonary trunk, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.3 (0.5)	4.8 (0.5)
. transposition of great vessels, L (F)	4.3 (0.5)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)
Litters affected, n (%)	1 (4.3)	3 (14.3)	1 (4.5)	2 (8.7)	13 (13.0)
Fetus affected, n (%)	1 (0.5)	3 (1.6)	1 (0.5)	2 (0.9)	14 (1.5)
Affected fetuses/Litter, Mean %	0.4	1.5	0.4	1.4

n: number; No statistically significant differences vs. controls. HCD Evreux: Historical Control Data-maximum value of mean litter and fetal incidences in New Zealand White Rabbits (control data collected from five studies at Charles River Laboratories Evreux between 2019 and 2020)

 

FETAL SKELETAL CARTILAGES

The distribution of significant differences in fetal and litter incidences of cartilage findings is presented in Table 9.

Table 9: Mean Litter (L) and Fetal (F) Incidences of Skeletal Cartilages

Dose level (mg/kg bw/day)	0	15	50	130	HCD CRL Evreux
Number of litters	23	21	22	23	100
Number of fetuses	219	189	182	213	949
Metacarpal bone
. cartilage of metacarpal bone: present, L (F)	30.4 (12.3)	38.1 (11.1)	40.9 (11.5)	47.8 (14.1)	52.4 (21.2)
Forepaw phalanx
. cartilage of median phalanx: present, L (F)	47.8 (20.5)	38.1 (16.9)	45.5 (11.0)	52.2 (23.9)	84.2 (40.0)
Pelvis
. cartilage of pelvic girdle: present, L (F)	8.7 (0.9)	0.0 (0.0)	4.5 (2.7)	21.7 (2.3)	21.1 (2.2)
Sternebra
. cartilage of sternebra(e): present L (F)	100.00 (83.1)	100.0 (83.1)	95.5 (79.7)	100.0 (82.6)	62.0 (48.9)
Litters affected, n (%)	23 (100.0)	21 (100.0)	21 (95.5)	23 (100.0)	62 (62.0)
Fetus affected, n (%)	195 (89.0)	166 (87.8)	152 (83.5)	192 (90.1)	512 (54.0)

n: number; Bold characters: test item-related. HCD Evreux: Historical Control Data-maximum value of mean litter and fetal incidences in New Zealand White. Rabbits (control data collected from five studies at Charles River Laboratories Evreux between 2019 and 2020); No statistical significance.

 

FETAL SKELETAL VARIATIONS

The distribution of significant differences in fetal and litter incidences of skeletal variations is presented in Table 10.

Table 10: Mean Litter (L) and Fetal (F) Incidences of Skeletal Variations (%)

Dose level (mg/kg bw/day)	0	15	50	130	HCD CRL Evreux
Number of litters	23	21	22	23	100
Number of fetuses	219	189	182	213	949
Sternebra
. unossified 6^th sternebra, L (F)	17.4 (4.1)	9.5 (1.1)	13.6 (3.3)	26.1 (3.8)	33.3 (6.7)
. incomplete ossification 6^th sternebra, L (F)	69.6 (21.0)	66.7 (16.9)	54.5 (24.7)	78.3 (36.2^#)	88.9 (27.0)
Metacarpal bone
. unossified 1^st metacarpal, L (F)	13.0 (1.8)	9.5 (1.1)	9.1 (1.1)	21.7 (7.0**)	38.1 (8.4)
. incomplete ossification of 1^st metacarpal, L (F)	30.4 (11.0)	33.3 (10.1)	40.9 (11.0)	43.5 (8.9)	42.9 (20.7)
Forepaw phalanx
. incomplete ossification 5^th median phalanx, L (F)	34.8 (11.0)	28.6 (4.8)	27.3 (5.5)	39.1 (10.8)	61.1 (18.2)
. unossified 5^th median phalanx, L (F)	34.8 (10.5)	33.3 (13.2)	31.8 (5.5)	52.2 (14.6)	73.7 (30.0)
Pelvis
. pubis: incomplete ossification, L (F)	8.7 (0.9)	0.0 (0.0)	4.5 (2.2)	21.7 (2.3)	22.2 (2.4)
Litters affected, n (%)	23 (100.0)	21  (100.0)	22 (100.0)	23 (100.0)	100 (100.0)
Fetus affected, n (%)	210 (95.9)	180 (95.2)	175 (96.2)	208 (97.7)	909 (95.8)

n: number; Bold characters: test item-related. HCD Evreux: Historical Control Data-maximum value of mean litter and fetal incidences in New Zealand White. Rabbits (control data collected from five studies at Charles River Laboratories Evreux between 2019 and 2020); Statistically significant vs. controls for the number of fetuses or litters: **: p<0.01 and ^#: p<0.001.

 

FETAL SKELETAL MALFORMATIONS

The distribution of the fetal and litter incidences of skeletal malformations is presented in Table 11.

Table 11: Mean Litter (L) and Fetal (F) Incidences of Skeletal Malformations

Dose level (mg/kg bw/day)	0	15	50	130	HCD CRL Evreux
Number of litters	23	21	22	23	100
Number of fetuses	219	189	182	213	949
Head-skull
. parietal, split L (F)	8.7 (0.9)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	0.0 (0.0)
. nasal: split, L (F)	0.0 (0.0)	4.8 (0.5)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)
. skullcap: hole, L (F)	0.0 (0.0)	4.8 (0.5)	4.5 (0.5)	0.0 (0.0)	5.6 (0.6)
Thoracic vertebrae
. fused, L (F)	4.3 (0.5)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)
. absent hemivertebra(e), L (F)	4.3 (0.5)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	0.0 (0.0)
Lumbar vertebra(e)
. absent, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.3 (0.5)	9.1 (1.0)
Caudal vertebra(e)
. misaligned, L (F)	4.3 (0.5)	4.8 (2.1)	4.5 (0.5)	0.0 (0.0)	25.0 (2.7)
Sternebra
. fused, L (F)	4.3 (0.5)	19.0 (2.1)	4.5 (0.5)	13.0 (1.9)	14.3 (1.4)
Rib
. branched, L (F)	0.0 (0.0)	4.8 (0.5)	0.0 (0.0)	4.3 (0.5)	0.0 (0.0)
. fused, L (F)	0.0 (0.0)	0.0 (0.0)	4.5 (0.5)	0.0 (0.0)	4.5 (0.5)
Litters affected, n (%)	5 (21.7)	6 (28.6)	5 (22.7)	4 (17.4)	21 (21.0)
Fetus affected, n (%)	5 (2.3)	10 (5.3)	5 (2.7)	5 (2.3)	26 (2.7)
Affected fetuses/Litter, Mean %	2.3	3.9	2.4	2.6

n: number; No statistically significant differences vs. controls. HCD Evreux: Historical Control Data-maximum value of mean litter and fetal incidences in New Zealand White Rabbits (control data collected from five studies at Charles River Laboratories Evreux between 2019 and 2020)

 

SUMMARY TABLE

Table 12: Summary table of relevant endpoints

Dose level (mg/kg bw/day)	0	15	50	130
Pregnant/total dams	23/24	21/24	22/24	23/24
-        Total number of early resorptions -        Mean number of early resorptions per female	4   0.2	3   0.1	10   0.5	13*   0.6
-        Total number of late resorptions -        Mean number of late resorptions per female	2   0.1	5   0.2	6   0.3	9   0.4
-        Dams with abortion, early deliveries, stillbirths and/or resorptions only -        Dams with dead fetuses	0 0	0 0	2 0	0 0
Mean percentage of pre-implantation loss (%)	16.7	17.8	19.3	11.6
-        Number of females with post-implantation loss -        Mean percentage of post-implantation loss (%)	6   2.6	5   4.2	10   6.9	10   9.3
Body weight on day 29 (g)	3988	4034	3981	3935
Body weight gain day 6-29 (g) In brackets, percentage difference (%) vs. controls	+363	+363 (0)	+318 (-12*)	+293 (-19*)
Gravid uterine weight (g) In brackets, percentage difference (%) vs. controls	586	543 (-7)	500 (-15)	535 (-9)
Mean number of live fetuses per animal	9.5	9.0	8.3	9.3
Mean fetal body weight males (g) In brackets, percentage difference (%) vs. controls	43.2	42.9 (-1)	42.9 (-1)	40.0 (-7)
Mean fetal body weight females In brackets, percentage difference (%) vs. controls	41.7	41.7 (0)	41.3 (-1)	38.5 (-8)
Mean fetal body weight (sexes combined) In brackets, percentage difference (%) vs. controls	42.3	42.3 (0)	42.2 (0)	39.3 (-7)
Variations, fetus affected, n (%) -        External -        Soft tissue -        Skeletal	0 (0) 65 (29.7) 210 (95.9)	0 (0) 66 (34.9) 180 (95.2)	1 (0.5) 74* (40.7) 175 (96.2)	3 (1.4) 92** (43.2) 208 (97.7)
Malformations, fetus affected, n (%) -        External -        Soft tissue -        Skeletal	0 (0.0) 1 (0.5) 5 (2.3)	0 (0.0) 3 (1.6) 10 (5.3)	3 (1.6) 1 (0.5) 5 (2.7)	1 (0.5) 2 (0.9) 5 (2.3)
Cartilages, fetus affected, n (%)	195 (89.0)	166 (87.8)	152 (83.5)	192 (90.1)

Bold characters: Statistically significant difference vs. controls: *: p<0.05, **: p<0.01

Applicant's summary and conclusion

Conclusions:

The No Observed Effect Level (NOEL) for maternal parameters was considered to be
15 mg/kg bw/day, based on the absence of any test item-related findings at this dose level and the No Observed Adverse Effect Level (NOAEL) was considered to be 130 mg/kg bw/day, based on the transient and statistically significant but non adverse lower mean body weight gain and food consumption at this dose level,

The No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 130 mg/kg bw/day, based on the absence of adverse effects at this dose level.

Executive summary:

A prenatal developmental toxicity study via oral gavage was performed in rabbits with test substance diethylaminopropylamine. The study was carried out according to GLP and to OECD test guideline No. 414 (25 June 2018). Three groups of 24 time-mated female New-Zealand White rabbits received the test item Diethylaminopropylamine (as pH-neutralized dose formulations, once daily by the oral route (gavage) at dose levels of 15, 50 or 130 mg/kg bw/day from Day 6 to Day 28 p.c. inclusive. A control group of 24 time-mated females received the vehicle (drinking water treated by reverse osmosis) under the same experimental conditions. A constant dosage volume of 2 mL/kg bw/day was used.

Actual concentrations of the test item in the dose formulations analyzed during the study were within an acceptable range of variations (-4.5% to +5.2%) when compared with the nominal concentrations.

The relevant endpoints are summarised in Table 12 of section "Any other information on results incl. tables". At hysterectomy on Day 29 p.c., 23/24, 21/24, 22/24 and 23/24 females were pregnant with live fetuses in the groups treated at 0, 15, 50 and 130 mg/kg bw/day, respectively. No test item-related deaths or clinical signs were recorded. Lower body weight gain was recorded between Days 6 and 29 p.c. at 50 and 130 mg/kg bw/day (-12% and -19% vs. controls, respectively; not statistically significant). These differences, which were especially due to continuous (at 50 mg/kg bw/day) or transient (at 130 mg/kg bw/day with p<0.05 on 2 interval days) lower body weight gain, did not impact the body weight on Day 29 p.c. and were considered to be non-adverse. This finding was associated with transient, non-adverse, slightly lower food consumption between Days 6 and 9 p.c. (-19%; p<0.05) at 130 mg/kg bw/day when compared with controls. No effects were observed at 15 mg/kg bw/day on body weight and food consumption. No effects were noted on gravid uterus weight, carcass weight or net body weight changes at any dose level. There were no test item-related macroscopic or microscopic findings at any dose level.

A slight increased mean post-implantation loss was noted from 50 mg/kg bw/day (up to 9.3% at 130 mg/kg bw/day vs. 2.6% in controls). As these variations were not statistically significant and were within the range of the HCD (min: 7.3 - max: 13.2%), they were not considered to be adverse. No other effects on the hysterectomy parameters were noted at any dose level. A minimally, not statistically significant and non-adverse lower mean fetal body weight was recorded at 130 mg/kg bw/day (-7% vs. controls) that correlated with minor findings of delayed ossification. No effects were noted on sex ratio at any dose level. No test item-related variations or malformations were observed at external examination at any dose level. Although a minor visceral abnormality (i.e. absence of brachiocephalic trunk) was noted from 50 mg/kg bw/day and minor findings of delayed ossification were noted in fetuses with higher litter and/or fetal incidences from 50 mg/kg bw/day (i.e. incomplete ossification of the 1^st metacarpal) and at 130 mg/kg bw/day [i.e. unossification or incomplete ossification of the 6^th sternebra(e), the 1^st metacarpal and the 5^th forepaw median phalanx, and/or incomplete ossification of pubis], no test item-related soft tissue or skeletal malformations were observed at any dose level.

Based on the results obtained in this study:

• the No Observed Effect Level (NOEL) for maternal parameters was considered to be
  15 mg/kg bw/day, based on the absence of any test item-related findings at this dose level and the No Observed Adverse Effect Level (NOAEL) was considered to be 130 mg/kg bw/day, based on the transient and statistically significant but non adverse lower mean body weight gain and food consumption at this dose level,


• the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 130 mg/kg bw/day, based on the absence of adverse effects at this dose level.