3-aminopropyldiethylamine

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

1-There is an OECD 421 guideline study performed accroding to GLP (Charles River, 2022)

The objective of this study was to evaluate the potential toxic effects of the test item (as pH-neutralized dose formulations) following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 13 post-partum (p.p.). This OECD TG 421 compliant study, conducted following GLP principles, provided initial information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Three groups of 10 male and 10 female Sprague-Dawley rats received the test item daily by oral route (gavage) at dose levels of 50, 150 and 500 mg/kg bw/day. Males were treated for 2 weeks before mating, throughout mating and then until the day before euthanasia (i.e. after a minimum treatment period of 4 consecutive weeks). Females were treated for 2 weeks before pairing, throughout mating and gestation periods, until Day 13 p.p. inclusive.

Another group of 10 males and 10 females received the vehicle only (drinking water treated by reverse osmosis) under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg bw/day was used.

 

The test item concentrations in the dose formulations analyzed during the study were satisfactory (within ± 10%) except for group 4 females (-13%, end of the study).

One lactating female given 500 mg/kg bw/day was euthanized prematurely on Day 4 p.p. after ante mortem signs of poor health (emaciated appearance, round back, ventral decubitus, hypoactivity, half closed eyes). male had a high number of dead pups during the first days of lactation. Macroscopic and microscopic findings observed for this female were considered to have contributed to moribundity and most probably related to the test item administration although not adverse and not associated with degeneration (stomach with brown deposits correlating with focal erosion and vacuolation of the mucosa and muscularis; kidneys with tan discoloration correlating with microscopic tubular vacuolation).

There were no adverse clinical signs, a transient and statistically significant effect on body weight gain at 500 mg/kg bw/day of male animals before mating, near -30% vs. control, p<0.01 but not statistically significant in females was recorded but no test item-related on mean food consumption. No treatment related effect was noted during pregnancy on body weight and food consumption.

There were no test item-related effects on the estrous cycle during the pre-mating period. There were no effects on mating index and all groups had comparable pre-coital time intervals (number of days taken to mate). No effects on fertility index, litter data or on mean duration of gestation were detected. There were no effects on reproductive and litter data.

At all dose levels, there were no test item treatment effects on the anogenital distance. There were no areolae or nipples in male pups on Day 12 p.p. There were no effects on the incidences of pups found dead or cannibalized and no test item-related effects on pup viability at all dose levels.

Mean TSH or T4 plasma concentration levels measured in test-item treated parent and pups were comparable to that of the controls.

No test item-related microscopic findings were observed in the testes, epididymides, ovaries (with oviducts), or thyroids (with parathyroids).

Test item-related, non-adverse microscopic observations were noted in isolated females at 150 and 500 mg/kg bw/day in the kidneys and thymus which were examined because of macroscopic abnormalities. They consisted of minimal tubular vacuolation in the kidneys from one female at 150 mg/kg bw/day and minimal or slight lymphocyte decreased cellularity and/or apoptosis/necrosis in the thymus from one female per group at 150 and 500 mg/kg bw/day. Changes in the thymus had to be interpreted with caution in the absence of microscopic examination of other animals in the study; they were most likely secondary to stress, although a direct role of the test item could not be excluded.

Non adverse microscopic observations were noted in isolated females at 150 and 500 mg/kg bw/day in the kidneys (tubular vacuolation) and thymus (lymphocyte decreased cellularity and/or apoptosis/necrosis) which were examined because of macroscopic abnormalities.

Under the experimental conditions of the study, the dose of 150 mg/kg bw/day was considered to be the No Observed Adverse Effect Level (NOAEL) for parental toxicity (the dose level of 500 mg/kg bw/day exceeded the MTD (Maximal Tolerated Dose) since 1/8 females was euthanized during lactation with test item-related clinical signs and non adverse histopathological findings), and the dose of 500 mg/kg bw/day was considered to be the NOEL for reproductive performance (mating, fertility and delivery).

 

2-There is an extended one generation study performed according to OECD 443 (Charles River, 2022):

The test item, Diethylaminopropylamine (as pH-neutralized dose formulations; batch No. I133306836), was administered daily by oral gavage at dose levels of 0, 40, 150, or 450 mg/kg/day to sexually-mature male and female rats (parental (P) generation) starting 2 weeks before mating and continuously through mating, gestation and weaning of their pups (F1). At weaning, the F1 generation was also exposed to the same dose levels of the test item and was assigned to Cohorts of animals for reproductive/developmental toxicity, developmental neurotoxicity or developmental immunotoxicity testing.

The high dose level was selected based on a prematurely euthanized female at 500 mg/kg/day during lactation (severe clinical signs and test item treatment related macroscopic and microscopic findings) in a previous OECD 421 study and because the OECD 443 study duration is longer than the OECD 421 study.

1. a) Systemic toxicity evaluation:

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity (excluding

reproductive and developmental toxicity endpoints) was considered to be 450 mg/kg/day based on the absence of adverse findings in both sexes.

The No Observed Effect Level (NOEL) for systemic toxicity (excluding reproductive and developmental toxicity endpoints) was considered to be lower than 40 mg/kg/day based on test item related effects at histopathology in the spleen from 40 mg/kg/day in P males.

1. b) Reproductive/developmental toxicity testing:

• in P generation animals, there were no adverse effects on estrus cycles, mating (including precoital time), fertility, delivery, or on F1 pups during lactation (survival, clinical signs, development).


• in Cohort 1A and/or 1B animals, there were no adverse effects on sexual maturation, estrus cycles, mating (including precoital time), fertility, delivery, or on F2 pups up to PND 4 (survival, clinical signs, development).

Therefore the NOAEL for reproductive/developmental toxicity was considered to be
450 mg/kg/day, and the NOEL was considered to be 40 mg/kg/day because of the trend towards a delayed first estrous after vaginal opening and the delay in vaginal opening at 450 mg/kg/day in F1 females, and of the dose-related delay in balanopreputial separation from 150 mg/kg/day in F1 males potentially related to the test item.

1. c) Developmental neurotoxicity testing:

• in Cohort 2A and 2B: there was no evidence of adverse developmental neurotoxicity at any dose level. Therefore the NOAEL for developmental neurotoxicity was considered to be 450 mg/kg/day.

1. d) Developmental immunotoxicity testing:

• in Cohort 1A: there were no test item treatment-related findings on lymphocyte subtyping and no test item-related effects on mean lymphoid organ weights or on histopathology of lymphoid organs,


• in Cohort 3: there was no evidence of adverse immunotoxicity potential at any dose level.

Therefore the NOAEL for developmental immunotoxicity was considered to be 450 mg/kg/day.

 

 

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 September 2020 to 16 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

Principles of method if other than guideline:

and with reference to: ECHA decision No. CCH-D-2114457570-49-01/F.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
Justification of the study design was provided in the ECHA decision No. CCH-D-2114457570-49-01/F.
- Premating exposure duration for parental (P0) animals: 2 weeks before mating. In the ECHA
decision, ECHA noted that ten week premating exposure duration is required in case there is no substance specific information in the dossier supporting shorter premating exposure duration as advised in the ECHA Guidance on information requirements and chemical safety assessment, Chapter R.7a. In this specific case, animals of Cohort 1B are mated to produce the F2 generation. Since the premating exposure duration will be 10 weeks for these Cohort 1B animals, the fertility parameters will be covered, allowing an evaluation of the full spectrum of effects on fertility in these animals. However, the premating period shall not be shorter than two weeks and must be sufficiently long to reach a steady-state in reproductive organs as advised in the ECHA Guidance.
- Basis for dose level selection: based on two previously performed studies, which are 1) a 2-week preliminary study,study, a 90-day toxicity study, a developmental study, and an OECD 421 study, all in Sprague-Dawley rats (for details see section "Details on study design").
- Inclusion/exclusion of extension of Cohort 1B: in the ECHA decision, ECHA concluded that Cohort 1B must be extended to include mating of the animals and production of the F2 generation, because the uses of the registered substance is leading to significant exposure of professionals and there are indications of modes of action related to endocrine disruption from an available study (90-day repeated dose toxicity study, with doses at 50, 250, and 750 mg/kg/day) resulting in an increase of the oestrous cycle length.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: In the ECHA decision, ECHA concluded that the developmental neurotoxicity cohorts 2A and 28 need to be conducted because there is a particular concern on (developmental) neurotoxicity based on the results from the above-identified rn vivo studies on the registered substance itself and on one analogous substance (3-dimethylaminopropiononitrile).
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: In the ECHA decision, ECHA concluded that the developmental immunotoxicity Cohort 3 needs to be conducted because there is a particular concern on (developmental) immunotoxicity based on the results from the above-identified in vivo study on the registered substance itself.
- Route of administration: the oral route was selected as it is the appropriate route of administration for substances except gases to focus on the detection of hazardous properties on reproduction as indicated in ECHA Guidance on information requirements and chemical safety assessment (version 6.0, July 2017) Chapter R.7a. Since the substance to be tested is a liquid, ECHA concluded that testing should be performed by the oral route.
- Species: In the ECHA decision, ECHA considers that the rat is the preferred species, and that therefore testing should be performed in rats. The Sprague-Dawley strain was selected since background data from previous studies are available at Charles River Laboratories Evreux.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Sponsor
- No correction factor was used
- The pH of the dose formulations (groups 2 to 4) was adjusted to 8.0 (± 0.5) using a solution of hydrochloric acid

Species:

rat

Strain:

Sprague-Dawley

Remarks:

(RjHan: SD)

Details on species / strain selection:

The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study. The Sprague-Dawley strain was selected as background data from previous studies are available at Charles River Laboratories Evreux.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) Males 11 wks; (P) Females 10 wks
- Weight at study initiation: (P) Males: 470 g (range: 422 g to 505 g) ; Females: 268 g (range: 246 g to 287 g)
- Fasting period before study: not indicated
- Housing: P and 1B: individually in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust; 1A, 2A, 3 animals: group housed in polycarbonate cages with stainless steel lids (in 2000P cages: up to 5/sex per cage) containing autoclaved sawdust. Each cage contained two objects (rat hut, nylabone and/or cocoon) for environmental enrichment. Towards end of gestation and during lactation, autoclaved wood shavings were provided to females and their litter as nesting material.
- Diet (e.g. ad libitum): SSNIFF rat/mouse pelleted maintenance diet, batch Nos. 57266070 and 81971534 (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly.
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water filtered with a 0.22 um filter.
- Contamination analyses: No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which may be expected to interfere with or prejudice the outcome of the study
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS (target)
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%,
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 17 Sept 2020 To: 16 March 2021

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

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

Details on exposure:

FORMULATION PROCEDURE:
- 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) with concentrated HCl (34%).
- Frequency of preparation: Test item dose formulations: based on test item dose formulation stability (Study No. 42758 AHS) 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
P and F1 generation:
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 5 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 then throughout the administration.

Details on mating procedure:

P generation:
For scheduling purposes (to avoid excessive numbers of animals at necropsy), the mating period started over 4 different days (1st day: first six surviving males/females, 2nd day: following up to six surviving males/females and unmated pairs, 3rd day: following up to six surviving males/females and unmated pairs, 4th day: last following surviving males/females and unmated pairs).

P generation and F1 generation Cohort 1B:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day GD 0
- After 14 days of unsuccessful pairing the animals were separated without further opportunity for mating
- After successful mating each pregnant female was caged: individually
- Parturition: Females were allowed to litter normally and rear their progeny until weaning (P generation), or until Day 4 p.p. (F1 generation). Any sign of a difficult or prolonged parturition (dystocia) was recorded. The morning when parturition is completed was designated Day 1 p.p. The length of gestation was calculated. Any abnormalities in nesting behavior or nursing performance were recorded.

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 prior to dose formulation analysis. Checked parameters, acceptance criteria and obtained results are described in the validation report.
- Determination of test item concentrations in dose formulations: 8 dosages: once in each P-premating, P-mating, P gestation, P lactation, F1-postweaning, F1-premating, F1-gestation and F1 lactation periods. A sample was taken from control and test item dose formulations and analyzed using the validated method.
- Acceptance criterion: Measured concentration = nominal concentration ± 15%.

Duration of treatment / exposure:

P ANIMALS
- P Males (at least 10 weeks of treatment) were treated daily for 2 weeks before mating, during the mating period (up to 2 weeks), and until euthanasia (after weaning of the pups).
- P Females (at least 8 to 10 weeks of treatment) were treated daily for 2 weeks before mating, during the mating period (up to 2 weeks), during gestation, during lactation until weaning of the pups (PND 21), or until euthanasia for females with no evidence of mating or no delivery (25-27 days after the last day of the mating period.

F1 GENERATION
- Cohort 1A were treated daily from weaning (PND 22) until euthanasia (on PND 91 to 95),
- Cohort 1B were treated daily. Males were treated from weaning (PND 22) for at least 10 weeks before mating, during the mating period (up to 2 weeks) and until after euthanasia of F2 pups (on PND 4). Females were treated from weaning (PND 22) for at least 10 weeks before mating, during the mating period (up to 2 weeks), during gestation and during lactation until PND 4 inclusive, or until euthanasia for females with no delivery,
- Cohort 2A were treated daily from weaning (PND 22) until euthanasia (on PND 76 to 78),
- Cohort 3 were treated daily from weaning (PND 22) until 4 days after KLH injection, when these animals were euthanized (between PND 60 and 64).

Frequency of treatment:

Once daily, 7 days a week

Dose / conc.:

40 mg/kg bw/day

Remarks:

group 2 low dose

Dose / conc.:

150 mg/kg bw/day

Remarks:

group 3 mid dose

Dose / conc.:

450 mg/kg bw/day

Remarks:

group 4 high dose

No. of animals per sex per dose:

P: 24/sex/dose
Cohort 1A: 20/sex/dose
Cohort 1B: 20/sex/dose
Cohort 2A: 10/sex/dose
Cohort 2B: 10/sex/dose
Cohort 3: 10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: on the basis of the results of a) a 2-week preliminary study, b) a 90-day toxicity study, c) a developmental study, and d) an OECD 421 study, all in Sprague-Dawley rats.
The high dose level was selected based on a prematurely euthanized female at 500 mg/kg bw/day in the previous OECD 421 study and because the OECD 443 study duration is longer than the OECD 421 study. The low-dose and mid-dose were selected using a ratio representing approximately a 3-fold interval (i.e. 40 and 150 mg/kg/day).
Indeed, 1/8 females euthanized during lactation given 500 mg/kg/day was euthanized prematurely on Day 4 p.p. after ante mortem signs of poor health (emaciated appearance, round back, ventral decubitus, hypoactivity, half closed eyes). Macroscopic and microscopic findings observed for this female were considered to have contributed to moribundity and most probably related to the test item administration although not adverse and not associated with degeneration (stomach with brown deposits correlating with focal erosion and vacuolation of the mucosa and muscularis; kidneys with tan discoloration correlating with microscopic tubular vacuolation).with clinical signs and non adverse histopathological findings), and the dose of 500 mg/kg/day was considered to be the NOEL for reproductive performance (mating, fertility and delivery).
Therefore :
• based on the high number of females (4 with poor clinical signs with one of them prematurely sacrificed) at 750 mg/kg/day on the 90 day toxicity study,
• based on this prematurely sacrified female given 500 mg/kg/day in the OECD 421 and the recorded histopathological findings described above:
• it was decided to slightly lower the top dose-level to be used in the OECD 443 to avoid any high incidence of mortality because of the longer exposure duration: , 450 mg/kg/day was therefore selected as the high-dose level based on the prematurely euthanized female at 500 mg/kg/day recorded in the OECD 421 study and because the study duration is longer than the latter one. The low-dose and mid-dose were selected using a ratio representing approximately a 3-fold interval (i.e. 40 and 150 mg/kg/day).

Rationale for dose-levels selection was based on the following below studies:
a) in the 2 week-oral repeated preliminary toxicity study (42759 TSR (Papineau (2015)), male and female Sprague-Dawley rats received Diethylaminopropylamine (as pH-neutralized dose formulations) daily, by gavage, at dose levels of 100, 300 or 1000 mg/kg/day for 2 weeks. At 1000 mg/kg/day, in life observations were limited to ptyalism in both sexes, and lower body weight gain at treatment initiation resulted in a lower final body weight in males. Histopathology findings consisted of minimal to slight hyperplasia of squamous cells, associated with minimal to slight hyperkeratosis within the forestomach in all males and females given 1000 mg/kg/day,
b) in the 90 day-repeated oral toxicity study (42760 TCR (Papineau (2015)), male and female Sprague-Dawley rats were treated daily by the oral route gavage with the test item, Diethylaminopropylamine, at 50, 250 or 750 mg/kg/day for 13 weeks, followed by a 6-week treatment-free period. Clinical signs of poor condition were observed at the dose level of 750 mg/kg/day in four females, which induced the premature euthanasia of one of them (Week 11) more severely affected. No other adverse effects were observed in the study. At the end of the treatment period, microscopic vacuoles were seen in the kidneys (correlated with tan discoloration), brain (in choroid plexus), pars nervosa (pituitary gland), spleen, mesenteric lymph node and/or GALT in males and females treated at 750 mg/kg/day and at a lesser extent, at 250 mg/kg/day in isolated females. There was also a non-adverse orthokeratotic hyperkeratosis in the forestomach from males and females treated at 750 mg/kg/day and increased severity and incidence of lymphoid atrophy in the thymus from males and surviving females treated at 750 mg/kg/day which correlated with small thymus and lower weights and may be related in part with stress. Consequently, the NOAEL was established at 750 mg/kg/day in males and 250 mg/kg/day in females,
c) in the rat development oral study (42762 RSR (Papineau (2016)), pregnant female Sprague-Dawley rats were treated daily by the oral route gavage with the test item, Diethylaminopropylamine, at 50, 250 or 750 mg/kg/day from Days 6 to 20 post coitum. Maternal toxicity was recorded at =250 mg/kg/day (one found dead animal on GD 20 at 750 mg/kg/day, clinical signs at both doses but adverse at 750 mg/kg/day, body weight and food consumption were affected with higher severity at 750 mg/kg/day) and NOAEL for maternal toxicity was set at 50 mg/kg/day. The NOAEL for embryo-fetal toxicity and teratogenicity was considered to be 50 and 250 mg/kg/day, respectively, in a context of marked (250 mg/kg/day) to severe (750 mg/kg/day) maternal toxicity. It is to be noted that incomplete ossifications of cervical vertebrae were observed at 750 mg/kg/day,
d) in the OECD 421 (47470 RSR (Chevalier (2019)), Diethylaminopropylamine was administered daily by oral gavage to male and female Sprague-Dawley rats for 15 days before mating, during mating, and (for females) throughout gestation and until PND 13 at dose levels of 0, 50, 150 or 500 mg/kg/day. Under the experimental conditions of the study, the dose of 150 mg/kg/day was considered to be the NOAEL for parental toxicity (1/8 females euthanized during lactation with clinical signs and non adverse histopathological findings), and the dose of 500 mg/kg/day was considered to be the NOEL for reproductive performance (mating, fertility and delivery).

CONSTITUTION OF THE F1 GENERATION
On PND 22, pups from all available litters (up to 20 litters per group) were assigned to three cohorts of animals.
- Cohort 1A = Reproductive/developmental toxicity testing;
- Cohort 1B = Reproductive/developmental toxicity testing with extension to produce an F2 generation;
- Cohort 2A = Developmental neurotoxicity testing
- Cohort 2B = Developmental neurotoxicity testing on PND 22 without any dosing or in vivo tests;
- Cohort 3 = Developmental immunotoxicity testing.
Pups were selected randomly (manual randomization), with the exception that obvious runts (animals with a body weight more than two standard deviations below the mean pup weight of the respective litter) were not included, as they were unlikely to be representative of the treatment group.
The pups selected to constitute the F1 cohorts (with exception of pups allocated to Cohort 2B euthanized on PND 22) were identified by an implanted microchip at weaning. PND 22 was designated Day 1 of the F1 generation.

F1 GENERATION COHORTS
- Cohort 1A animals were selected (1/sex/litter/group if possible) for assessment of general toxicity and effects on their reproductive system. The estrous cycle stage was determined for all females in Cohort 1A from a fresh vaginal lavage as follows:
• after the onset of vaginal patency, until the first cornified smear was recorded (estrous), in order to determine the time interval between these two events,
• at least a period of 2 weeks towards the end of the treatment period (same calendar dates for all females).
- Cohort 1B animals were selected (1/sex/litter/group if possible) for follow-up assessment of reproductive performance (by mating F1 animals) and to potentially obtain additional histopathology data. Males and females of the same dose group were cohabited (avoiding the pairing of siblings) for up to 2 weeks or until mating, beginning on PND 90-94;
- Cohort 2A animals were selected (1/litter/group) for neurobehavioral testing on PND 64-65 followed by neurohistopathology assessment when they were adults,
- Cohort 2B animals were selected (1/litter/group) for neurohistopathology assessment on PND 22 without any dosing or in vivo tests;
- Cohort 3 animals were selected for immunotoxicity testing.

F2 GENERATION
The F2 litters were terminated on Day 4 p.p.

Parental animals: Observations and examinations:

MORBIDITY AND MORTALITY (P and F1 generation): Yes
- Time schedule: Each animal, once a day during the acclimation period (P animals only) and at least twice a day during the treatment period, including weekends and public holidays.

CAGE SIDE OBSERVATIONS (P and F1 generation): Yes
- Time schedule: once a day during the before treatment period (P animals only), and from the start of the treatment period, each animal twice a day (before and after treatment).

DETAILED CLINICAL OBSERVATIONS (P and F1 generation): Yes
- Time schedule: once a week.
- Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also evaluated.

BODY WEIGHT (P, Cohorts 1A, 1B, 2A and 3): Yes
- Time schedule: Males: once during pretest (P animals only), on the first day of treatment (Study Day 1), then at least once a week until euthanasia. Females: once during pretest (P animals only, on the first day of treatment (Study Day 1), then once a week (P, Cohorts 1A, 1B, 2A and 3) until mated (P, Cohort 1B), on GD (Gestational Day) 0, 4, 7, 10, 14, 17 and 20 and, on PND 1, 4, (P, Cohort 1B) and then on PND 7, 14 and 21 (P females).

FOOD CONSUMPTION (P, Cohorts 1A, 1B, 2A and 3): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- For the F1 generation, food consumption determination started after weaning from the first day of treatment onwards.
- During the mating period (P and 1B animals), food consumption was not measured for males or females.

WATER CONSUMPTION : No

HEMATOLOGY AND COAGULATION (P and Cohort 1A): Yes
Blood samples were collected from the orbital sinus of 10 animals/sex/dose on the day of necropsy under light isoflurane anaesthesia.
- Animals fasted: Yes, overnight period of at least 14 hours
- Parameters examined:
Erythrocytes (RBC), Mean cell volume (MCV), Packed cell volume (hematocrit) (PCV), Hemoglobin (HB), Mean cell hemoglobin concentration (MCHC), Mean cell hemoglobin (MCH), Thrombocyte (platelet) count (PLT), Total leucocyte count (WBC), Differential white cell count with cell morphology (neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L), large unstained cells (LUC), monocytes (M)), Reticulocytes (RTC), Prothrombin time (PT), Fibrinogen (FIB), Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY (P generation and Cohort 1A): Yes
Blood samples were collected from from the orbital sinus of 10 animals/sex/dose on the day of necropsy under light isoflurane anaesthesia.
- Animals fasted: Yes, overnight period of at least 14 hours
- Parameters examined:
Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT), Total bilirubin (TOT.BIL), Total cholesterol (CHOL), Triglycerides (TRIG), Alkaline phosphatase (ALP), Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Total proteins (PROT), Albumin (ALB), Albumin/globulin ratio (A/G), Bile acids (BIL.AC).

URINALYSIS (P generation and Cohort 1A): Yes
For urine collection, the animals were individually placed in metabolism cage an overnight period for at least 14 hours. The urine was collected onto thymol crystals.
- parameters examined:
Volume (Volume), pH (pH), Specific gravity (SP.GRAV), Proteins (PROT), Glucose (GLUC), Ketones (CETO), Bilirubin (BILI), Nitrites (NITR), Blood (hemoglobin) (BLOOD), Appearance (APP), Color (COLOR).

THYROID HORMONE ANALYSIS (P, Cohort 1A): Yes
Blood samples were taken (between 07:30 and 10:00 a.m.) into tubes containing K3-EDTA as anticoagulant.
- at termination from the first ten surviving males/group and the first ten surviving females/group (P generation and Cohort 1A animals) (approximately 0.5 mL of blood was collected from the orbital sinus under isoflurane anesthesia), for measurements of thyroid hormone (T4) and thyroid stimulating hormone (TSH) levels

Oestrous cyclicity (parental animals):

For P and 1B females, the estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning, during the 2 weeks of the premating period, and during the mating period, until the females were mated or the mating period had ended.

Vaginal smears were examined daily for all females in Cohort 1A, after the onset of vaginal patency, until the first cornified smear was recorded (estrous), in order to determine the time interval between these two events. Estrous cycles for all females in Cohort 1A were also monitored for a period of 2 weeks (from PND 79, same calendar dates for all females) before the end of the treatment period.

Sperm parameters (parental animals):

Parameters examined in surviving P and Cohort 1A males from the control and high-dose groups (groups 1 and 4): epididymis weight, epididymal sperm motility, epididymal sperm morphology, and epididymal sperm count.
In addition, the following was also examined for group 2 and 3: Sperm motility (P and Cohort 1A males), sperm count (Cohort 1A males).
The left testis of all P and Cohort 1A males (groups 1 to 4) was sampled and stored at -20°C but no sperm count evaluation was performed.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes (F1 pups), no (F2 pups: terminated on PND 4)
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible)

PARAMETERS EXAMINED
The following parameters were examined in F1 litters:
number and sex of pups, stillbirths, live births, postnatal mortality, clinical signs, abnormal behavior and external abnormalities, pup weight (on PND 1, 4, 7, 14, 21), anogenital distance (AGD) on PND 1, presence of nipples/areolae in male pups on PND 12.
The following parameters were examined in F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, clinical signs, abnormal behavior and external abnormalities, pup weight (on PND 1 and 4)

SEXUAL DEVELOPMENT (F1)
- All males were observed each day from PND 35 or 36. (i.e. Day 14 or 15 of F1 generation, until cleavage of the balanopreputial groove (preputial separation) was observed (when not observed before: until euthanasia, for Cohorts 1A, 2A and 3 animals, or until mating for Cohort 1B animals).
- All females were observed each day from PND 25 (i.e. Day 4 of F1 generation), until vaginal opening was observed [when not observed before: until euthanasia, for Cohorts 1A (or until monitoring of estrous cycles towards the end of the treatment period for Cohort 1A females with persistent vaginal thread), 2A and 3, or until mating for Cohort 1B animals].
The body weight of each animal was recorded on the day of the positive measurement. Any abnormalities of genital organs, such as persistent vaginal thread, hypospadias or cleft penis, were noted.

THYROID HORMONE ANALYSIS (F1 Culled Pups and pups not selected for cohorts, F2 Pups): Yes
Blood samples were taken (between 07:30 and 10:00 a.m.) into tubes containing K3-EDTA as anticoagulant.
• on PND 4 from F1 culled pups and from F2 pups euthanized (as much blood as possible
was collected by decapitation under isoflurane anesthesia and then pooled per litter), for potential measurements of thyroid hormone (T4) and thyroid stimulating hormone (TSH) levels,
• on PND 22 from F1 pups not selected for Cohorts (at least 0.25 mL per pup was collected from the vena cava immediately after euthanasia; 10 animals/sex/group), into individual tubes for potential measurements of thyroid hormone (T4) and thyroid stimulating hormone (TSH) levels,
• on PND 4 from F2 pups euthanized (as much blood as possible was collected by decapitation under isoflurane anesthesia and then pooled per litter), for potential measurements of thyroid hormone (T4) levels.
- Plasma samples obtained on PND 4 from pups were kept at -80°C but not analysed.
- Fasting: No

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
Cohort 2A: animals were selected for neurobehavioral testing (auditory startle test on PND 23, functional observation battery and motor activity on PND 64-65) followed by neurohistopathology assessment when they were adults.
Cohort 2B animals were selected for neurohistopathology assessment at weaning (PND 22).

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Cohort 1A: 10/sex/group (1 pup/litter, as much as possible) were selected for nvestigation of pre- and post-natally induced immunotoxic effects. Splenic lymphocyte subpopulation were analysed (T lymphocytes, CD4+ and CD8+ T lymphocytes, B lymphocytes, Natural Killer (NK) cells and NKT cells).
Cohort 3: animals were selected for immunotoxicity testing. On PND 55-59, an intravenous injection of Keyhole Limpet Hemocyanin (KLH) was performed and specific IgM anti-KLH antibodies were measured (specific ELISA method developed and validated at CRL Evreux) to evaluate the primary antibody response 5 days after immunization.

Postmortem examinations (parental animals):

SACRIFICE (P generation and Cohort 1A, 1B)
At schedule euthanasia and with the exception of females with viable lactating pups (P generation and Cohort 1B), all animals were deprived of food for an overnight period of at least 14 hours.
On completion of the treatment period, all surviving P and Cohort 1B animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
- P females: on PND 23-24, females which did not deliver: on Days 24-26 p.c., female with no evidence of mating: 27 days after the end of the mating period
- Cohort 1A males: on PND 91-95
- Cohort 1A females: on PND 92-95
- Cohort 1B males: after euthanasia of the F2 progeny
- Cohort 1B females: on PND 4

GROSS NECROPSY (P generation and Cohort 1A, 1B)
- A complete macroscopic post-mortem examination was performed on all P and F1 Cohort 1A, 1B animals.
- Gross necropsy consisted of external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
The numbers of implantation sites were recorded for females euthanized on PND 23-24 (P generation).

HISTOPATHOLOGY / ORGAN WEIGHTS (P generation and Cohort 1A, 1B)
- Only animals euthanized as scheduled
- Organ weights: Adrenals, brain (including medulla/pons cerebellar and cerebral cortex), epididymides, heart (except for 1B), kidneys, liver (except for 1B), ovaries (with oviducts), pituitary gland, prostate (dorso-lateral and ventral parts combined), seminal vesicles (including coagulating glands and their fluids), spleen, testes, thymus, thyroid with parathyroids (post-fixation), uterus (with cervix).
- Tissues examined (P and 1A animals; groups 1 and 4 only): Macroscopic lesions, adrenals, brain (including medulla/pons cerebellar and cerebral cortex), cecum, colon, duodenum, epididymides, esophagus, eyes with optic nerve, gut-Associated Lymphoid Tissue (GALT), heart, ileum, jejunum, kidneys, liver, lungs with bronchi, lymph nodes (mandibular and mesenteric), mammary gland area (males and females), ovaries (with oviducts), pituitary gland, prostate (dorso-lateral and ventral parts combined), rectum, sciatic nerve, seminal vesicles (including coagulating glands and their fluids), skeletal muscle, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroid with parathyroids (post-fixation), trachea (with thyroid and parathyroids attached), urinary bladder, uterus (with cervix), vagina, vas deferens (males).
- Tissues examined (groups 2 and 3): spleen (P animals), kidneys (Cohort 1A animals),
- Tissues examined (Cohort 1B; groups 1 and 4 only): macroscopic lesions, spleen.

Postmortem examinations (offspring):

SACRIFICE (F1 Cohorts 2A, 2B and 3, F2 pups)
At schedule euthanasia and with the exception of females with viable lactating pups (P generation and Cohort 1B), all animals were deprived of food for an overnight period of at least 14 hours.
All surviving F1 animals (pups and adults) and F2 pups were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
- F1 offspring not selected as parental animals were sacrificed on PND 22.
- F1 animals (Cohort 2A): between PND 76 and 78
- F1 animals (Cohort 2B): on PND 22
- F1 animals (Cohort 3): 5 days after KLH injection and after blood sampling (i.e. between PND 60 and 64)
- F2 pups: on PND 4

GROSS NECROPSY (F1 Cohorts 2A, 2B and 3, F2 pups)
- A complete macroscopic post-mortem examination was performed on all F1 animals (including F1 pups culled on PND 4 and not selected F1 pups on PND 22), F2 pups and prematurely euthanized or found dead pups.
- Gross necropsy consisted of external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS (Cohorts 2A, 2B and 3)
- Only animals euthanized as scheduled
- Organ weights: Adrenals, brain (including medulla/pons cerebellar and cerebral cortex), epididymides, heart, kidneys, liver , ovaries (with oviducts), pituitary gland, prostate (dorso-lateral and ventral parts combined), seminal vesicles (including coagulating glands and their fluids), spleen, testes, thymus, thyroid with parathyroids (post-fixation), uterus (with cervix).
- Tissues examined (Cohort 2A, 2B, 3; groups 1 and 4 only): macroscopic lesions, brain (only Cohort 2A and 2B), eyes with optic nerve (only Cohort 2A), sciatic nerve (only Cohort 2A), skeletal muscle (only Cohort 2A), spinal cord (only Cohort 2A), thyroid with parathyroid (only Cohort 2A females).
- Organ weights (F1 pups not selected for cohorts): brain, spleen, thymus (up to 10 pups/sex/group from as many litters as possible)

NEUROHISTOPATHOLOGY (Cohort 2A and 2B):
- Cohort 2A: between PND 76 and 78, Cohort 2B: PND 22.
- Group 1 and 4 (all measurements, group 2 and 3 (Cohort 2A males: all measurements; Cohort 2B: hippocampus L4-3 only).
- Multiple sections were examined from the brain: olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum. For Cohort 2A, the eyes (retina and optic nerve) and samples of peripheral nerve, muscle and spinal cord were also examined.

Statistics:

- Body Weight, Food Consumption and Reproductive Data:
Data were compared by one-way variance analysis and Dunnett's test, (mean values being considered as normally distributed, variances being considered as homogeneous) or by Fisher’s exact probability test (proportions).
- Organ Weights:
PATHDATA software was used (level of significance of 0.05 or 0.01) according to a specific sequence.
- Splenic Lymphocyte Immunophenotyping:
Statistical analysis was performed using the SAS Enterprise Guide software.
- Number of Primordial Follicles/Corpora Lutea, Ano-genital Distance, Number of Nipples and Areolaes, Time of Preputial Separation/Vaginal Opening, Time to First Estrous After Vaginal Opening/Patency, Seminology, Hematology, Coagulation, Blood Biochemistry, Urinalysis, Thyroid Hormones, Anti KLH IgM, Auditory Startle Reflex Test, Motor Activity, Post-implantation Loss, Sex Ratio, Live Birth, Viability and Lactation Indexes:
Citox software was used according to a specific sequence.
See attached pdf file with details on the specific sequences for the above mentioned parameters.

Reproductive indices:

- Post-implantation loss: ((Number of implantation sites - Number of live pups) / Number of implantation sites) x 100 ;
- Mating index: (Number of mated animals / Number of paired animals) x 100 ;
- Fertility index: (Number of pregnant female partners / Number of mated pairs) x 100 ;
- Gestation index: (Number of females with live born pups / Number of pregnant females) x 100

Offspring viability indices:

Live birth index: (Number of live pups on PND 1 / Number of delivered pups) x 100 ;
Viability index on PND 4: (Number of surviving pups on PND 4 (before culling) / Number of delivered pups) x 100 ;
Lactation index: (Number of surviving pups on PND 21 / Number of surviving pups on PND 4 (after culling)) x 100 ;
AGD/cube root of body weight ratio: AGD / ³vBody weight

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

(Text Table 11 and 12 in study report)
In the P generation, ptyalism (hypersalivation) was noted mainly after treatment in test item-treated males (not in control males) and in more test item-treated females than control females. The highest incidence was at the high-dose of 450 mg/kg bw/day (almost all animals in the group were affected). This clinical sign was considered to be test item-related but non-adverse (common finding observed after oral gavage with test item dose formulations).
Piloerection was observed in all test item-treated male groups, with no dose-relationship and with a limited incidence per group. This clinical sign can be spontaneously observed in laboratory rats, therefore a relationship with the test item treatment was considered unlikely.
The other clinical signs observed in the test item-treated P generation (reflux at dosing, hunched posture, burrowing activity, alopecia/thinning of air, chromodacryorrhea, chromorhynorrhea, scabs, wounds, exteriorized penis, cryptorchidism, soiled urogenital region, soft feces and vaginal discharge) were noted with limited incidence or incidence similar to or lower than that of controls, without a dose-relationship and/or as common background findings in laboratory rats. They were not considered to be test item-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no unscheduled deaths in test item-treated P Generation animals. In controls, one female was prematurely euthanized on GD 24 (Study Day 44) due to difficulties to deliver.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Pre- and post mating periods (Text Table 13 in study report) :
At 450 mg/kg bw/day, when compared with controls, mean body weight was statistically significantly lower in males from Day 8 (down to -6%, p<0.001) and mean body weight change over the treatment period was statistically significantly lower mainly due to effects at the beginning of the treatment period. These findings were considered to be test item-related and non-adverse (low magnitude).
There were no effects on mean body weight or mean body weight change in males at 40 and 150 mg/kg bw/day until termination, or in females during the pre-mating period. The statistically significant higher mean body weight gain in males at 40 mg/kg bw/day in Week 4 was considered to be incidental in the absence of a dose-relationship.

Pregnancy period (Text Table 14 in study report) :
At 450 mg/kg bw/day and when compared to controls, mean body weight gain was lower on
Days 0 to 4 p.c. (+19g vs. +25 g in controls, p<0.01). A test item relationship cannot be ruled out but taking into account the amplitude of the changes, the transiency of the effect and the absence of an effect on mean body weight, this finding was considered to be non-adverse.
When compared to controls, there were no effects on mean body weight at 40 or 150 mg/kg bw/day during the pregnancy period.

Lactation period (Text Table 15 in study report) :
At 450 mg/kg bw/day and when compared to controls, mean body weight gain was higher over the lactation period (+45g vs. +34 g in controls, p<0.05). A test item relationship cannot be excluded but taking into account the amplitude of the changes and absence of an effect on mean body weight, this finding was considered to be non-adverse.
When compared to controls, there were no effects on mean body weight at 40 or
150 mg/kg bw/day during the lactation period. The statistically significant higher mean body weight gain at 150 mg/kg bw/day on PNDs 4 to 7 was considered to be incidental in the absence of a dose relationship.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Pre- and postmating periods (Text Table 16 in study report) :
When compared to controls and despite the single statistically significant difference at 450 mg/kg bw/day (week 5), there were no dose-related effects on mean food consumption in P generation males or females. Therefore, any relationship to the test item-treatment was considered to be unlikely.

Pregnancy period (Text Table 17 n study report) :
There were no effects on mean food consumption during the gestation period.

Lactation period (Text Table 18 in study report) :
When compared to controls, there were no effects on mean food consumption in P generation females during the lactation period.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

(Text Table 69 in study report)
There were no test item-related effects on coagulation in P generation animals. In both sexes at 450 mg/kg bw/day, when compared to controls, mean eosinophil count was statistically significantly lower (about -50%, p<0.01). This was observed together with a trend towards lower mean count of most other white blood cell subpopulations in males, hence the statistically significant lower mean total white blood cell count at this dose level in males. Despite the low magnitude of the changes, a test item treatment effect cannot be excluded, but the findings were considered as non-adverse. In females, the statistically significant difference noted in mean reticulocyte relative count at 450 mg/kg bw/day was considered to be not toxicologically signficant in the absence of effects on the other red blood cell parameters, of similar effects in males and of statistical significant difference in mean reticulocyte absolute count.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

(Text Table 70 in study report)
There were no test item treatment-related effects on blood biochemistry in P generation females.
In males at 450 mg/kg bw/day, mean glucose level was statistically significantly lower than in controls (-11%, p<0.05). Despite the low magnitude of the change, a test item treatment effect cannot be excluded, but the effect was considered as non-adverse. The statistically significant differences recorded at 150 mg/kg bw/day in males (K+) and at 40 mg/kg bw/day in females (A/G) had no dose relationship. Therefore, they were considered to be incidental.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

(Text Table 71 in study report)
When compared to controls and in the absence of statistical significance or a dose-relationship, there were no test item-treatment effects on quantitative urinalysis parameters in P generation animals. There were no test item treatment-related effects on qualitative and semi-quantitative urinalysis parameters. At 150 mg/kg bw/day, one male had yellow/brown, turbid urine with a high urinary pH and levels of proteins and blood. This type of urine was found only in this mid-dose P animal and was therefore not ascribed to the test item treatment.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

(Text Table 81 in study report)
Test item-related changes were noted in the spleen (pigment). Minimal dose-related brown pigment was seen in enlarged macrophages in males treated at = 40 mg/kg bw/day and in females treated at = 150 mg/kg bw/day. The Perls staining performed in one control male and one male treated at 450 mg/kg bw/day with minimal pigment demonstrated no clear differences (i.e. similar Perls-positive pigment in both animals) probably because of the low magnitude of this pigment in test item-treated animals. No definitive conclusion could be drawn with the special staining.
There was a good correlation between the vaginal smears and the histopathological examination of estrus cycle in all examined females. There were no test item-related findings in the reproductive tract of males and females.
The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls. This included the minimal accumulation of hyaline droplets, urothelium hyperplasia, infiltrate of mononuclear cells and vacuolation of tubules, basophilic tubules, dilated pelvis and cortex mineralization in the kidneys seen with minimally increased incidence in some test item treated animals when compared to controls. These changes were considered to be part of the spontaneous background.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormones (Text Table 72 and 73 in study report):
When compared to controls and despite the statistically significant higher mean T4 level in males at 40 mg/kg bw/day, a test item treatment relationship was considered to be unlikely in the absence of a dose-relationship.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

(Text Table 19 in study report)
There were no test item-related effects on mean estrous cycle parameters.
At 450 mg/kg bw/day and when compared to controls, the mean number of estrous cycles was lower (-10%, p<0.05). This difference was mainly due to 1/24 high-dose female that was blocked in diestrous, and 2/24 control females with two abberant cycles of 2 days, and was therefore considered not to be test item-related.
At the same dose, the mean number of days of diestrous was higher than in controls (+39%, p<0.01). A test item-relationship was considered to be unlikely when looking at the individual data and in the absence of any statistically significant effects on the distribution of the other estrous stages or of any test item-related effects on mean duration/number of estrous cycles or P generation fertility. Moreover, the mean data was within the HCD range.
There were no test item-related effects at 40 or 150 mg/kg bw/day on mean estrous cycle parameters.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

(Text Table 79 in study report)
When compared to controls and HCD, there were no test item-related effects on epididymal sperm parameters (motility, morphology or sperm numerations). There was one male in each test item groups with a low number of motile spermatozoa or no motile spermatozoa (50%, 1,5% or 0% at 40, 150 or 450 mg/kg bw/day, respectively, associated at 450 mg/kg bw/day with a very low number of spermatozoa and very low percentage of morphologically normal spermatozoa). A test item relationship was considered unlikely in view of the isolated incidence per group. Besides, this had no impact on male fertility as these 3 males mated and conceived.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

(Text Table 20- and 21 in study report)
There were no test item-related effects on mating (including the mean number of days taken to mate) or fertility data. All pairs mated within 5 days. The statistically significant higher mean number of pregnant females at 40 mg/kg bw/day was considered to be incidental in the absence of a dose-relationship and as there was no decrease compared to controls.
When compared to controls, there were no test item-effects in mean delivery data, including the duration of gestation, as there was no statistical significance and/or as the mean data were within the reference control range.
At enumeration of corpora lutea in the ovary of high-dose group and control group, there were no statistical differences with a mean number of corpora lutea of 9.67 (±2.33) and 11.91 (±5.69) per animal respectively. There were no statistical differences between the high-dose and control groups, with a mean number of 9.96 (±8.08) and 9.16 (±7.01) primordial follicles per animal on PCNA-stained slides respectively.

Key result

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A (Text Table 33 and 34 in study report):
Ptyalism was noted after treatment in some animals of both sexes at 450 mg/kg bw/day from Days 11 to 70. This finding was observed after treatment and at the high-dose only, therefore it was considered to be test item-related but non-adverse (commonly observed after gavage administration). There were no test item-related clinical signs at 40 or 150 mg/kg bw/day. Other findings (reflux at dosing, chromodacryorrhea, chromorhynorrhea, alopecia, thinning of hair, tail bent, bent head, nodosity, scabs, wounds, enophthalmos, opacity, hunched posture, piloerection, cryptorchidism) were observed with a low and/or similar incidence as in controls, with an absence of dose-relationship, and/or are commonly observed in this species/strain of animals. They were therefore not considered to be test item treatment-related.

Cohort 1B (Text Table 43 and 44 in study report):
Ptyalism was noted, mainly after treatment, in a few animals at 40 and 150 mg/kg bw/day and in more than half the animals of both sexes at 450 mg/kg bw/day (from Days 21 to 109 at the high-dose). This finding is commonly observed after gavage with test item dose formulations and was considered to be test item treatment-related (dose-related) but not adverse. There were no test item-related clinical signs at 40 mg/kg bw/day. Other findings (reflux at dosing, chromodacryorrhea, chromorhynorrhea, alopecia, thinning of hair, abnormal growth of teeth, tail bent or shortened, nodosity, scabs, wounds, soiled body parts, vaginal discharge, hunched posture, piloerection, exteriorized penis, loud breathing and soft feces) were observed with a low and/or similar incidence to controls, with no dose-relationship, and/or are commonly observed in this species/strain of animals. They were therefore not considered to be test item treatment-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Cohort 1A:
There were no unscheduled deaths in Cohort 1A males. In Cohort 1A females, one female at150
mg/kg bw/day was prematurely euthanized on Study Day 45 on humane grounds. On the day of
euthanasia, loud and abdominal breathing were observed and the female had lost 13% of its body weight in 2 days. The cause of moribundity was not evident at pathology, but a test item relationship was excluded in view of the isolated occurrence of this unscheduled euthanasia and of the lack of test item-related changes at macroscopic or microscopic examination. No other unscheduled deaths were recorded in Cohort 1A females at any dose level.

Cohort 1B:
There were no unscheduled deaths in Cohort 1B males. Two unscheduled deaths recorded among Cohort 1B females. At 150 mg/kg bw/day one female was prematurely euthanized due to delivery difficulties on GD 24 (Study Day 96). This female showed pallor of extremities, ventral recumbency, tremors, dyspnea, half-closed eyes and soiled urogenital region on that day. In the bedding, there were 5 live, 1 dead and 1 cannibalized pup. Histopathology concluded that this animal had a congenital malformation of the right abdominal organs (kidney, ureter, uterine horn), agenesis and secondary reproduction trouble. At 450 mg/kg bw/day one female, after having delivered on GD 23, was prematurely euthanized on PND 1 due to dead litter (Study Day 100). This female showed piloerection, hunched posture and pallor of extremities before its euthanasia. These two isolated premature deaths due to reproduction troubles were not considered to be related to the test item administration. No other unscheduled deaths were recorded in Cohort 1B females at any dose level.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A (Text Table 35 in study report):
There were no adverse test item-related effects on mean body weight and mean body weight change in Cohort 1A. At 450 mg/kg bw/day, when compared to controls, mean body weight gains were lower in both sexes in the first (+41 g in males vs. +51 g in controls; + 30 g in females vs. +43 g in controls; p<0.01) and/or second week of dosing (+64 g in males vs. +70 g in controls, p<0.01) followed in females by higher mean body weight gains from Week 3. Mean body weights at the beginning of Weeks 2 and 3 correlated with the low body weight gains and were lower (p<0.01) than controls in both sexes. These effects were considered to be test item related as they were observed at the high dose and in both sexes. However, in view of the slight differences from controls, taking into account that high-dose male mean body weight was already at -6% from controls on Day 1, and taking into account that the effects on mean body weights were transient, the findings were considered as nonadverse. Mean body weight in males stayed statistically lower than in controls until the end of the dosing period, but the differences from controls were similar to those on Day 1 in the second half of the dosing period. Mean body weights in females became similar to controls again in Week 4. There were no effects on mean body weight or mean body weight change at 40 and 150 mg/kg bw/day.

Cohort 1B:
Pre- and post mating period (Text Table 45 in study report):
There were no adverse test item-related effects on mean body weight and mean body weight change. At 450 mg/kg bw/day, when compared to controls, mean body weight gains were lower in both sexes in the first (+36 g in males vs. +49 g in controls; +32 g in females vs. +43 g in controls; p<0.001) and/or second week of dosing (+58 g in males vs. +66 g in controls, p<0.01), followed, in females, by higher mean body weight gains in Weeks 3 and 4 [respectively: +43 g (p<0.001) and +26 g (p<0.01) vs. +34 g and +21 g in controls]. Mean body weight gains in males tended to be lower than in controls again in the last 2 weeks of the dosing period. Mean body weights at the beginning of Weeks 2 and 3 correlated with the low body weight gains and were lower (-9 to -16%, p<0.001) than controls in both sexes. These effects were considered to be test item-related as they were observed at the high dose and in both sexes. However, in view of the slight differences from controls and taking into account that high-dose male mean body weight was already at -5% vs. controls on Day 1, the effects were considered as non-adverse. Mean body weight in males stayed statistically lower than in controls until the end of the dosing period (-9% vs. controls at the end of the dosing period, p<001), but the differences from controls were minimal at the end of the dosing period compared to Day 1 (-5% vs. controls on Day 1). Mean body weights in females were similar to those of controls from Week 4. There were no effects on mean body weight or mean body weight changes in the low- and mid-dose groups. The few statistically significantly higher mean body weight gains when compared to controls were transient and considered as incidental.

Pregnancy period (Text Table 46 in study report):
There were no test item-related effects on mean body weight or mean body weight change in Cohort 1B females during the pregnancy period.

Lactation period (Text Table 47 in study report):
There were no test item-related effects on mean body weight or mean body weight change in Cohort 1B females at the beginning of the lactation period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A (Text Table 36 in study report): Correlating with the effects seen on mean body weight, mean food consumption of Cohort 1A was lower (-18%, p<0.01) in females at 450 mg/kg bw/day in the first week of dosing when compared to controls. A similar trend was noted in males (-16%) at the same dose level but with no statistical significance vs. controls. These findings were considered as test item-related but non-adverse as they were transient and slight. In the following weeks, the differences from controls were =10%. These differences were therefore not considered to be test item related, including the statistical significance noted in males in Week 9. There were no effects on mean food consumption at the low and mid-dose groups. Some cage food consumption data were found to be abnormally high in test item groups (Week 10 in males and Weeks 6 and 8 in females), but this did not impact the interpretatio n of this study parameter.

Cohort 1B (Text Table 48 in study report):
Pre- and post mating period:
Correlating with the effects seen on mean body weight, mean food consumption was lower in both sexes at 450 mg/kg bw/day in the first (-14% or -15%, p<0.001) and/or second (-13%, p<0.001, in males) week of dosing when compared to controls. These findings were considered as test item-related and non-adverse as they were transient and slight. No effects were considered to be test item-related in the following weeks despite the statistical significance of the differences in males from Week 8 at 450 mg/kg bw/day, as the differences from controls were = 10%. There were no effects on mean food consumption in the low- and mid-dose groups.

Pregnancy period:
There were no test item-related effects on mean food consumption in Cohort 1B females during the pregnancy period.

Lactation period:
There were no test item-related effects on mean food consumption in Cohort 1B females during the lactation period.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A (Text Table 74 in study report):
There were no test item-related effects on coagulation in Cohort 1A animals at any dose level. The differences from controls were considered to be of no toxicological significance even though a test item treatment effect cannot be excluded as they were recorded generally in both sexes and at the high dose level. The differences were of low magnitude, were within the HCD, did not correlate with other hematology parameters or histopathological findings in Cohort 1A animals, and/or the directions of the changes were not clinically significant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A (Text Table 75 in study report):
The differences from controls noted at 450 mg/kg bw/day in mean glucose, cholesterol, and albumin levels and ALP activity were considered to be of no biological significance. They were of low magnitude, within the HCD, and often observed in one sex only. The statistically significant differences in Cohort 1A animals noted in mean urea and chloride levels were recorded with no dose relationship and were not observed in both sexes at the same dose level. Therefore, a test item-relationship was considered to be unlikely.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A (Text Table 76 in study report):
There were no test item-related effects on qualitative or semi-quantitative urinary parameters in Cohort 1A animals at any dose level. From 150 mg/kg bw/day onwards in males, there was a doserelated higher mean urinary volume when compared to controls (up to +64%, p<0.05). Taking into account the low amplitude of the changes, this finding was probably test item-related but was considered to be non-adverse. There were no test item-related effects on mean urinary volume in female at any dose level. There were no test item-related effects on mean urine specific gravity or pH at any dose level despite the statistically significant difference in specific gravity.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A:
There were no test item-related organ weight changes. The few observed organ weight changes were not considered to be test item-related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings. This included the increased absolute and relative-to-body spleen weights recorded in females treated at 450 mg/kg/day (+16%; p<0.01 in absolute weight only). There were no correlates at microscopic examination.

Cohort 1B:
There were no test item-related organ weight changes. The few organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings. Increased absolute and/or relative-to-body spleen weights were recorded in females treated at = 150 mg/kg bw/day (up to +20%; p<0.05 or 0.01). This correlated with congestion at microscopic examination and gross enlargement at necropsy. These changes were considered to be related to the agonal process and unrelated to the test item administration. There was no significant increase of microscopic pigment in test item-treated animals when compared to controls. The increased relative-to-body adrenal gland, epididymides, seminal vesicles, spleen and testes weights in males treated at 450 mg/kg bw/day were considered to be related to the decreased final body weight (-9%; p<0.01).
The decreased absolute and relative-to-body thymus weights recorded in females treated at 450 mg/kg bw/day (-21% in relative weights; p<0.01) had no correlates at macroscopic examination and were not seen in the males from 1B cohort or in the animals from other cohorts. There was one female treated at 450 mg/kg bw/day with lower thymus weight than the other females from the group (outlier). Consequently this difference was considered not to be related to the test administration.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A:
There were no test item-related findings. The few isolated gross observations were considered to be consistent with spontaneous findings encountered in rats of these strain and age. This included the black discoloration in the stomach from 5/20 females treated at 450 mg/kg/day (correlated in 1 female with mucosal erosion/hemorrhage) which are common background
findings.

Cohort 1B:
There were no test item-related findings. The few isolated gross observations were considered to be consistent with spontaneous findings encountered in rats of these strain and age. This included the black discoloration in the stomach from 1/20 males treated at 450 mg/kg bw/day, the small prostate from 1/20 males treated at 450 mg/kg bw/day and the shortened tail from 1/20 males treated at 450 mg/kg bw/day which had no microscopic correlates and/or are common background findings. There were also enlarged spleen in males and females treated at = 40 mg/kg bw/day that correlated with congestion in males and females. It is noteworthy that one control male had also an enlarged spleen. The microscopic correlated congestion was considered to be an agonal change not related to the test item administration.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A (Text Table 82 in study report):
Test item-related changes were noted in the kidneys in males treated at 450 mg/kg bw/d
ay, characterized by:
. vacuolation of tubules suggestive of lipidosis in 5/20 males, mainly in cortex (unilateral, grouped, convoluted proximal tubules with enlargement of cells because of clear vacuoles),
. increased severity and incidence of tubular basophilia when compared to controls,
. increased severity and/or incidence of pelvis dilatation when compared to controls,
. slight degeneration/necrosis of tubules (medulla) in one high-dose male, associated with he
morrhage and urothelial ulceration.
The remaining microscopic findings were not considered to be associated with the test item adm
inistration because these findings were consistent with spontaneous and background findings
described in the literature, the findings were distributed randomly among groups, and/or their app
earance was similar to changes found in controls. This included the minimal mononuclear cell infiltrates seen in the lungs from 4/20 males treated at 450 mg/kg bw/day that can be seen in untreated rats. There were no test item-related changes in the male and female reproductive organs. There was a good correlation between the vaginal smears and the histopathological examination of estrus cycle in all examined females.

Cohort 1B:
There were no test item-related findings. The few microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.
This included the minimal to slight congestion seen in the spleen from males and females treated at = 40 mg/kg bw/day and that correlated with increased organ weights and macroscopic enlargement. It was considered to be an agonal change.

Other effects:

no effects observed

Description (incidence and severity):

Cohort 1A (Text Table 77 and 78 in study report):
In Cohort 1A animals and when compared to controls and HCD, there were no test item-related effects on mean thyroid hormones levels (T4 and TSH).

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

(Text Table 37 and 38 in study report)
At 450 mg/kg bw/day and when compared with controls and HCD, there was a trend towards a delayed first estrous after vaginal opening in Cohort 1A females, with especially 3/20 individuals above the control range. This finding was considered to be test item-related, but non-adverse in the absence of effects on reproductive performance in Cohort 1B. There were no effects on mean time of the first estrous after the onset of vaginal patency at 40 or 150 mg/kg bw/day. There were no test item-related effects on mean estrous cycle parameters in Cohort 1A females at the end of the dosing period.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

(Text Table 80 in study report)
In Cohort 1A, in the absence of statistical significance vs. controls and/or of a dose-relationship, no effects on sperm analysis parameters (motility, morphology, sperm numeration) were considered to be test item-related.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1B (Text Table 49 and 50 in study report):
There were no test item-related effects on mating and fertility data of Cohort 1B.There were no test item-related effects on the mean delivery data and mean sex ratio. At 450 mg/kg bw/day, when compared to controls and HCD, there was a trend towards a higher mean sex ratio on PND 1 and consequently on PND 4 in F2 pups (+18% vs. controls, no statistical significance). The difference from controls was partially due to 2 control litters with less than 15% of males on Day 1 (control group mean at 49.4% when excluding these 2 litters). Besides, all individual data at 450 mg/kg bw/day [33.3; 76.9] were within or similar to the control range [10.0; 76.5]. A test item effect was therefore considered to be unlikely
At enumeration of corpora lutea in the ovary of Cohort 1A high-dose and control groups, there were no differences, with a mean number of corpora lutea of 22.25 (±7.30) and 19.30 (±6.75) per animal respectively, reaching no statistical significance. At enumeration of primordial follicles in the ovary of Cohort 1A high-dose and control groups, there were no differences, with a mean number of primordial follicles of 10.18 (±5.81) and 10.63 (±6.49) per animal respectively, reaching no statistical significance.

Key result

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F1 pups during lactation period (Text Table 26 in study report) :
No relevant differences were noted between controls and test item-treated groups in the number of pups with findings on PND 1. The findings observed during lactation were with a low or similar incidence when compared to controls, with no dose relationship and/or are common findings in rat pups of this species maintained under experimental conditions similar to this study. They were therefore not ascribed to the test item treatment.

Cohort 2A (Text Table 58 and 59 in study report):
Ptyalism was noted after treatment in 3/20 animals at 450 mg/kg bw/day, on 1 day only. This finding was similar to the clinical signs seen after treatment in the Cohort 1 and is commonly observed after gavage with test item dose formulations. A test item effect was therefore considered to be likely and the effect was considered to be non-adverse. There were no test item-related clinical signs at 40 or 150 mg/kg bw/day. The other findings noted in test item groups (soiled urogenital region, scabs, tail bent, reflux at dosing and chromodacryorrhea) were observed with a limited incidence and no dose-relationship, and are commonly observed in this species/strain of animals. They were therefore not considered to be test item-related.

Cohort 3 (Text Table 63 and 64 in study report):
Ptyalism was noted after treatment in a 2/20 animals at 450 mg/kg bw/day, on 1 day only. This finding was similar to the clinical signs seen after treatment in Cohort 1, and this sign is commonly observed after gavage with test item dose formulations. A test item effect was therefore considered to be likely and the sign was considered to be non-adverse. There were no test item-related clinical signs at 40 or 150 mg/kg bw/day. The other findings noted in test item groups (chromodacryorrhea, chromorhynorrhea, scabs) were observed with a limited incidence and no dose-relationship, and are commonly observed in this species/strain of animals. They were therefore not considered to be test item-related.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F1 pups during lactation period (Text Table 22-24 in study report):
When compared to controls and reference control data, and despite the statistically significantly higher number of pups found dead or cannibalized by PND 4 at 150 mg/kg bw/day, there were no test item-treated effects on P generation offspring mortality (no dose-relationship and within the reference control range).
At necropsy of found dead pups, an absence of milk and autolysis were noted in a few pups from test item-treated groups, but with no dose-relationship. This can be observed in control groups as seen in previous OECD 443 studies and was not ascribed to the test item treatment.
There were no effects on PND4 viability or lactation indexes.

Cohort 2A: There were no unscheduled deaths in Cohort 2A females. In Cohort 2A males, one male in control and one male at 150 mg/kg/day were found dead on Study Day 55 and 42, respectively (accidental death was noted in the raw data for the male at 150 mg/kg/day). Before euthanasia, no relevant clinical signs or effects on body weight change were observed. These deaths did not correlate with the test item (control animal or no dose-relationship and apparently accidental). No other unscheduled deaths were recorded in Cohort 2A males at any dose level.

Cohort 3:
There were no unscheduled deaths in Cohort 3 males. In Cohort 3 females, one female at 40 mg/kg bw/day was prematurely euthanized on Study Day 34 on human grounds (limb broken accidently). This death was therefore not test item-related and no other unscheduled deaths were recorded in Cohort 3 females at any dose-level.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1 pups during lactation period (Text Table 27 in study report) :
There were no test item treatment-related effects on F1 pup mean body weight and body weight change during lactation at any dose level.

Cohort 2A (Text Table 60 in study report):
There were no adverse test item-related effects on mean body weight and mean body weight change in Cohort 2A. At 450 mg/kg bw/day, when compared with controls, mean body weight gain was lower, mainly in males, in the first week of dosing (+39 g in males vs. +50 g in controls, p<0.01; +37 g in females vs. +41 g in controls, p<0.05). Statistically significant, lower mean body weights in males at the beginning of Weeks 2 and 3 (respectively: -11% and -8%, p<0.05) correlated with the low body weight gain. These effects were considered to be test item-related as they were observed at the high dose, in both sexes and also occurred in Cohort 1. However, in view of the slight and transient differences from controls, the effects were considered as non-adverse. There were no effects on mean body weight or mean body weight change at 40 and 150 mg/kg bw/day.

Cohort 3 (Text Table 65 in study report):
In Cohort 3 at 450 mg/kg bw/day, when compared to controls, mean body weight gains were lower in the first (+37 g vs. +47 g in controls, p<0.01) and fifth (+51 g vs. +60 g in controls, p<0.05) weeks of dosing in males, and the first (+26 g vs. +42 g in controls, p<0.01) and second (+49 g vs. +55 g in controls, no statistical significance) weeks of dosing in females, followed, in females, by higher mean body weight gains in Weeks 3 (+46 g vs. +35 g in controls, p<0.01) and 5 (+27 g vs. +22 g in controls, no statistical significance). The lower mean body weight in males on Day 1 (PND 22) (-9% vs. controls, p<0.05) was considered to be incidental in the absence of effects on F1 pup mean body weight during lactation. However, at the beginning of Weeks 2 and 3, the difference from controls was more pronounced (-12% to -18% vs. controls, p<0.01) in both sexes. This correlated with the concomitant low mean body weight gains. These effects were considered to be test item-related as they were observed at the high dose and in both sexes. However, taking into account that the effects on mean body weight were transient, they were considered to be non-adverse. Mean body weight in males was also statistically lower than in controls in the last 2 weeks of the dosing period, but the differences from controls were similar to those on Day 1. Mean body weights in females became similar to those of controls again in Week 4. There were no effects on mean body weight or mean body weight change at 40 and
150 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Cohort 2A:
There were no test item-related effects on mean food consumption in Cohort 2A animals. Some cage data in controls (Weeks 3 and 4) were considered to be abnormal and abberent, but this did not impact the interpretation of this parameter.

Cohort 3 (Text Table 66 in study report):
There were no adverse test item-related effects on mean food consumption in Cohort 3. Food consumption tended to be slightly lower in 1/2 cages of females at 450 mg/kg bw/day in the first 3 weeks of dosing when compared with the 2 control cages. This finding could be test item-related as it correlated with the concomitant lower mean body weight gains and was similar to the effects seen in Cohort 1. However, the finding was considered to be non-adverse as it was transient and slight. Some cage data were found to be abnormally high or low (in Weeks 3 and 4 in male controls, and Weeks 1, 2 and 5 in test item-treated females), but this did not impact the interpretation of this study parameter.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

(Text Table 31 and 32 in study report)
There were no adverse test-item treatment related effects on mean age and mean body weight at balanopreputial separation in F1 males. There was a dose-related delay in mean age at balanopreputial separation from 150 mg/kg bw/day (+3 or +4% vs. controls, p<0.01) in the F1 generation when compared to controls and/or HCD, in absence of relevant differences in mean body weight (data similar to controls and not dose-related). A test item treatment effects could not be excluded but this finding was considered to be non-adverse in view of the slight magnitude of the change (<5%) and especially in the absence of effects on reproductive performance in Cohort 1B. One to three males had no or incomplete balanopreputial separation until termination in each test item-group, but there was no dose-relationship. There were no effects on balanopreputial separation at the low-dose group.

At 450 mg/kg bw/day and when compared with controls and HCD, the mean age of vaginal opening was delayed (+9% vs. controls, p<0.01) in F1 females in the presence of a statistically significant change in mean body weight (+7% vs. controls, p<0.01) on the day of occurrence. This finding was therefore considered to be test item-related, but non-adverse in the absence of effects on reproductive performance in Cohort 1B. There were no test item-related effects on vaginal opening at the low- and mid-dose groups in F1 females. One or two females had an incomplete vaginal opening (vaginal thread left) in each group, including in controls.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

(Text Table 28 in study report)
There were no test item-related effects on PND 1 mean anogenital distance (AGD) and normalized AGD in males or females. At 40 mg/kg bw/day, the statistically significantly higher mean normalized AGD vs. controls in females was considered as incidental in the absence of a dose-relationship and as it was within HCD.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no observable nipples or areolae in male pups examined on PND 12.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related organ weight changes in Cohort 2A, 2B or 3, nor in non selected F1 pups on PND 22. The few observed organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings. In Cohort 2A this included the increased absolute and relative-to-body thyroid gland weights in females treated at 450 mg/kg bw/day because there were no microscopic correlates and such change was not seen in the other cohorts.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

(Text Table 29 and 30 in study report)
On PND 4, the most relevant macroscopic finding was an abnormal color of the liver in one pup at 150 mg/kg bw/day (greenish liver) and in 5 pups among 3 litters at 450 mg/kg bw/day (brownish liver) vs. none in the controls. A test item treatment effect was considered to be unlikely as this finding was not seen in pups euthanized on PND 22 or in the F1 generation (Text Table 30 in study report) .
At weaning among the pups non-selected for the F1 generation, there were no test item treatment-related findings in any group. The few findings noted were also recorded in controls, observed with a low incidence or in isolated pups, noted with no dose relationship and/or are common findings in this species and strain maintained under experimental conditions similar to this study. This included the changes seen in one male at 450 mg/kg bw/day that had emaciation, dilatation of brain ventricles with translucent content, secondary deformed pituitary gland, and thinned, soft cranium bone with translucent content in cranial cavity. These changes were considered to be congenital with no relationship to test item administration.

There were no test item-related findings observed in Cohort 2A, 2B or 3, nor in non selected F1 pups on PND 22. The few isolated gross observations were considered to be consistent with spontaneous findings encountered in rats of these strain and age. This included the following. In Cohort 2A, the black discoloration in the stomach from 3/10 females treated at 450 mg/kg bw/day that correlated with microscopic hemorrhage. In Cohort 2B, the red thymus and deformed tail in 1/10 females treated at 450 mg/kg bw/day. In Cohort 3, irregular color in the kidneys, small prostate, small epididymides from 1/10 males treated at 450 mg/k bwg/day, and the red thymus at 150 and 450 mg/kg bw/day, granular, irregular color in kidneys at 150 and/or 450 mg/kg bw/day and small kidneys, black discoloration in the stomach and black content/dilatation of the ureters from isolated females treated at 450 mg/kg bw/day. In non selected F1 pups on PND 22, the changes seen in one male at 450 mg/kg bw/day that had emaciation, dilatation of brain ventricles with translucent content, secondary deformed pituitary gland, and thinned, soft cranium bone with translucent content in cranial cavity. These changes were considered to be congenital (hydrocephalus) with no relationship to test item administration.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At microscopic examination of macroscopic lesions in Cohort 2A, 2B and 3, there were no test item-related findings. The few microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormones (Text Table 78 in study report):
In PND 22 pups and when compared to controls, there were no test item-related effects on mean thyroid hormone levels (T4 and TSH) despite the statistically significant differences, as there was no dose-relationship.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Neurobehavioral tests (Text Table 61 and 62 in study report):
In the auditory startle test, there were no test item treatment-related effects on mean latency times, mean amplitude of the responses at any dose level or on habituation. Females dosed at 40 mg/kg bw/day did not seem to become habituated, but in the absence of a dose-relationship this was not ascribed to the test item.
In the Functional Observation Battery (FOB), Cohort 2A animals were tested once between PND 64 and 65 (Study Days 43 and 44). When compared to controls, there were no test item-related effects on reactivity to manipulation or to different stimuli. There were 2/10 females at 150 mg/kg bw/day with increased forelimbs grip strength and 3/10 females at 450 mg/kg bw/day with decreased forelimbs grip strength, vs. none in controls. As these effects were opposite between the two groups, and in the absence of correlating clinical signs or findings in the other FOB parameters, a test item-related effect on forelimbs grip strength was considered unlikely.
In the absence of a dose relationship and/or statistical significance in the motor actrivity test and as group means were within the HCD range, an effect of the test item treatment on the mean number of horizontal movements and rearing was considered to be unlikely.

Neurohistopathology (Text Table 83-85 in study report)
There were no test item-related brain weight differences in Cohort 2A or 2B. The few observed differences were minimal and/or not dose-related. There were no test item-related microscopic findings in the multiple brain sections (olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum). There were no effects in the eyes (retina and optic nerve), peripheral nerve, skeletal muscle or spinal cord.
In Cohort 2A males treated at = 40 mg/kg bw/day had various lower measurements when compared to controls in cortex, hippocampus and cerebellum (up to -12%; p<0.01 or 0.05) except for the cortex level L3 4+8 (statistical significance not reached). Similar differences were not seen in 2A females. Although poorly dose-related, the relationship to test item administration of these differences was considered to be ambigous according to Garman, 2016.
There were no statistically significant differences between Cohort 2B controls and animals treated at 450 mg/kg bw/day for the measurements performed on cortex, hippocampus and cerebellum. A decision tree is included in the article of Garman et al (2016) to ease the assessment of the potential biological relevance of test article-related findings. A clear evidence of neurotoxicity potential requires that an agent induces either overt neuroanatomic lesions that can be readily identified by qualitative evaluation or morphometric measurements or demonstrable neurobehavioral dysfunctions. The influence on morphometric measurements must involve a more complex pattern than a single significant different value at only one time point. Instead, patterns of dose-dependent morphometric alterations attributed to neurotoxicity would include aberrations in 1 or more measurements that either i) affect animals at both PND 22 and PND 70, especially if the effect is greater in the adult, or ii) occur in both sexes at PND 70. Here, the relationship to test article administration was considered to be “ambiguous” in view of the combination of i) absence of treatment-related microscopic alteration in the nervous system and ii) statistically significant differences in linear measurements that appear to be inconsistent across age (seen only in cohort 2A- PND 75-90) and sex (seen only in males) with poor dose-relationship, and iii) behavioral effects that are inconsistent across dose, age and sex.
Garman RH, Li AA, Kaufmann W, Auer RN and Bolon B (2016) Recommended methods for brain processing and quantitative analysis in rodent developmental neurotoxicity studies. Toxicol Pathol 44(1), pp 14-42

Developmental immunotoxicity:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A (Text Table 39-42 in study report):
Immunophenotyping analyses by flow cytometry on dissociated spleen from Cohort 1A rats were successfully performed using an immunophenotyping panel to assess the impact of the test item administration on the different spleen-derived lymphocyte subpopulations. No statistically significant differences from controls were found in females or males at any dose level. Under the experimental conditions used in this study, no significant differences were found in terms of relative count (%) or absolute count (cells/mg of spleen) in animals from test item-treated groups when compared to controls. No test item treatment-related impact was observed for the evaluated spleen-derived lymphocyte subpopulations.

Cohort 3 (Text Table 67 and 68 in study report):
In Cohort 3 an ELISA was performed on rat serum samples to determine anti-KLH IgM concentrations after injection of KLH via the intravenous route. The mean anti-KLH IgM serum level in controls was more than 10-fold higher on PND 61 compared to PND 56, showing that there was a humoral response inducted by the KLH immunization as expected.
On PND 61, a slight tendency towards lower PND 61/PND 56 ratio was noted in test item male groups when compared to the control male group. However, there was no clear dose-relationship, this tendency was not observed in female groups, and there was no statistical significance vs. controls at any dose level, demonstrating that there was no significant impact of the test item on the primary humoral response.

Pathology discussion:
There was no test item-related mortality.
Brown pigment of low magnitude (minimal severity) in macrophages was noted in the spleen from males treated at = 40 mg/kg bw/day and females treated at = 150 mg/kg bw/day in P generation. It was also recorded in one control female. This change was not test item-related in any other cohorts. This pigment was suggestive of hemosiderin but no definitive conclusion could be drawn since there were no difference with Perls staining performed in the spleen from one control and on high-dose male. In view of the low severity and nature of this change, it was considered to be non-adverse.

Several findings were seen in the kidneys from cohort 1A males treated at 450 mg/kg bw/day, characterized by vacuolation of tubules, increased severity/incidence of tubular basophilia, pelvis dilatation, and degeneration/necrosis of tubules (this latter lesion was noted in only one high-dose male). These lesions were considered to be non-adverse in view of their morphology, their limited distribution (only a few grouped tubules with vacuolation), their low magnitude and incidence, and since the affected animals had no noteworthy clinical signs.

At the quantitative evaluation of the ovarian corpora lutea or primordial follicles in P generation and 1A cohort, no statistically significant differences could be evidenced.
There were no other alterations in the reproductive organs from females from both generations (ovaries, uterus, vagina), in agreement with the evaluation of the corresponding vaginal smears performed the day of necropsy.

At neurohistopathology of cohort 2B (PND 22), there were no test item-related changes.

At neurohistopathology of cohort 2A (PND 75-90), there were no differences between controls and high-dose groups for the measurements performed in females while there were lower cortex, hippocampus and cerebellum measurements in males treated at = 40 mg/kg bw/day when compared to controls (up to -12%; p<0.01 or 0.05) although poorly dose-related.

Based on the integrative weight-of-evidence approach of Garman et al (2016), ambiguous evidence of developmental neurotoxicity in the central nervous system was concluded because of the following reasons:
1/ These statistically significant differences in linear cortex, hippocampus and cerebellum measurements in males treated at = 40 mg/kg bw/day appeared to be inconsistent: similar difference was not seen in 2A females, or in the males and females from cohort 2B. In addition the magnitude of the changes was poorly dose-related.
2/ There was an absence of test item treatment-related gross abnormalities (brain size reduction readily visible as a gross lesion), brain weight changes and microscopic alteration in the central nervous system, either in P generation (n=24 per group/sex), 1A (n=20), 2A (n=10) or 2B (n=10) F1 cohorts, either in males or females. No microscopic degenerative abnormalities as dysplasia, hypoplasia, hypo/-dysmyelination or ectopia, or neuronal necrosis, myelinic edema or vacuolation were present.
3/ There was no evidence of test item treatment-related nervous behavioral effects (e.g. in responses in auditory startle test, FOB or motor activity). At in-life examination, no nervous clinical signs were noted, or the findings noted are commonly seen in the rats kept under laboratory conditions.
Even if direct effects on the nervous system cannot be completely ruled out, these decreases in morphometric measurements of juveniles from neuropathology cohort in conjunction with no clinical nervous signs, no gross or microscopic changes were considered to be isolated.

In conclusion, there were lower cortex, hippocampus and cerebellum measurements in males of cohort 2A treated at =40 mg/kg bw/day when compared to controls. The relationship to test item was considered to be “ambiguous” according to the decision tree mentioned in the reference article of Garman et al (2016).

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

450 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

(Text Table 54 and 55 in study report)
No relevant differences were noted between controls and test item-treated groups in the number of pups with findings on PND 1. The findings observed for the test item groups were with single incidences and with no dose relationship. Moreover, they are common findings in rat pups of this species maintained under similare experimental conditions to those of this study. They were therefore not ascribed to the test item treatment.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

(Text Table 51-53 in study report)
When compared with controls and reference control data, there were no test item-treated effects on F1 generation offspring mortality (no dose-relationship and no statistical significance). The higher number of pups that died at 150 mg/kg bw/day was due particularly to one litter with eight dead or cannibalized pups by PND 2. At necropsy of found dead pups, absence of milk and autolysis were noted in a few pups or litters from all groups with no dose-relationship. This was not ascribed to the test item treatment.
There were no effects on viability on PND 4 at any dose level.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item treatment-related effects on F2 pup mean body weight or body weight change at the beginning of lactation at any dose level.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

(Text Table 57 in study report)
All findings recorded in euthanized as scheduled F2 pups are commonly observed in this species/strain of animals at this age and were of low incidence. They were therefore not considered to be test item-related.

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

450 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Chemical Analysis of the Dose Formulations

The dose formulations prepared for the P-premating, P-mating, P-gestation, P-lactation, F1-postweaning, F1-premating, F1-gestation and F1-lactation periods were all found to be within the acceptance criteria (measured concentrations at -6.7 to +7.5% of the theoretical concentrations; ± 15% required). No test item was found in the control dose formulations for the P-mating, P-gestation, P-lactation, F1-premating, and F1-lactation periods. A small quantity of test item  was found in the control dose formulation for the P-premating, F1-postweaning, and F1-gestation periods, but it was below the limit of quantification and considered as negligible.

Conclusions:

The test item, Diethylaminopropylamine (as pH-neutralized dose formulations; batch No. I133306836), was administered daily by oral gavage at dose levels of 0, 40, 150, or 450 mg/kg/day to sexually-mature male and female rats (parental (P) generation) starting 2 weeks before mating and continuously through mating, gestation and weaning of their pups (F1). At weaning, the F1 generation was also exposed to the same dose levels of the test item and was assigned to Cohorts of animals for reproductive/developmental toxicity, developmental neurotoxicity or developmental immunotoxicity testing.
The high dose level was selected based on a prematurely euthanized female at 500 mg/kg/day during lactation (severe clinical signs and test item treatment related macroscopic and microscopic findings) in a previous OECD 421 study and because the OECD 443 study duration is longer than the OECD 421 study.
a) Systemic toxicity evaluation:
The No Observed Adverse Effect Level (NOAEL) for systemic toxicity (excluding
reproductive and developmental toxicity endpoints) was considered to be 450 mg/kg/day based on the absence of adverse findings in both sexes.
The No Observed Effect Level (NOEL) for systemic toxicity (excluding reproductive and developmental toxicity endpoints) was considered to be lower than 40 mg/kg/day based on test item related effects at histopathology in the spleen from 40 mg/kg/day in P males.
b) Reproductive/developmental toxicity testing:
• in P generation animals, there were no adverse effects on estrus cycles, mating (including precoital time), fertility, delivery, or on F1 pups during lactation (survival, clinical signs, development).
• in Cohort 1A and/or 1B animals, there were no adverse effects on sexual maturation, estrus cycles, mating (including precoital time), fertility, delivery, or on F2 pups up to PND 4 (survival, clinical signs, development).
Therefore the NOAEL for reproductive/developmental toxicity was considered to be
450 mg/kg/day, and the NOEL was considered to be 40 mg/kg/day because of the trend towards a delayed first estrous after vaginal opening and the delay in vaginal opening at 450 mg/kg/day in F1 females, and of the dose-related delay in balanopreputial separation from 150 mg/kg/day in F1 males potentially related to the test item.
c) Developmental neurotoxicity testing:
• in Cohort 2A and 2B: there was no evidence of adverse developmental neurotoxicity at any dose level. Therefore the NOAEL for developmental neurotoxicity was considered to be 450 mg/kg/day.
d) Developmental immunotoxicity testing:
• in Cohort 1A: there were no test item treatment-related findings on lymphocyte subtyping and no test item-related effects on mean lymphoid organ weights or on histopathology of lymphoid organs,
• in Cohort 3: there was no evidence of adverse immunotoxicity potential at any dose level.
Therefore the NOAEL for developmental immunotoxicity was considered to be 450 mg/kg/day.

Executive summary:

The objective of this study was to provide general information concerning reproductive and developmental effects of the test item, Diethylaminopropylamine (as pH-neutralized dose formulations), that may occur as a result of pre- and post-natal exposure, and to evaluate the systemic toxicity, following daily oral administration (gavage), in pregnant and lactating females and young and adult offspring.

In the assay, sexually-mature male and female rats (parental (P) generation) were exposed to graduated doses of the test item starting 2 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups were selected and assigned to cohorts of animals for:

• reproductive/developmental toxicity testing (cohort 1),


• developmental neurotoxicity testing (cohort 2),


• developmental immunotoxicity testing (cohort 3).

Methods

Dosing

P generation:

Three groups of 24 male and 24 female Sprague-Dawley rats (P generation) received the test item, Diethylaminopropylamine (as pH-neutralized dose formulations; batch No. I133306836), at 40, 150 or 450 mg/kg/day daily for 2 weeks prior to pairing, during pairing, through gestation and lactation until weaning of the F1 pups [PND (post-natal day) 21]. The test item was administered orally (gavage, 5 mL/kg). The high dose level was selected based on a prematurely euthanized female at 500 mg/kg/day during lactation (severe clinical signs  and test item treatment related macroscopic and microscopic findings) in a previous OECD 421 study and because the OECD 443 study duration is longer than the OECD 421 study.

A control group of 24 males and 24 females received the vehicle alone (drinking water treated by reverse osmosis), under the same experimental conditions, and acted as a reference control group.

F1 generation:

The test item was administered orally, by gavage (5 mL/kg), to groups of 20 rats/sex (Cohorts 1A and 1B) or 10 rats/sex (Cohorts 2A and 3) at 0 (drinking water treated by reverse osmosis), 40, 150 or 450 mg/kg/day. There was no direct dosing in Cohort 2B animals (euthanized on PND 22). The dosing schedules were the following:

• Cohort 1A were treated daily from weaning (PND 22) until euthanasia (on PND 91 to 95),


• Cohort 1B were treated daily. Males were treated from weaning (PND 22) for at least 10 weeks before mating, during the mating period (up to 2 weeks) and until after euthanasia of F2 pups (on PND 4). Females were treated from weaning (PND 22) for at least 10 weeks before mating, during the mating period (up to 2 weeks), during gestation and during lactation until PND 4 inclusive, or until euthanasia for females with no delivery,


• Cohort 2A were treated daily from weaning (PND 22) until euthanasia (on PND 76 to 78),


• Cohort 3 were treated daily from weaning (PND 22) until 4 days after KLH injection, when these animals were euthanized (between PND 60 and 64).

Examination of Parental, F1 and/or F2 pups

Clinical signs and mortality were checked daily. Food consumption and/or body weight were recorded at designated intervals.

Estrous cycle stages were determined daily for each P female during the pre-mating period and until mated, and for each Cohort 1B female during the mating period. P generation and
Cohort 1B males and females were paired until mated or until 14 days had elapsed. Females from the P generation and Cohort 1B were allowed to deliver normally and rear their progeny until weaning (P generation) or PND 4 (Cohort 1B). Pregnancy and litter parameters were recorded.

During lactation, the F1 and F2 pups were observed daily for survival and clinical signs. Body weight was measured at designated intervals and the sex-ratio was recorded. In F1 pups, the size of each litter was adjusted on PND 4 to obtain ten pups per litter. Pup physical and/or reflex development was assessed at designated time-points.

Examination of F1 Generation

Clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated intervals. All animals were observed for balanopreputial separation (males) or vaginal opening (females).

Cohort 1A: animals were selected for assessment of general toxicity and effects on their reproductive system. Estrous cycle stages were determined daily for all females after the onset of vaginal patency, until the first cornified smear was recorded (estrus), and for 2 weeks before the end of the dosing period.

Cohort 1B: animals were selected for follow-up assessment of reproductive performance (by mating F1 animals) and to potentially obtain additional histopathology data.

Cohort 2A: animals were selected for neurobehavioral testing (auditory startle test on
PND 23, functional observation battery and motor activity on PND 64-65) followed by neurohistopathology assessment when they were adults.

Cohort 2B: animals were euthanized on PND 22 without any dosing or in vivo tests.

Cohort 3: animals were selected for immunotoxicity testing. On PND 55-59, an intravenous injection of Keyhole Limpet Hemocyanin (KLH) was performed and specific IgM anti-KLH antibodies were measured to evaluate the primary antibody response 5 days after immunization.

Terminal examinations

A macroscopic post-mortem examination was performed on all P and F1 animals (including F1 pups culled on PND 4 and F1 pups not selected on PND 22) and F2 pups. Special attention was paid to the reproductive organs. Organs were weighed wet as soon as possible after dissection. A microscopic examination was performed on all macroscopic lesions, many tissues (complete list) from animals in the control and high-dose groups, and a few tissues from low-and
mid-dose groups.

P Generation and Cohort 1A: 10 animals/sex/group were fasted (food only) overnight prior to blood sampling (for hematology, coagulation and blood biochemistry) and urine collection (for urinalysis) at termination. Sperm cell evaluations were performed on males: motility, morphology, sperm cell head count in epididymal tissues.

Thyroid hormone levels (P Generation, F1 pups and Cohort 1A animals): Thyroid hormone (T4) and Thyroid Stimulating Hormone (TSH) were measured in blood samples collected on PND 4 from F1 culled pups (pool/litter), on PND 22 from 10 F1 pups/sex/group not selected for Cohorts and at termination from 10 males/group and 10 lactating females/group (P and Cohort 1A animals).

Cohort 1A: splenic lymphocyte subpopulation analysis was performed on 10 animals/sex/group.

Cohorts 2A and 2B: neurohistopathology was performed for all high-dose and control animals of each sex following completion of neurobehavioral testing (between PND 76 and 78) for Cohort 2A animals and on PND 22 for Cohort 2B animals. Neurohistopathology was also performed for some measurements in low- and mid-dose Cohort 2A and 2B males.

Results

The test item concentrations in the dose formulations analyzed during the study remained within an acceptable range of variations when compared with the nominal values (± 15% of the nominal concentrations required). No significant amount of test item was detected in the control dose formulations.

P Generation

• Mortality: there were no test item-related unscheduled deaths.


• Clinical signs: in both sexes the test item induced non-adverse hypersalivation, with a poor dose-relationship between 40 and 150 mg/kg/day, but with the highest incidence at 450 mg/kg/day.


• Mean body weight, mean body weight change and mean food consumption: there were no adverse effects.


• Estrous cycle, mating, fertility and delivery data: there were no test item-related effects on mean duration and number of estrous cycles, on mating (including the mean number of days taken to mate) or on fertility.


• Pathology: there were no test item-related mortality, organ weight differences, or gross or microscopic findings, except non-adverse pigment in the spleen in males from 40 mg/kg/day and in females from 150 mg/kg/day. At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and the control groups.


• P generation offspring (pre-weaning F1 pups and non-selected pups on PND 22): there were no test item-treated effects on mortality, survival indexes, clinical observation, body weight, body weight gain, normalized mean anogenital distance on PND1 or macroscopic post-mortem There were no nipples or areolae in male pups examined on PND 12. There were no test item-related organ weight differences (brain, spleen, thymus) on PND 22.

F1 Generation: Cohorts 1A and 1B

• Mortality: there were no test item-related unscheduled deaths.


• Clinical signs: the test item induced non-adverse hypersalivation in both sexes at 450 mg/kg/day, and to a lesser extend at 40 and 150 mg/kg/day (Cohort 1B only). There were no test item-related clinical signs at 40 mg/kg/day in females.


• Body weight, body weight change and food consumption: there were no adverse effects.


• Estrous cycles, first estrous (Cohort 1A): there were no test item-related effects on estrous cycle at the end of the dosing period. However, there was a trend towards a delayed first estrous after vaginal opening in Cohort 1A females dosed at 450 mg/kg/day when compared with controls and historical control data (4.5 2.8 days in controls, no statistical significance). This was considered to be test item-related but non-adverse in the absence of effects on reproductive performance in Cohort 1B. There were no effects on mean time of the first estrous after the onset of vaginal patency at 40 or 150 mg/kg/day.


• Mating, fertility and delivery (Cohort 1B): there were no test item-related effects on mating (including the mean number of days taken to mate), fertility or delivery data.


• Lymphocyte subtyping (Cohort 1A): there were no test item treatment-related effects.


• F1 generation offspring (Cohort 1B): there were no test item-related effects on neonatal mortality, survival index, clinical observations, body weight, body weight gain or macroscopic post-mortem


• Pathology: there were no test item-related mortality, organ weight differences, or gross or microscopic findings in Cohorts 1A and 1B, except the non-adverse findings in the kidneys in Cohort 1A males at 450 mg/kg/day, characterized by vacuolation of tubules, increased severity and incidence of tubular basophilia and pelvis dilatation, and slight degeneration/necrosis of tubules in only one high-dose male. At quantitative evaluation of primordial follicles or corpora lutea in Cohort 1A, there were no differences between the high-dose and the control groups.

F1 Generation: Cohorts 2A

• Mortality: there were no test item-related unscheduled deaths.


• Clinical signs: a test item effect was considered likely for the ptyalism seen in
  3/20 animals at 450 mg/kg/day on one day for each animal, and non-adverse. There were no test item-related clinical signs at 40 or 150 mg/kg/day.


• Body weight, body weight change and food consumption: there were no adverse effects.


• Neurobehavioral testing (auditory startle test, functional observation battery, motor activity): there were no test item-related effects.


• Pathology: there were no test item-related mortality, organ weight differences, or gross or microscopic findings. At neurohistopathology, there were no test item-related changes in females, while lower cortex, hippocampus and cerebellum measurements were noted in males from 40 mg/kg/day when compared to controls. The relationship of these differences to test item administration was considered to be ambiguous.

F1 Generation: Cohort 2B

There were no test item-related organ weight differences, gross or microscopic findings.

At neurohistopathology, there were no test item-related changes.

F1 Generation: Cohort 3

• Mortality: there were no test item-related unscheduled deaths.


• Clinical signs: a test item effect was considered likely for the ptyalism seen in 2/20 animals at 450 mg/kg/day on one day for each animal, and non-adverse. There were no test item-related clinical signs at 40 or 150 mg/kg/day.


• Body weight, body weight change and food consumption: there were no adverse effects.


• Quantification of anti-KLM IgM response: the test item did not significantly impact the primary humoral response in any group.

F1 Generation: all Cohorts

Sexual development: at balanopreputial separation in F1 males, there was a dose-related delay in mean age from 150 mg/kg/day (mean age at 49.3 and 50.0 at 150 and 450 mg/kg/day respectively, vs. 48.0 in controls, p<0.01) in the absence of relevant effects on mean body weight; a test item treatment effects could not be excluded but considered as non-adverse in view of the slight magnitude of the change and of the mean age within or close to the Historical control data, and in the absence of effects on reproductive performance in Cohort 1B. In F1 females, the mean age of vaginal opening was delayed at 450 mg/kg/day (38.1 vs. 34.8 days in controls, p<0.01) in the presence of a change in mean body weight (+7% vs. controls, p<0.01) on the day of occurrence, and was considered to be test item-related. Even if these observations were abnormal, they were considered to be non-adverse as they had no negative consequences  on reproductive performance as seen in Cohort 1B, with especially all high-dose Cohort 1B females having being mated with no delay, and then pregnant.

P and F1 Generation:

• Hematology, coagulation, blood biochemistry and urinalysis: there were no adverse effects on P generation or Cohort 1A animals.


• Thyroid hormones: there were no test item-related effects in P generation, in Cohort 1A animals or in F1 pups not selected on PND 22.


• Sperm analysis: there were no effects in P generation or Cohort 1A animals.

Conclusion

The test item, Diethylaminopropylamine (as pH-neutralized dose formulations; batch No. I133306836), was administered daily by oral gavage at dose levels of 0, 40, 150, or 450 mg/kg/day to sexually-mature male and female rats (parental (P) generation) starting 2 weeks before mating and continuously through mating, gestation and weaning of their pups (F1). At weaning, the F1 generation was also exposed to the same dose levels of the test item and was assigned to Cohorts of animals for reproductive/developmental toxicity, developmental neurotoxicity or developmental immunotoxicity testing.

The high dose level was selected based on a prematurely euthanized female at 500 mg/kg/day during lactation (severe clinical signs and test item treatment related macroscopic and microscopic findings) in a previous OECD 421 study and because the OECD 443 study duration is longer than the OECD 421 study.

1. a) Systemic toxicity evaluation:

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity (excluding

reproductive and developmental toxicity endpoints) was considered to be 450 mg/kg/day based on the absence of adverse findings in both sexes.

The No Observed Effect Level (NOEL) for systemic toxicity (excluding reproductive and developmental toxicity endpoints) was considered to be lower than 40 mg/kg/day based on test item related effects at histopathology in the spleen from 40 mg/kg/day in P males.

1. b) Reproductive/developmental toxicity testing:

• in P generation animals, there were no adverse effects on estrus cycles, mating (including precoital time), fertility, delivery, or on F1 pups during lactation (survival, clinical signs, development).


• in Cohort 1A and/or 1B animals, there were no adverse effects on sexual maturation, estrus cycles, mating (including precoital time), fertility, delivery, or on F2 pups up to PND 4 (survival, clinical signs, development).

Therefore the NOAEL for reproductive/developmental toxicity was considered to be
450 mg/kg/day, and the NOEL was considered to be 40 mg/kg/day because of the trend towards a delayed first estrous after vaginal opening and the delay in vaginal opening at 450 mg/kg/day in F1 females, and of the dose-related delay in balanopreputial separation from 150 mg/kg/day in F1 males potentially related to the test item.

1. c) Developmental neurotoxicity testing:

• in Cohort 2A and 2B: there was no evidence of adverse developmental neurotoxicity at any dose level. Therefore the NOAEL for developmental neurotoxicity was considered to be 450 mg/kg/day.

1. d) Developmental immunotoxicity testing:

• in Cohort 1A: there were no test item treatment-related findings on lymphocyte subtyping and no test item-related effects on mean lymphoid organ weights or on histopathology of lymphoid organs,


• in Cohort 3: there was no evidence of adverse immunotoxicity potential at any dose level.

Therefore the NOAEL for developmental immunotoxicity was considered to be 450 mg/kg/day.

 

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

450 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and accoroding to guideline443

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Study duration:

subchronic

Effects on developmental toxicity

Description of key information

There are two main developmental studies performed in rats and rabbits according to GLP and OECD 414 guideline, two preliminary studies in rats and rabbits and a maximum tolerated dose study in rabbits.

1-OECD 414 main developemental study in rats (CitoxLab, 2016)

The potential toxic effects of Diethylaminopropylamine, on the pregnant female and on embryonic and fetal development was evaluated following daily oral administration (gavage) to pregnant female rats from implantation until the day before scheduled hysterectomy [Day 6 to Day 20 post-coitum (p.c.)inclusive] (Papineau, 2016). This GLP study was carried out according to OECD test guideline No. 414 (22 January 2001). Three groups of 24 time-mated female Sprague-Dawley rats received Diethylaminopropylamine (as pH-neutralized dose formulations), by the oral route (gavage), at dose-levels of 50, 250 or 750 mg/kg/day, once daily from Day 6 until Day 20 p.c.. Another group of 24 time-mated rats received the vehicle only, drinking water treated by reverse osmosis, under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg/day was used. The test item concentrations in the dose formulations were determined. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 21p.c., the females were sacrificed and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities (including cartilage). The stomach of all dams was sampled and preserved in 10% buffered formalin.

The concentrations of the test item in the dose formulations (-1% to +4%) remained within the acceptable range of variations (± 10%) when compared to the nominal concentrations. At study termination on Day 21p.c., there were 24, 21, 23 and 18 females with live fetuses in the groups given 0, 50, 250 or 750 mg/kg/day, respectively. On Day 20p.c., one pregnant female at 750 mg/kg/day was found dead. From 250 mg/kg/day, ptyalism, sneezing, chromorhinorrhea and/or dacryhorrhea were considered to be test item treatment-related but of minor toxicological importance. At 250 and 750 mg/kg/day, 4/24 and 19/23 dams, respectively, showed clinical signs (round back, emaciated appearance, piloerection, loud breathing and/or reddish vaginal discharge) which were considered to be test item treatment-related and adverse at 750 mg/kg/day. An adverse body weight loss was observed in females given 750 mg/kg/day at the beginning of the treatment period (-16 g on Day 6- 9 p.c., p<0.001), which resulted in a lower body weight gain until the end of the treatment period (+97 gvs.+149 g in controls, during the period of Days 6 to 21 p.c. p<0.001).Body weight gain was slightly lower in females given 250 mg/kg/day (+5 gvs.+15 g in controls, on Days 6 to 9 p.c., p<0.01), but returned towards control values from Day 9p.c..Therefore, toxicologically significant effects were observed on terminal body weight in females given 750 mg/kg/day (-12%versuscontrols on Day 21p.c., p<0.001). Body weight changes correlated with lower mean food consumption (21%vs. controls between Days 6 and 9p.c. at 250 mg/kg/day, and -9 to -63%vs.controls between Days 6 and 18p.c.at 750 mg/kg/day). These findings were considered to be adverse at 750 mg/kg/day. Spleen enlargement, raised focus on the spleen and colored focus on the stomach wall were observed only in females given the high dose-level. Therefore, a relationship to the test item treatment cannot be excluded. At 750 mg/kg/day, statistically significantly lower mean net weight change (+8.5 g vs. +42.0 in controls, p<0.001) and lower carcass weight (-10% vs. controls, p<0.001) were noted. At 250 and 750 mg/kg/day, mean gravid uterus weights were 9% and 18% lower than controls, respectively. This correlated with a lower mean number of live fetuses at both dose levels (11.4 and 11.2 vs.13.2 in Historical Control Data (HCD), respectively) and with a lower mean fetal body weight at 750 mg/kg/day. At 250 and 750 mg/kg/day, total resorption in one female at each dose level and higher mean post-implantation loss (15.5 and 20.0vs.3.8 in HCD, respectively) were also observed. At 750 mg/kg/day, higher mean number of early resorptions (2.1vs.0.6 in controls, p<0.001) was noted, as along with lower mean net weight change and lower carcass weight.

All these findings were considered to represent embryo toxicity from 250 mg/kg/day.

At 750 mg/kg/day and when compared with controls, the lower mean fetal body weights were statistically significant (p<0.05). This finding, which was considered to be of minor toxicological importance, was not of a similar amplitude in both sexes as differences were mainly due to the lower body weight of male fetuses (p<0.05). The sex ratio was unaffected by the test item treatment at any dose-level. There were no test item treatment-related variations at external/oral cavity examination of the fetuses. At 750 mg/kg/day, two fetuses, from two different litters had anasarca, cleft palate, short trunk, short tail and/or anal atresia together with acaudia, for which a relationship to the test item treatment could not be ruled out. There were no test item-related variations and malformations at soft tissue examination. At 750 mg/kg/day and when compared with controls, there were higher litter and fetal incidences of fetuses with incomplete ossification of cervical vertebra(e) centrum). From 250 mg/kg/day and taking into account the percentage of litter with fetuses of abnormalities of the axial skeleton, a test-item treatment related effect cannot be excluded.

On the basis of the results obtained in this study:

. the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 50 mg/kg/day (based on clinical signs, food consumption, body weight, net body weight change and/or gravid uterus weight from 250 mg/kg/day and mortality at 750 mg/kg/day),

. the NOAEL for embryo-fetal toxicity and teratogenicity was considered to be 50 and 250 mg/kg/day, respectively, (in a context of marked (250 mg/kg/day) to severe (750 mg/kg/day) maternal toxicity).

 

Preliminary Range finding study in rats (CitoxLab, 2015)

The potential toxic effects of diethylaminopropylamine, on the pregnant female and on embryonic and fetal development was evaluated in a range-finding study following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (Day 6 to Day 20post-coituminclusive), in order to select dose-levels for a further main study (Papineau, 2015).

Three groups of eight time-mated female Sprague-Dawley rats were given the test item at 100, 300 or 1000 mg/kg/day by oral gavage once daily from Days 6 to20 p.c.. Another group of eight time-mated rats received the vehicle, drinking water treated by reverse osmosis, under the same experimental conditions and acted as a control. All animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded regularly. After sacrifice, a macroscopic post-mortem examination was performed on the females on Day 21p.c.. Hysterectomy was performed and the numbers and distribution of corpora lutea, implantation sites, early and late resorptions, uterine scars, and live and dead fetuses were recorded. The fetuses were sexed, weighed and examined for oral cavity and external abnormalities.

On Day 21p.c., all control females and females given 100 or 300 mg/kg/day and 6/8 females given 1000 mg/kg/day were pregnant with live concepti.

Unscheduled mortality occurred in 1/8 females at 1000 mg/kg/day. At final sacrifice, transient ptyalism was observed in all females given 1000 mg/kg/day associated with piloerection in 1/7 females. Reflux at dosing was noted on rare occasions in 2/8 and 1/7 females given the test item at 300 or 1000 mg/kg/day, respectively. A body weight loss was observed in females given 1000 mg/kg/day at the beginning of the treatment period (-13 g) whereas slight lower body weight gain was recorded in females given 300 mg/kg/day (+9 g vs. +13 g in controls); this was followed with lower body weight gain until Day 15p.c.at 1000 mg/kg/day leading to a statistically significantly lower body weight gain throughout the whole study period (+105 g vs. +140 g in controls). Body weight gain of females given 300 mg/kg/day returned to control values from Day 9p.c.. These differences were accompanied with reduced food consumption (-11% at 300 mg/kg/day over the first 3 days of the treatment period and -13 to -52% at 1000 mg/kg/day between Days 6 and 15p.c., compared to controls). There were no effects of treatment with the test item at 100 mg/kg/day. At necropsy on terminal sacrifice, there were no test item-related gross findings. When compared with the control group, dams given 1000 mg/kg/day had a lower gravid uterus weight observed in high-dose dams (-9% compared to controls). Lower net weight change (+16 g vs. +41 g in controls) was considered to be a consequence of the lower body weight gain in these females. Gestation parameters were not impacted by the test item treatment at any dose-levels. There was no effect on the fetal body weight and on the percentage of male fetuses (sex ratio). There were no treatment-related fetal external malformations or variations in any groups.

 

3- Main developmental reproduction study in rabbits (Charles River, 2021)

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.

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.

Preliminary developmental study in rabbits (Charles River, 2020)

The objective of this preliminary study was to evaluate the potential toxic effects of the test item, Diethylaminopropylamine (as pH-neutralized dose formulations), on the pregnant female and on embryonic and fetal developments, following daily oral administration (gavage) to pregnant female rabbits from implantation to the day before scheduled hysterectomy [Day 6 to Day 28 post-coitum (p.c.) inclusive], in order to select dose levels for a further main study (study plan No. 47474 RSL). The dose-levels of 100, 300 and 750 mg/kg/day were selected based on the maximum tolerated study in rabbits.

At hysterectomy, on Day 29 p.c., all surviving females were pregnant with live fetuses at termination.

At 300 and 750 mg/kg/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, coldness 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.

No mortality was observed at 100 mg/kg/day.

No test item-related clinical signs were reported in the 4/8 surviving females at 300 mg/kg/day or in females at 100 mg/kg/day.

At 300 mg/kg/day, severe reduction in food intake (down to -82% vs. controls) was noted between Days 6 and 15 p.c. (p<0.01 or p<0.001) followed with a low food consumption in surviving females until the end of the treatment period (down to -54%). This correlated with body weight loss (-193 g in cumulative; p<0.05 or p<0.001) and low body weight gain the same intervals, leading to a low body weight gain over the treatment period (+70 g vs. +405 g in controls; p<0.01). Body weight was slightly affected from Day 12 p.c. (down to -10% vs. controls).

At 100 mg/kg/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/day in the stomach, at ≥ 300 mg/kg/day in the kidneys and at 750 mg/kg/day in the intestines and liver.

At 300 and 100 mg/kg/day, low net body weight changes (-393.3 and -269.2 g vs. -187.2 g in controls, respectively) was noted. At 300 mg/kg/day, low gravid uterus weight (-22% vs. controls) and fetal weight (-11% vs. controls) were observed.

There were no effects on hysterectomy data at 100 or 300 mg/kg/day and no effects on fetal body weight at 100 mg/kg/day.

There were no external variations or malformations at fetal examination in the 100 and 300 mg/kg/day groups.

Maximum tolerated dose study in rabbits (Charles River, 2020)

The objective of this preliminary study was to evaluate the potential toxic effects of the test item, Diethylaminopropylamine (as pH-neutralized dose formulations), following daily oral administration (gavage) in the non-pregnant female rabbit, and to establish a Maximum Tolerated Dose (MTD), in order to assist the selection of dose levels for a further preliminary embryo-fetal development study to be performed in this species (Report No. 47473 RSL).

Three non-pregnant female New Zealand White rabbits received the test item, Diethylaminopropylamine (batch No. I133306836), at successive doses of 100, 300 and 1000 mg/kg/day in the vehicle (drinking water treated by reverse osmosis) by oral route (gavage) once daily for 7, 7 and 4 days, respectively (under dose volumes of 3, 3 and 5 mL/kg, respectively).

Assessment of toxicity was based on mortality, clinical observation, body weight, food consumption and gross pathology (thoracic and abdominal organs).

After 4 days at 1000 mg/kg/day, all females were prematurely euthanized for absence of food consumption from the start of the dosing period. One out of three females showed coldness to the touch, decreased activity and ventral recumbency prior to the euthanasia on Day 4 as well as body weight loss (-5 to -8%). At necropsy, findings were observed in the cecum (distended with feces, brown liquid content), stomach (brown liquid content), liver (enlarged and firm, tan discolored), gall bladder (dilated) and kidneys (enlarged and tan discolored).

At 300 mg/kg/day, test-item related effects were limited to a transient body weight loss and reduction of food consumption. These effects did not impacted the body weight and the food intake after 7 days of dosing.

At 100 mg/kg/day, there were no test item-related effects.

Under the experimental conditions of the study, the dose level of 1000 mg/kg/day given orally (gavage) was considered to have exceeded the MTD in New Zealand White female rabbits (premature euthanasia after 4 days of dosing, clinical signs of poor health in 1/3 females, body weight loss, no food consumption).

There were no adverse effects up to 300 mg/kg/day based on were limited to a transient body weight loss and reduction of food consumption at 300 mg/kg/day and no effet at 100 mg/kg/day .

Therefore and under the experimental condition of this study, the MTD was considered to be within the [300; 1000] mg/kg/day interval.

 

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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)

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

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.)

Dose / conc.:

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

Remarks:

Low-dose group

Dose / conc.:

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

Remarks:

Mid-dose group

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).

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".

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.

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

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

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

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

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, le Genest-Saint-Isle, France.
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: 308 g (range: 256 g to 373 g)
- Fasting period before study: no
- Housing: individually housed in polycarbonate cages
- Diet: SSNIFF R/M-H pelleted maintenance diet, ad libitum
- Water: tap water (filtered with a 0.22 µm filter), ad libitum
- Acclimation period: 4 or 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed and mixed with the required quantity of vehicle
The pH of the dose formulation was adjusted to 8.0 (± 0.5) with a solution of Hydrochloric acid [30 35%, TraceSELECT® Ultra, for ultratrace analysis (96208 Fluka)]. The volume of HCl added was recorded for each concentration
The final pH was checked and recorded by using a pH-meter

VEHICLE
- Concentration in vehicle: 10, 50 and 150 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas Chromatography with FID detection (GC-FID)
Once on Days 6, 13 and 19 p.c.

Details on mating procedure:

The females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as Day 0 p.c..

Duration of treatment / exposure:

From Day 6 to Day 20 p.c., inclusive.

Frequency of treatment:

Daily

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-levels were selected based on a preliminary study for effects on embryo-fetal development by the oral route in rats (Papineau, 2016b; CiToxLAB France/Study No. 42761 RSR).

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS:
From arrival, each animal was observed once a day as part of the routine examinations.
From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT:
The body weight of each female was recorded on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c. (see § Study plan adherence).

FOOD CONSUMPTION:
The quantity of food consumed by each female was recorded for the following intervals:
Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c. (see § Study plan adherence).

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day 21
- Organs examined: principal thoracic and abdominal organs.
- The weight of the gravid uterus was recorded for each pregnant female (with at least one live fetus) at hysterectomy

Ovaries and uterine content:

The ovaries and uterus of the females were examined to determine:
. number of corpora lutea,
. number and distribution of dead and live fetuses,
. number and distribution of early and late resorptions,
. number and distribution of uterine scars,
. number and distribution of implantation sites.

The following classification was used to record:
. uterine scar: uterine implantation without implant,
. early resorption: evidence of implant without recognizable embryo,
. late resorption: dead embryo or fetus with external degenerative changes,
. dead fetus: non-live fetus with no external degenerative changes.

For apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique (Salewski, 1964).

A gross evaluation of placentas was also undertaken.

Fetal examinations:

- External examinations: Yes: half per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No

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 Fischer exact probability test (proportions).

Indices:

- For each pregnant female:
. body weight change for different intervals
. net body weight (presented as carcass weight)
. net body weight change
- For each litter:
. total number of resorptions
. total number of dead fetuses
. % of dead fetuses per litter
. total number of live fetuses
. % of live fetuses per litter
. % of pre-implantation loss
. % of post-implantation loss
. average fetal body weight
- For each group:
. mean and standard deviation: body weight, body weight change, food consumption, number of corpora lutea, number of implantation sites, number of fetuses and fetal body weight with litter as the experimental unit,
. number of animals affected: clinical signs, necropsy findings, fetal observations (number of fetuses, number of litters),
. % of animals affected: fetal observations (% of fetuses, % of litters),
. mean % per litter of fetuses affected,
. % of pre-implantation loss relative to the number of corpora lutea (mean of pre-implantation loss per litter),
. % of live fetuses and % of dead fetuses (relative to total number of fetuses),
. % of male fetuses (relative to total number of live fetuses),
. mean, standard deviation and % relative to the number of implantation sites: resorptions plus uterine scars, uterine scars, early resorptions, late resorptions,
. % of post-implantation loss relative to the number of implantation sites (mean of post-implantation loss per litter), % of dams affected.

Historical control data:

Available

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ptyalism was observed in one female given 250 mg/kg/day and in most of the surviving females given 750 mg/kg/day from Day 9 p.c. up to Day 20 p.c.. Sneezing, chromorhinorrhea and/or dacryhorrhea were transiently noted on some occasions at all dose-levels. Reddish vaginal discharge observed at 50 mg/kg/day was isolated and a relationship to the test item treatment was considered to be unlikely. These signs were considered to be of minor toxicological significance.
From 250 mg/kg/day, test item-related clinical signs were mainly observed from Day 11 or 12 p.c and consisted of round back, emaciated appearance, piloerection, loud breathing and/or reddish vaginal discharge. These clinical signs had dose-related increased incidence and were considered to be adverse.
Short/broken teeth, area of hair loss, and cutaneous lesions on different parts of the body were observed in 1 of 24 females at each of the following dose-levels: 0, 50 or 250 mg/kg/day. These findings were considered to be unrelated to the test item treatment, as they were reported on a limited number of occasions and/or are findings commonly observed in rats of this strain and age.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no unscheduled deaths in control, 50 and 250 mg/kg/day groups.
At 750 mg/kg/day, female E28593 was found dead on Day 20 p.c.. Prior to death, this female showed piloerection, round back, emaciated appearance, dyspnea and chromorhinorrhea. A body weight loss of 39 g was recorded between Days 6 and 18 p.c. together with reduced food consumption. At macroscopic post-mortem examination, no abnormalities were observed. This female was pregnant (1 early resorption and 9 dead fetuses for 13 corpora lutea).
This unscheduled death was considered to be test item treatment related.

- Net weight change: Table 4
At 750 mg/kg/day, when compared with controls, females had a low mean gravid uterus weight ( 18%, p<0.05) together with a low net weight change (+9 g vs. +42 g in controls, p<0.001). This was associated with a lower mean carcass weight (-10%, p<0.001) and correlated with the lower mean female body weight gain, number of fetuses and fetal body weight recorded at this dose-level.
These observations confirmed that there was a toxicologically significant effect of the test item on the maternal body weight and the gravid uterus weight.
At 250 mg/kg/day, when compared with controls, females had a slightly lower mean gravid uterus weight ( 9%). This was associated with the lower mean number of fetuses recorded at this dose-level.
There were no test item-related effects at 50 mg/kg/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Body weight and body weight change: Table 2
At 750 mg/kg/day, when compared with controls, mean body weight loss was noted over the first 3 days of treatment (-16 g vs. +15 g, p<0.001), followed with a lower mean body weight gain until the end of the treatment period. (+11 to +44 g vs. +20 to +51 g) resulting in a lower body weight gain over the whole dosing period (+97g vs. +149 g in controls, p<0.001).
Effects on mean body weight change at 750 mg/kg/day contributed to a lower terminal mean body weight gain (-12% vs. controls, p<0.001). These effects were attributed to the test item treatment and correlated with reduced mean food consumption between Days 6 and 18 p.c. (see below). They were therefore considered to be adverse.
At 250 mg/kg/day, when compared with controls, low mean body weight gain was noted over the first 3 days of treatment (+5 g vs. +15 g, p<0.01) with a return to control values thereafter.
At 50 mg/kg/day, there were no effects on mean body weight or mean body weight change.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See Table 3.
At 750 mg/kg/day, when compared to controls, a low mean food consumption (down to -63% on Days 6 to 9 p.c., p<0.001) was recorded between Days 6 and 15 p.c..
At 250 mg/kg/day, a comparable trend was observed over the first three days of the study, but to a lesser extent (-21% vs. controls during the period of Days 6 to 9 p.c., p<0.001; with a return to control values thereafter).
These differences correlated with the lower mean body weight gain and/or the mean body weight loss also observed at 250 or 750 mg/kg/day, respectively. Therefore, they were considered to be adverse at 750 mg/kg/day.
At 50 mg/kg/day there were no effects of treatment with the test item.

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

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Spleen enlargement, raised focus on the spleen and colored focus on the stomach wall were observed only in females given the high dose-level. Therefore a relationship to the test item treatment was not excluded.
All other macroscopic observations (i.e. granular surface of the spleen, absence of kidney, ureter, ovary and uterine horn, serous cyst on ovary, dilation of uterine horn, uterine horn with colored or serous content, enlarged cervix, colored content in cervix and/or transverse vaginal septum) were not attributed to the test item treatment as they were reported both in control and test item-treated animals at comparable incidences and/or are findings commonly observed in rats of this strain and age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

See Table 5.
At 250 and 750 mg/kg/day, there were higher mean post-implantation loss (15.5% and 20.0% vs. 4.6%, respectively, with p<0.05 at 750 mg/kg/day) and lower mean number of live fetuses (11.4 and 11.2 vs. 13.5, respectively). In addition all values were outside the limits of the Historical Control Data.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

See Table 5.
At 250 and 750 mg/kg/day, one female in each group had total resorption.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

See Table 5.
A higher mean number of early resorptions were recorded at 750 mg/kg/day (2.1 vs. 0.6 in controls, p<0.001).

Dead fetuses:

effects observed, treatment-related

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Dose descriptor:

NOAEC

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

early or late resorptions

food consumption and compound intake

mortality

organ weights and organ / body weight ratios

pre and post implantation loss

Abnormalities:

effects observed, treatment-related

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Table 6
In the 750 mg/kg/day group, when compared with controls, mean fetal body weight was lower ( 5%, p<0.05). This finding was attributed to the test item treatment since mean value was below the lower limit of the HCD and correlated with the lower mean gravid uterus weight recorded at this dose-level. As this difference was minimal, this was considered to be of minor toxicological importance.
In the 50 and 250 mg/kg/day groups there were no effects on mean fetal body weight.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio was unaffected by the test item treatment at any dose-level.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

. Variations: Table 7
There was no test-item treatment related increase of the frequencies of external variations.
Malrotated paw was observed at comparable incidence at 750 mg/kg/day and at the upper limit of the historical control data, and malrotated hindlimb was also observed in one control fetus. Therefore, these findings were considered to be unrelated to the test item treatment.
. Malformations: Table 8
There were no fetal external malformations in the 0, 50 and 250 mg/kg/day groups.
In the 750 mg/kg/day group, two fetuses from two different litters had external malformations:
. fetus E28579-07 (fetal body weight: 4.13 g): anasarca, cleft palate, short trunk (absent cervical vertebra(e) at skeletal examination) and short tail,
. fetus E28588-10 (fetal body weight: 3.58 g): anal atresia and acaudia.
These findings affecting the trunk/anus and/or tail were observed in two multi-malformed fetuses from two different litters with body weights significantly lower than the other fetuses from the same litter and sex. If a test item treatment related effect cannot be ruled out, it is most probably secondary to the fetal toxicity in a context of severe maternal toxicity.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

. Cartilage: Table 11
In the 750 mg/kg/day group and when compared with controls, there were higher litter and fetal incidence of fetuses with cartilage of cervical vertebra(e) present. This finding associated with a delayed centrum ossification as described in the § Variations (Cervical vertebra(e): incomplete ossification of centrum) was considered to be test item treatment-related but non-adverse (the cartilage/bone structure still present).
. Variations: Table 12
Up to 250 mg/kg/day, there were no test-item treatment related skeletal variations.
In the 750 mg/kg/day group and when compared with controls, there were higher litter and fetal incidences of fetuses with incomplete ossification of cervical vertebra(e) centrum. This finding associated with higher litter and fetal incidence of cervical vertebra(e) with cartilage (see § Cartilage) was therefore considered to be test item treatment-related but non-adverse (ossification delay).
When compared with controls and HCD, the fetal and litter incidences of caudal vertebra(e) with unossified centrum (1.8% and 5.6%, respectively) were higher than control (0% and 0%, respectively) and HCD (2.0% and 7.1%, respectively). Therefore finding was considered to be test-item treatment related but non-adverse.
. Malformations: Table 13
In the 750 mg/kg/day, two litters had a malformed fetus: E28579-07 with absent cervical vertebra, fused sternebrae, absent rib(s) and fused metacarpal bones and E28574-13 with absent thoracic vertebra(e) and rib(s).
In the 250 mg/kg/day group, three litters had one malformed fetus: E28559-04 with absent thoracic vertebra and absent rib(s), E2849-09 with absent lumbar vertebra and E28550-11 with supernumerary lumbar vertebra.
In the 50 mg/kg/day group, one litter had 5 malformed fetuses: E28544-05, -09, -11, -13 and -15 with absent lumbar vertebra and E28544-05 and-13 with absent rib.
In the control group, one litter had one malformed fetus: E28503-03 with absent lumbar vertebra(e).
Taking into account the number and percentage of fetus and litter affected in all groups (including controls), a test-item treatment related effect cannot be demonstrated.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

. Variations: Table 9
In the 750 mg/kg/day group, the incidences of absent innominate artery (3.9% and 16.7%, fetal and litter incidences respectively) were higher than the upper limit of the HCD (0.8% and 5.0%, respectively). Taking into account the amplitude of the incidences (litter and fetal) of this non-adverse finding at 750 mg/kg/day, a test item treatment related effect cannot be excluded.
When compared with controls and HCD, there were tendencies toward increased fetal and litter incidences of dilated renal pelvis and thymus with reddish foci at 750 mg/kg/day and dilated ureter in all test item treated-groups. However as these variations were not dose-related, within and/or closed to HCD values, they were considered not to be test-item treatment related.
There were no test-item treatment related increase of the frequencies of soft tissue variations in the 0, 50 and 250 mg/kg/day groups.
Other soft tissue variations (i.e. kidney with paleness and short innominate artery) observed at comparable incidence in control and test item-treated groups were considered to be unrelated to the test item treatment.
. Malformations: Table 10
There was no test item treatment-related increase in the frequencies of soft tissue malformations.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

Key result

Dose descriptor:

NOAEL

Remarks:

embryotoxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

changes in litter size and weights

other: post-implantation losses

Abnormalities:

effects observed, treatment-related

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Table 1: Pregnancy status

Dose-level(mg/kg/day)	0	50	250	750
Number of females	24	24	24	24
Non-pregnant females	0	3	0	4
Females with total resorption	0	0	1	1
Females with live fetuses at term	24	21	23	18

Table 2: Mean body weight and body weight changes (g)

Dose-level (mg/kg/day)	0	50	250	750
Body weight (g)
Day 6p.c.	310	310	309	307
	-	(0)	(0)	(-1)
Day 9p.c.	326	323	315	292^#
	-	(-1)	(-3)	(-10)
Day 12p.c.	346	346	333	303^#
	-	(0)	(-4)	(-12)
Day 15p.c.	365	366	353	319^#
	-	(0)	(-3)	(-13)
Day 18p.c.	408	410	394	355^#
	-	(0)	(-3)	(-13)
Day 21p.c.	459	459	442	404^#
	-	(0)	(-4)	(-12)
Body weight change (g)
Days 6 - 9p.c.	+15	+13	+5**	-16^#
Days 9 - 12p.c.	+20	+23	+19	+11**
Days 12 - 15p.c.	+20	+20	+20	+16
Days 15 - 18p.c.	+43	+44	+40	+36
Days 18 - 21p.c.	+51	+49	+48	+44
Days 6 - 21p.c.	+149	+149	+132	+97^#
	-	(0)	(-11)	(-35)

Statistical significance; **: p<0.01 and^#: p<0.001.

p.c.:post-coitum.

-: not applicable.

( ): in brackets, percentage differencevs.controls.

 

Table 3: Mean food consumption (g/animal/day)

Dose-level (mg/kg/day)	0	50	250	750
. Days 6 - 9p.c.	24	24	19^#	9^#
	-	(0)	(-21)	(-63)
. Days 9 - 12p.c.	27	27	24	17^#
	-	(0)	(-11)	(-37)
. Days 12 - 15p.c.	28	29	27	23^#
	-	(+4)	(-4)	(-18)
. Days 15 - 18p.c.	32 -	33 (+3)	31 (-3)	29 (-9)

Statistical significance; ^#: p<0.001.

p.c.:post-coitum.

-: not applicable.

( ): in brackets, percentage differencevs.controls.

 

Table 4: Mean carcass, net change and gravid uterus weights (a) (g)

Dose-level (mg/kg/day)	0	50	250	750
Gravid uterus weight	107 -	103 (-4)	97 (-9)	88* (-18)
Carcass weight	352 -	356 (+1)	345 (-2)	316^# (-10)
Net weight change from Day 6.p.c.	+42	+46	+36	+9^#

Statistical significance:*: p<0.05 and^#: p<0.001.

( ): in brackets, percentage differencevs.controls.

p.c.:post-coitum.

(a): weights are rounded values.

-: not applicable.

 

Table 5: Hysterectomy data 

Dose-level (mg/kg/day)	0	50	250	750	HCD
Number of pregnant females at hysterectomy	24	21	24	19	194
Number of females with live fetuses at termination	24	21	23	18	185
Number of females with total resorption	0	0	1	1	93
Mean number ofcorpora lutea	15.4	14.8	14.9	15.1	[14.4; 16.6]
Mean number of implantation sites	14.2	13.4	13.2	14.1	[12.9; 14.6]
Mean pre-implantation loss (%)	6.9	8.2	11.8	6.7	[6.5; 12.7]
Mean number of live fetuses	13.5	12.7	11.4	11.2	[12.5; 14.0]
Dead fetuses (%)	0.0	0.0	0.0	0.0	[0.00; 0.10]
Mean number of implantation scars	0.0	0.0	0.5	0.7	/
Mean number of early resorptions	0.6	0.7	0.9	2.1^#	/
Mean number of late resorptions	0.1	0.0	0.3	0.0	/
Mean post-implantation loss (%)	4.6	6.0	15.5*	20.0*	[1.5; 6.2]

Statistically significant *: p<0.05, #: p<0.001.

HCD: Historical Control Data (control data collected from nine studies covering a period ranging from March 2013 to June 2014), [min.; max.].

/: not reported in HCD.

 

Conclusions:

In term of maternal toxicity, the following test-item treatment related findings were recorded:
- Mortality: at 750 mg/kg/day, one found dead pregnant female on Day 20 p.c with previous severe clinical signs, body weight loss and reduced food consumption.
- Clinical signs: from 250 mg/kg/day, round back, emaciated appearance, piloerection, loud breathing and/or reddish vaginal discharge from Day 11 or 12 p.c..
- Body weight and body weight change: at 250 mg/kg/day, low body weight gain over the first 3 days of treatment confirmed at 750 mg/kg/day with body weight loss followed with a lower body weight gain until the end of the treatment period.
- Food consumption: reduced at 250 mg/kg/day (-21% vs. controls during the period of Days 6 to 9 p.c; with a return to control values thereafter) and at 750 mg/kg/day (down to -63% vs. controls on Days 6 and 15 p.c.).
- Net weight change: low gravid uterus weight at 250 mg/kg/day ( 9% vs. controls) associated with a low number of fetuses and at 750 mg/kg/day ( 18% vs. controls) together with a low net weight change (+9 g vs. +42 g in controls, p<0.001) and carcass weight (-10% vs. controls).

In term of upon gestation and/or embryo/fetal development, the following test-item treatment related findings were recorded:
- Hysterectomy data: at 250 and 750 mg/kg/day, total resorptions, high mean post-implantation loss (15.5% and 20.0% vs. 4.6% in controls, respectively), low number of live fetuses (11.4 and 11.2 vs. 13.5 in controls, respectively); high number of early resorptions at 750 mg/kg/day (2.1 vs. 0.6 in controls).
- Fetal examination: when compared with controls and Historical Control Data, dose-related increases in the incidences of skeletal abnormalities from 250 mg/kg/day (and external abnormalities at 750 mg/kg/day), in a context of marked to severe fetal toxicity, involving mainly the axial skeleton.
Overall, in a context of marked (250 mg/kg/day) or severe (750 mg/kg/day) maternal toxicity, there was a series of fetal abnormalities impacting mainly the axial skeleton.

Executive summary:

The potential toxic effects of the test item, Diethylaminopropylamine, on the pregnant female and on embryonic and fetal development was evaluated following daily oral administration (gavage) to pregnant female rats from implantation until the day before scheduled hysterectomy [Day 6 to Day 20 post-coitum (p.c.)inclusive]. This GLP study was carried out according to OECD test guideline No. 414 (22 January 2001). Three groups of 24 time-mated female Sprague-Dawley rats received the test item, Diethylaminopropylamine(as pH-neutralized dose formulations), by the oral route (gavage), at dose-levels of 50, 250 or 750 mg/kg/day, once daily from Day 6 until Day 20 p.c.. Another group of 24 time-mated rats received the vehicle only,drinking water treated by reverse osmosis, under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg/day was used.The test item concentrations in the dose formulations were determined.The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recordedat designated intervals. On Day 21p.c., the females were sacrificed and submitted to a macroscopicpost-mortemexamination. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities (including cartilage). The stomach of all dams was sampled andpreserved in 10% buffered formalin.

The concentrations of the test item in the dose formulations (-1% to +4%)remained within the acceptable range of variations(± 10%)whencompared to the nominal concentrations. At study termination on Day 21p.c.,there were 24, 21, 23 and 18 females with live fetuses in the groups given 0, 50, 250 or 750 mg/kg/day, respectively. On Day 20p.c.,one pregnant female at 750 mg/kg/day was found dead. From 250 mg/kg/day, ptyalism, sneezing,chromorhinorrhea and/or dacryhorrheawere considered to be test item treatment-related but of minor toxicological importance. At 250 and 750 mg/kg/day, 4/24 and 19/23 dams, respectively, showed clinical signs (round back, emaciated appearance, piloerection, loud breathing and/or reddish vaginal discharge) which were considered to be test item treatment-related and adverse at 750 mg/kg/day. An adverse body weight loss was observed in females given 750 mg/kg/day at the beginning of the treatment period (-16 g on Day 6- 9 p.c.,p<0.001), which resulted in a lower body weight gain until the end of the treatment period (+97 g vs.+149 g in controls, during the period of Days 6 to 21 p.c.p<0.001). Body weight gain was slightly lower in females given 250 mg/kg/day (+5 gvs.+15 g in controls, on Days 6 to 9 p.c., p<0.01), but returned towards control values from Day 9p.c..Therefore, toxicologically significant effects were observed on terminal body weight in females given 750 mg/kg/day (-12% versus controls on Day 21p.c.,p<0.001). Body weight changes correlated with lower mean food consumption (21%vs. controls between Days 6 and 9p.c. at 250 mg/kg/day, and -9 to -63%vs.controls between Days 6 and 18p.c.at 750 mg/kg/day). These findings were considered to be adverse at 750 mg/kg/day. Spleen enlargement, raised focus on the spleen and colored focus on the stomach wall were observed only in females given the high dose-level. Therefore, a relationship to the test item treatment cannot be excluded. At 750 mg/kg/day, statistically significantly lower mean net weight change (+8.5 g vs. +42.0 in controls, p<0.001) and lower carcass weight (-10% vs. controls, p<0.001) were noted. At 250 and 750 mg/kg/day, mean gravid uterus weights were 9% and 18% lower than controls, respectively. This correlated with a lower mean number of live fetuses at both dose levels (11.4 and 11.2 vs.13.2 in Historical Control Data (HCD), respectively) and with a lower mean fetal body weight at 750 mg/kg/day. At 250 and 750 mg/kg/day, total resorption in one female at each dose level and higher mean post-implantation loss (15.5 and 20.0vs.3.8 in HCD, respectively) were also observed. At 750 mg/kg/day, higher mean number of early resorptions (2.1 vs. 0.6 in controls, p<0.001) was noted, as along withlower mean net weight change and lower carcass weight.

All these findings were considered to represent embryo toxicity from 250 mg/kg/day.

At 750 mg/kg/day and when compared with controls, the lower mean fetal body weights were statistically significant (p<0.05). This finding, which was considered to be of minor toxicological importance, was not of a similar amplitude in both sexes as differences were mainly due to the lower body weight of male fetuses (p<0.05). The sex ratio was unaffected by the test item treatment at any dose-level. There were no test item treatment-related variations at external/oral cavity examination of the fetuses. At 750 mg/kg/day, two fetuses, from two different litters had anasarca, cleft palate, short trunk, short tail and/or anal atresia together with acaudia, for which a relationship to the test item treatment could not be ruled out. There were no test item-related variations and malformations at soft tissue examination. At 750 mg/kg/day and when compared with controls, there were higher litter and fetal incidences of fetuses with incomplete ossification of cervical vertebra(e) centrum). From 250 mg/kg/day and taking into account the percentage of litter with fetuses of abnormalities of the axial skeleton, a test-item treatment related effect cannot be excluded.

On the basis of the results obtained in this study:

.         the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 50 mg/kg/day (based on clinical signs, food consumption, body weight, net body weight change and/or gravid uterus weight from 250 mg/kg/day and mortality at 750 mg/kg/day),

.         the NOAEL for embryo-fetal toxicity and teratogenicity was considered to be 50 and 250 mg/kg/day, respectively (in a context of marked (250 mg/kg/day) to severe (750 mg/kg/day) maternal toxicity).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification is warranted for reproductive and developmental toxicity according to CLP criteria.