3-aminopropyldiethylamine

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

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.

Test material

Test material information

Constituent 1

Reference substance name:

3-aminopropyldiethylamine

EC Number:

203-236-4

EC Name:

3-aminopropyldiethylamine

Cas Number:

104-78-9

Molecular formula:

C7H18N2

IUPAC Name:

(3-aminopropyl)diethylamine

Test material form:

liquid

Details on test material:

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

Specific details on test material used for the study:

SOURCE 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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day

Remarks:

group 2 low dose

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day

Remarks:

group 3 mid dose

Doses / concentrations 3

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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Effect levels (P1)

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

Target system / organ toxicity (P1)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

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 (F1)

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.

Details on results (F1)

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).

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

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.

Effect levels (F2)

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

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.