2,2'-dimethyl-2,2'-azodipropiononitrile

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 July 2021 - 14 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Study performed according to the specifications described in:
• ECHA decision No. CCH-D-2114516984-40-01/F (https://echa.europa.eu/documents/10162/e837b1d8-09e7-9aaa-6381-38cbe16a8c41, accessed on 09 Jun 2021).

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-dimethyl-2,2'-azodipropiononitrile

EC Number:

201-132-3

EC Name:

2,2'-dimethyl-2,2'-azodipropiononitrile

Cas Number:

78-67-1

Molecular formula:

C8H12N4

IUPAC Name:

2,2'-dimethyl-2,2'-azodipropiononitrile

Test material form:

other: White powder

Details on test material:

Physical description: White powder
- Storage conditions: In the refrigerator set at +5°C
- Expire date: 03 July 2022
- Purity: 99.1%
- Lot/batch #: B300J1117
- Specific handling conditions: Keep in its original container or in polyethylene container with large opening. Strictly limit the quantities of product in the work area to those which are absolutely necessary for the work in hand (~ 10 g). Avoid producing test item dust
- Stability in the vehicle: According to Study No. 48855 VAS (Robin, 2021) (stability testing) for up to 10 days in corn oil.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free (COBS-VAF®)

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: Charles River Laboratories Italia, Calco, Italy
- Age at study initiation: (P) males and females were 7 and 6 wks old respectively.
- Weight at study initiation: (P) Males: 153-261 g; Females: 128-163 g; (F1).
- Fasting period before study: No
- Housing: individually housed, except during mating (males + females) and lactation (females + pups), in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing appropriate bedding. Each cage contained two objects (rat hut and nylabone/cocoon) for environmental enrichment. Cohorts 1A, 1B and 1C animals were group housed. They were housed in polycarbonate cages with stainless steel lids (in 2000P cages: up to 4/sex per cage) containing autoclaved sawdust (Le Comptoir des Sciures, Meyzieu, France).
- Diet: SSNIFF rat/mouse pelleted maintenance diet ad libitum (SSNIFF Spezialdiäten GmbH, Soest, Germany).
- Water: tap water (filtered with a 0.22 um filter) ad libitum
- Acclimation period: 8 days
The batches of diet, sawdust and wood shavings were analyzed by the suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which may be expected to interfere with or prejudice the outcome of the study.

ENVIRONMENTAL CONDITIONS SET TO MANTAIN
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 8 to 15. Filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h
IN-LIFE DATES: From: 29 July 2021 To: 7 Feb 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Done according to Study No. 48855 VAS (Robin, 2021) (stability testing) describing the preparation procedure for a range of concentrations [0.1 mg/mL - 10 mg/mL] covering the lowest and highest used in this study. The frequency of preparation dosing formulations was also based on test item dose formulation stability and vehicle expiry (i.e. for up to 10 days in corn oil after preparation of the formulations).
Dose formulations were stored in the refrigerator set at +5°C and protected from light.
Dose formulations (vehicle and test-item formulations) were stirred at least 30 minutes before administration. The formulations were maintained under continuous magnetic stirring throughout the dosing procedure

VEHICLE
- Justification for use and choice of vehicle: selected in agreement with the Sponsor, based on previous experimental work and/or preliminary study(ies)
- Concentration in vehicle: 0, 0.2, 0.6 and 3 mg/mL or the control low, mid and high dose group, respectively
- Amount of vehicle (if gavage): 5 mL/kg bw/day
- Lot/batch no.: MKCN9742 and MKCN3364

Details on mating procedure:

- M/F ratio per cage: 1
- Length of cohabitation: until mating occurred or 14 days had elapsed.
- Proof of pregnancy: presence of a vaginal plug or sperm in a vaginal lavage. The day of confirmed mating was designated as post-coitum (p.c) day 0.
- When mating had not occurred after 2 weeks, the animals were separated without further opportunity for mating.
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using High Performance Liquid Chromatography with UV detection (HPLC/UV) and a validated analytical procedure (Test Facility Study No. 48855 VAS) .
Eight dosages: once in each P-premating, P gestation, P lactation periods, and then five times spread over the F1 postweaning period. A sample was taken from control and test item dose formulations and analyzed using the validated method.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for solutions of target concentration.

Duration of treatment / exposure:

P: males and females 10 weeks before mating and during mating; males until the day before euthanasia (after weaning of the F1 pups); females during gestation and lactation (day 21 post-coitum); until euthanasia for females with no evidence of mating or no delivery (24-26 days after the last day of the mating period).
F1:
Cohort 1A: males and females from weaning day 22 post-natal until day 90-93
Cohort 1B: males and females from weaning day 22 post-natal until day 120-122
Cohort 1C: males and females from weaning day 22 post-natal until day 85-124 or until preputial separation was observed (males) and until day 126 or until vaginal opening was observed (females).

Frequency of treatment:

Once daily (pups during lactation were not dosed directly)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

3 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

15 mg/kg bw/day

No. of animals per sex per dose:

P: 24 males + 24 females per group
F1: 20 males + 20 females per group and per Cohort

Control animals:

yes, concurrent vehicle

Details on study design:

RATIONALE FOR DOSE LEVEL SELECTION:
The dose levels were selected on the basis of the results of the following previous studies:
-A 13-week toxicity study in rats (Covance reference No. 8266542, summary provided by the Sponsor): In this study, the test item was administered daily by gavage to males and females at dose levels of 0.5, 2 or 10 mg/kg bw/day for at least 90 days. The administration was well tolerated. There was no evidence of neurotoxicity. There were slight changes in some blood clinical chemistry and urine parameters at all dose levels. The liver and kidney were identified as target organs based on increased weights (at all dose levels in males) and microscopic findings of hepatocyte hypertrophy (at all dose levels in males and at 2 mg/kg bw/day in females) and focal nephropathy/tubular basophilia and hyaline droplets in males at all dose levels. The liver hypertrophy was considered as an adaptive change commonly associated with metabolic burden and the changes in the kidney were considered specific to the male rat as confirmed by immunohistochemical staining. The No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 10 mg/kg bw/day.
-An OECD 422 study in rats (Reference No. R-95-007, summary provided by the Sponsor): In this study, the test item was administered daily by gavage to males and females at dose levels of 2, 10 or 50 mg/kg bw/day until Day 43 for males and Day 4 post-partum for females. The test item had no effects on the copulation index or fertility index at 50 mg/kg bw/day or lower doses. The test item did not affect the length of the gestation period or delivery index in maternal animals, either. No abnormal parturition was observed. Abnormal nursing behavior was noted in 3 animals from the 50 mg/kg bw/day group. The findings in offspring showed no effects on the parturition index, live pup delivery index, overall delivery index, or the sex ratio and body weight on Day 0 at all dose levels. In the 50 mg/kg bw/day group, the offspring viability index and body weight on Day 4 of lactation showed a tendency to decrease. No offspring from the AZDN groups showed any morphological anomaly. Based on these results, the No-Observed-Effect-Level (NOEL) for reproductive and developmental toxicity was 50 mg/kg bw/day in males and 10 mg/kg bw/day in females and in pups.
-An OECD 414 study in rats (Covance reference No. 8266543, summary provided by the Sponsor): In this study, the test item was administered daily by gavage to pregnant females at dose levels of 1, 5 or 20 mg/kg bw/day from Day 6 p.c. to Day 19 p.c. Administration of the test item induced maternal toxicity from 5 mg/kg bw/day upwards based on transient effects of reduced body weight gain and reduced mean food intake while no effects on fetal development were observed. At 1 mg/kg bw/day, no adverse effects were observed in both parental and fetal development. There was no evidence of embryotoxicity at any dose level tested. The maternal NOAEL was 1 mg/kg bw/day based on a significant decrease in maternal body weight gain and food consumption. The embryo-fetal NOAEL was 20 mg/kg bw/day.
-AZDN - Reproduction/Developmental Toxicity Screening Test by Oral Route (Gavage) in Rats (Charles River Laboratories Evreux/Study No. 48747 RSR (Papineau)): In this OECD 421 study, the test item was administered daily by oral gavage to male and female Sprague-Dawley rats for 15 days before mating, during mating, throughout gestation and, for females, until Day 21 p.p., at dose levels of 0, 5, 20 or 50 mg/kg bw/day. No unscheduled deaths occurred in males. Two high dose females were prematurely euthanized or found dead before mating (on Study Day 8), due to poor clinical condition. Poor clinical conditions (emaciated appearance, piloerection, round back and/or hypoactivity) were observed in 1 out of 8 surviving females at 50 mg/kg bw/day during the pre-mating period. Ptyalism was also observed in test item treated males and females during the whole treatment period. At all dose levels and in both sexes, there were no adverse effects on body weight, body weight change or food consumption as changes observed in males and/or females were transient and/or of minimal to slight magnitude. There were no effects on estrous cycles, pairing, mating or fertility data. From 20 mg/kg bw/day and in male parents, liver centrilobular hepatocellular hypertrophy correlating with macroscopic enlargement and increased liver weight was observed in both sexes. At 50 mg/kg bw/day, there was also hepatocellular degeneration/necrosis and periportal vacuolation in males only. The hypertrophic changes were considered to be adverse at 50 mg/kg bw/day in view of their severity and association with degenerative changes. In the kidneys, in males only, hyaline droplet accumulation and adverse tubular degenerative changes consistent with a2µ globulin nephropathy were observed from 5 mg/kg bw/day. These findings are considered to be non-relevant for humans. At 50 mg/kg bw/day and in pups, there were adverse low mean body weights in males and females after culling (down to -16% vs. controls on Day 8 p.p., p<0.01). There were no effects on viability or lactation indexes and no effects on sex ratio. At pups' external examination, there were no areolae/nipples in male pups and no external abnormalities in any pups. There were no effects on mean plasma TSH or T4 concentration levels in male parents and Day 21 p.p. pups. Therefore, 15 mg/kg bw/day was selected as the high-dose level taking into account the increase of exposure duration in the OECD 443 and, the macroscopic and/or microscopic findings observed from 20 mg/kg bw/day. The low-dose and mid-dose were selected using ratios representing 3- to 5-fold intervals (i.e. 1 and 3 mg/kg bw/day).

Positive control:

N.A.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice a day (before and after treatment), at approximately the same time of day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a week until the end of the study, including at least once before the first treatment. During the treatment period, detailed clinical examination was performed after dosing and replaced the cage side observation. 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: Yes
- Time schedule for examinations: weekly including mating period; Mated females on day 0, 4, 7, 10, 14, 17 and 20 p.c. and, on Days 1, 4, 7, 14 and 21 p.p. (post-partum).

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly; Mated females for the intervals: Days 0-4, 4-7, 7-10, 10-14, 14-17 and 17-20 p.c. and during lactation for the intervals: Days 1-4, 4-7, 7-14, and 14-21 p.p. Food consumption was not measured for males or females during the mating period.

WATER CONSUMPTION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes at least 14 hours
- How many animals: 10 males + 10 females per group
- Parameters: Red blood cells (RBC), Mean cell volume (MCV), Haematocrit (PCV), Haemoglobin (HB), Mean cell haemoglobin concentration (MCHC), Mean cell haemoglobin (MCH), Thrombocyte (platelet) count (PLT), White blood cells (WBC), Differential white cell count with cell morphology [Neutrophils (N), Lymphocytes (L), Monocytes (M), Eosinophils (E), Basophils (B), Reticulocytes (RTC), large unstained cells (LUC)], Coagulation parameters [Prothrombin time (blood clotting time) (PT), Fibrinogen (FIB), Activated partial thromboplastin time (APTT)]

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: At least 14 hours
- How many animals: 10 males + 10 females per group
- Parameters checked : Sodium, Potassium, Chloride, Calcium, 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).

THYROID HORMONES: Yes
- Time schedule for collection of blood: at termination (between 07:30 and 10:00 a.m.)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: serum of 10 males + 10 females per group
- Parameters checked : TSH, T4

URINALYSIS: Yes
- Time schedule for collection of urine: on day of necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: At least 14 hours
- How many animals: 10 males + 10 females per group
- Parameters: Volume, pH, Specific gravity, Bilirubin, Protein, Ketones, Glucose, Blood, Appearance and Color.

Oestrous cyclicity (parental animals):

Daily vaginal lavage from 14 days prior to mating period and during mating until evidence of copulation was observed. On day of termination vaginal lavage of all surviving females to allow correlation with reproductive organs histopathology.

Sperm parameters (parental animals):

Sperm count in epididymides, sperm (progressive) motility, sperm morphology were measured on all P males from the control and high-dose groups (groups 1 and 4) from left epidydimis sampled after anesthesia and before euthanasia.
Sperm morphology was determined from a smear, after eosin staining, counting 200 spermatozoa per slide, including •Total number of sperm cells evaluated (n)
• Number of normal sperm cells (n)
• Number of normal sperm cells / Total number of sperm cells evaluated (%)
• Total number of abnormal sperm cells (n)
• Number of sperm cells with head abnormalities (n)
• Number of sperm cells with detached head (n)
• Number of sperm cells with abnormal midpiece (n).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- The size of each litter was adjusted by randomly culling extra pups to obtain as nearly as possible five males and five females per litter. Whenever necessary, partial adjustment (for example six males and four females) was permitted.

PARAMETERS EXAMINED IN F1 OFFSPRING:
- On day 1 p.p.: External visible abnormalities, cleft palate, subcutaneous hemorrhages, abnormal skin color or texture, presence of umbilical cord, lack of milk in the stomach, presence of dried secretion, sex of each pup, qualitative assessment of body temperature, activity, reaction to handling and anogenital distance (AGD) (all pups before culling; normalized to the cube root of body weight recorded on Day 1 p.p.),
- Body weight on Days 1, 4, 7, 14 and 21 p.p.; on the first day of treatment (Day 1); then at least once a week until euthanasia.
- Food Consumption was measured once a week from the first day of treatment until euthanasia.
- Sexual development was assessed from Day 35 p.p. until preputial separation or euthanasia in males, and from Day 25 p.p. until vaginal opening or euthanasia in females.
- Number of nipples and of areolae in male pups on Day 12 p.p.
- Animals were observed for clinical signs daily during lactation. After weaning, each animal was observed for clinical signs, at least twice a day.
- After weaning, detailed clinical examinations were performed on all animals at least once a week until the end of the study.
- Number and sex of pups, stillbirths, live births, postnatal mortality (at least twice a day)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes at least 14 hours
- How many animals: 10 males + 10 females of Cohort 1A
- Parameters: Red blood cells (RBC), Mean cell volume (MCV), Haematocrit (PCV), Haemoglobin (HB), Mean cell haemoglobin concentration (MCHC), Mean cell haemoglobin (MCH), Thrombocyte (platelet) count (PLT), White blood cells (WBC), Differential white cell count with cell morphology [Neutrophils (N), Lymphocytes (L), Monocytes (M), Eosinophils (E), Basophils (B), Reticulocytes (RTC), large unstained cells (LUC)], Coagulation parameters [Prothrombin time (blood clotting time) (PT), Fibrinogen (FIB), Activated partial thromboplastin time (APTT)]

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: At least 14 hours
- How many animals: 10 males + 10 females of Cohort 1A
- Parameters checked : Sodium, Potassium, Chloride, Calcium, 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).

THYROID HORMONES: Yes
- Time schedule for collection of blood: at termination (between 07:30 and 10:00 a.m.)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: serum of 10 males + 10 females of Cohort 1A
- Parameters checked : TSH, T4

URINALYSIS: Yes
- Time schedule for collection of urine: on day of necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: At least 14 hours
- How many animals: 10 males + 10 females of Cohort 1A
- Parameters: Volume, pH, Specific gravity, Bilirubin, Protein, Ketones, Glucose, Blood, Appearance and Color.

SPERM PARAMETERS: Yes
- Sperm count in epididymides, sperm (progressive) motility, sperm morphology were measured on all P and Cohort 1A males from the control and high-dose groups (groups 1 and 4) from left epidydimis sampled after anesthesia and before euthanasia.
Sperm morphology was determined from a smear, after eosin staining, counting 200 spermatozoa per slide, including •Total number of sperm cells evaluated (n)
• Number of normal sperm cells (n)
• Number of normal sperm cells / Total number of sperm cells evaluated (%)
• Total number of abnormal sperm cells (n)
• Number of sperm cells with head abnormalities (n)
• Number of sperm cells with detached head (n)
• Number of sperm cells with abnormal midpiece (n).

LYMPHOCYTE SUBTYPING: Yes
- For the investigation of pre- and post-natal induced immunotoxic effects, 10 males and 10 females from each group in Cohort 1A animals were subjected to a splenic lymphocyte subpopulation analysis [T lymphocytes, CD4+ and CD8+ T lymphocytes, B lymphocytes, Natural Killer (NK) cells and NKT cells] using one half of the spleen (weight determined at necropsy), the other half of the spleen was preserved for histopathological evaluation. The lymphocyte subtyping by flow cytometry followed the method validated in Study No. 44564 RDR (Hamel, 2020) and the internal procedures.

Postmortem examinations (parental animals):

SACRIFICE:
On completion of the treatment period, all surviving P animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
- Male animals: after weaning of the F1 progeny.
- Maternal animals: on Day 23 p.p. (Days 24-26 p.c. if they did not deliver [euthanasia by inhalation of carbon dioxide gas followed by cervical dislocation], 25 days after the end of the mating period for a female with no evidence of mating [euthanasia by inhalation of carbon dioxide gas followed by cervical dislocation])

GROSS NECROPSY
- A complete macroscopic post-mortem examination was performed on all P animals. Gross necropsy consisted of examination of the 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 corpora lutea and implantation sites were recorded for females euthanized on Day 23 p.p. The numbers of corpora lutea and implantation sites were recorded when possible for females euthanized on Days 24 26 p.c. due to no delivery.

HISTOPATHOLOGY / ORGAN WEIGTHS
- The organs specified in Table 2 (under ''Any other information on materials and methods incl. tables'') were weighed wet as soon as possible after dissection.
-A microscopic examination was performed on:
• all tissues listed in Table 2 (under ''Any other information on materials and methods incl. tables'') from animals of the control and high-dose groups (groups 1 and 4) from P generation.
• reproductive organs from animals that did not mate or conceive, from pregnant females that did not deliver, from females with abnormal estrous cycles and from males with abnormalities at sperm analysis, to investigate possible causes.
• all macroscopic lesions of all groups, including those from prematurely euthanized animals.

Postmortem examinations (offspring):

SACRIFICE:
Animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
- Pups whose mother died were ethanised as soon as possible,
- Pups not selected on Day 4 p.p.
- Pups not selected at weaning: on Day 22 p.p.
- On completion of the treatment period for all surviving F1 adults
• F1 males (Cohort 1A): on Days 90-93 p.p.
• F1 females (Cohort 1A): on Days 90-94 p.p.,
• F1 males and females (Cohort 1B): after euthanasia of Cohort 1A animals
• F1 males and females (Cohort 1C): after cleavage of the balanopreputial groove or vaginal opening. After euthanasia of Cohort 1A animals for animals with negative sexual development landmarks.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Moribund and prematurely euthanized pups were subjected to macroscopic post-mortem examination of the principal thoracic and abdominal organs.
- A complete macroscopic post-mortem examination was performed on all adult F1 animals. Gross necropsy included the same examinations performed for the P generation.

HISTOPATHOLOGY / ORGAN WEIGTHS
- The organs specified in Table 2 (under ''Any other information on materials and methods incl. tables'') were weighed wet as soon as possible after dissection.
-A microscopic examination was performed on:
• all tissues listed in Table 2 (under ''Any other information on materials and methods incl. tables'') from animals of the control and high-dose groups (groups 1 and 4) from cohort 1A. Microscopic examination in Cohort 1B was conducted if results from Cohort 1A are equivocal or in cases of suspected reproductive or endocrine toxicants.
• reproductive organs from from cohort 1A males with abnormalities at sperm analysis, to investigate possible causes.
• all macroscopic lesions of all F1 cohorts.

Statistics:

-Statistical analyses of body weight, food consumption and reproductive data was performed using Provantis or Reprotox softwares.
-PATHDATA software was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01) according to the sequence in section 5.12.2 of the attached study report.
- Statistical analysis of Splenic Lymphocyte Immunophenotyping was performed using the SAS Enterprise Guide software according to the sequence in section 5.12.3 of the attached study report.
- Provantis software was used to perform the statistical analyses (according to the sequence in section 5.12.4 of the attached study report) of: Number of Primary 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, Post-implantation Loss, Sex Ratio, Live Birth, Viability and Lactation Indexes.

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 Day 1 p.p. / Number of delivered pups) x 100,
• Viability index on Day 4 p.p. = (Number of surviving pups on Day 4 p.p. (before culling) / Number of delivered pups) x 100,
• Lactation index = [(Number of surviving pups on Day 21 p.p. / Number of surviving pups on Day 4 p.p. (after culling)] x 100,
• Normalized Ano-Genital Distance (Norm. AGD) = AGD / ³vBody weight.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs recorded in terminated as scheduled P Generation males (i.e. unscheduled deaths
excluded) are presented in table 3 under ''Any other information on results incl. tables'':
In males:
-At 15, 3 and 1 mg/kg bw/day, there were no test-item related adverse clinical signs.
Clinical signs recorded in terminated as scheduled P Generation females (i.e. unscheduled deaths excluded) are presented in table 4 under ''Any other information on results incl. tables''
In females:
-At 15 mg/kg bw/day, loud breathing and/or erected fur were recorded in one animal for 1 or 2 days. While a test-item relationship cannot be excluded, these findings were considered to be non-averse based on their incidence and duration.
-At 3 and 1 mg/kg bw/day, there were no test-item related adverse clinical signs.
Hypersalivation was observed in all treated groups. This finding is commonly observed after a gavage procedure and was considered to be test item treatment-related but not adverse.
Other findings observed with a low and/or similar incidence across groups (including controls) were those that are commonly observed in this species/strain of animals or in pregnant animals, and they were therefore not considered to be test item-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

-One mid-dose male was prematurely euthanized on Study Day 99. Lateral recumbency, chromodacryorrhea, chromorhynorrhea, erected fur, cold to the touch and abdominal breathing were observed before euthanasia. At necropsy, no observations were noted. This death was not considered to be test item related.
-One control female was prematurely euthanized on Day 24 p.c. (Study Day 97) for delivery difficulties. Thin appearance, pallor (extremities and eyes), hunched posture, erected fur and abdominal breathing were recorded before death. At necropsy and microscopic examination, there was an imperforate vagina. In addition, 4 dead fetuses and 8 scars were recorded in uterus.
-One mid-dose female was prematurely euthanized on Day 23 p.c. (Study Day 96) due to delivery difficulties. No clinical signs were observed before death. At necropsy, 8 scars were present in the right horn, and 2 dead fetuses, 3 placentae and 4 scars were present in the left horn. No macroscopic findings were noted. These isolated reproduction troubles (difficulties to deliver) were considered to be unrelated to the test item administration.
-One high-dose female was prematurely euthanized on Study Day 70 on humane grounds. Soiled coat, pallor (extremities and eyes), hunched posture, erected fur and swelling (shoulder and forelimb) were observed before euthanasia. At necropsy, induration, red discoloration and gelatinous left forelimb muscle was noted. This was considered to be of accidental origin (trauma).
-A second high-dose female was prematurely euthanized on Day 14 p.c. (Study Day 89). Pallor (eyes and extremities), erected fur, half-closed eyes, decreased activity, thin fur and vaginal discharge were observed before death. At necropsy, this female had 10 alive concepti and 9 early resorptions. There was red content in vagina that correlated with hemorrhage in lumen. These isolated reproduction troubles (difficulties to deliver) were considered to be unrelated to the test item administration.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

When compared to controls and despite a few statistically significant differences, there were no dose-related effects on mean body weight or mean body weight change in P generation males or females during the premating period.
Despite a statistically significant difference (+5 vs. +9 g at 15 mg/kg/day on Days 4 to 7 p.c. p<0.05), there were no adverse effects on mean body weight or mean body weight change in P generation females during the pregnancy period.
There were no adverse effects on mean body weight or mean body weight change in P generation females during the lactation period either. At 15 mg/kg/day, the overall net body weight gain was higher than controls (+43 vs.+23 g, p<0.001). This difference was considered to be test-item treatment-related but non-adverse (mean body weight returning to control values during the post-partum period).
Therefore, any relationship to a test item treatment was considered to be unlikely. See tables 18 to 20 in the attached study report.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

When compared to controls and despite a statistically significant difference (+13% at 15 mg/kg bw/day in males on Days 113 to 120, p<0.05), there were no dose-related effects on mean food consumption in P generation males or females during the premating period.
There were no effects on females mean food consumption during the pregnancy period and lactation periods either.
Therefore, any relationship to a test item treatment was considered to be unlikely. See tables 21 to 23 in the attached study report.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse effects on hematology or coagulation.
The few statistically significant differences (increased number of monocytes in high dose females and decreased APTT in high dose males) were recorded with no dose relationship, were not observed in both sexes and/or remained within the range of Historical Control Data. See table 3 under ''Any other information on results incl. tables''

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse effects on blood biochemistry in P generation animals.
The statistically significant differences in male potassium and cholesterol levels and female urea levels were recorded with no dose relationship, were not observed in both sexes and/or remained within the range of Historical Control Data. See table 3 under ''Any other information on results incl. tables''

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no effects on TSH or T4 plasma levels

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were noted in the kidneys, liver and thyroid glands (See also table 3 under ''Any other information on results incl. tables'')
- Kidneys
Minimal to marked, dose-related changes were noted in the kidneys of males treated at
= 1 mg/kg bw/day, including tubular degeneration/regeneration, increased severity/incidence of accumulation of hyaline droplets, tubular casts, tubular basophilia and infiltrate of mononuclear inflammatory cells. The increased severity/incidence of tubular dilatation was seen only at
15 mg/kg bw/day.
The tubular degeneration/regeneration was characterized by the presence of exfoliated tubular cells with hypereosinophilic cytoplasm and/or shrunken boundaries (degeneration/necrosis) and enlarged tubules with clear cytoplasm and thickened basal membrane. The basophilic tubules were characterized by tubular cells with basophilic cytoplasm.
In females, a few occasional findings were noted but were seen with similar incidence and severity in control and treated females.
- Liver
Minimal to moderate hepatocellular hypertrophy in centrilobular zone was noted at
= 1 mg/kg bw/day, together with minimal degeneration/necrosis at 15 mg/kg bw/day in 2/24 males and 1/24 females and minimal to slight microvesicular vacuolation (mainly periportal) at = 1 mg/kg bw/day.
- Thyroid glands
Minimal follicular hypertrophy was noted in 3/24 males treated at 15 mg/kg bw/day. Although seen with low incidence and severity, this change was considered to be test item-related in view of the increased thyroid gland weight and biological evidence (see discussion under ''details on results(P0)'').
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 fibrosis in the parathyroid from 1/22 males treated at 15 mg/kg bw/day, minimal infiltrate of mononuclear inflammatory cells in the heart from 3/24 males and
1/22 females treated at 15 mg/kg bw/day, minimal vacuolation of cortical adrenal glands in
1/24 males and 1/22 females treated at 15 mg/kg bw/day and the hemorrhage/necrosis in the uterus of 1/22 females treated at 15 mg/kg bw/day.
These observations were seen with minimally increased incidence in some test item treated animals when compared to controls. In view of their low incidence and of their common occurrence in untreated rats, these changes were considered to be part of the spontaneous background.

ENUMERATION OF CORPORA LUTEA IN HE-STAINED SLIDES:
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 7.10 (± 3.13) and 7.77 (± 4.08) per animal, respectively.

ENUMERATION OF THE NUMBER OF PRIMORDIAL FOLLICLES ON PCNA-STAINED SLIDES:
There were no statistical differences between the high-dose and control groups, with a mean number of 8.28 (± 4.68a) and 7.77 (± 5.95) primordial follicles per animal on PCNA-stained slides, respectively.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

See table 3 under ''Any other information on results incl. tables''

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no effects on motility, morphology and sperm cells count.

Reproductive performance:

no effects observed

Description (incidence and severity):

The following parameter were investigated: enumeration of corpora lutea in HE-stained slides , enumeration of the number of primordial follicles on PCNA-stained slides, pre-coital interval, mating index, pregnancies, fertility index, gestation length, gestation index, live litters born, live birth index, number of implantations, mean percent of post-implantation loss, number of pups delivered, litter size, mean number of live pups at birth, offspring bw and pup sex ratio.
See table 3 under ''Any other information on results incl. tables''

Details on results (P0)

PATHOLOGY DISCUSSION:

There was no test item-related mortality in this study.
In the P generation, adverse degeneration/regeneration was noted in the renal tubules of males treated at 3 or 15 mg/kg bw/day together with hyaline droplets accumulation (that correlated with increased kidney weights in males treated at = 1 mg/kg bw/day), tubular basophilia, casts, dilatation and inflammatory cells infiltrate. The inflammatory/degenerative lesions correlated with macroscopic kidney enlargement, granular aspect and tan discoloration at necropsy. At 1 mg/kg bw/day, the renal findings were considered to be non-adverse in view of their morphology, their limited distribution and their low magnitude and incidence.
The hyaline droplet accumulation was probably consistent with Alpha-2-u-globulin although no immunohistochemical staining was performed and was highly suggestive of hyaline droplet nephropathy, a male rat specific change of no relevance to human.
In the liver, hepatocellular hypertrophy and periportal vacuolation were seen at = 1 mg/kg/day in males and females, that correlated with increased weight and accentuated lobular pattern at necropsy in males treated at 15 mg/kg/day. Isolated minimal hepatocellular degeneration/necrosis was also noted at 15 mg/kg/day (2/24 males and 1/24 females). Although there were no noteworthy clinical pathology correlates and in spite of the low magnitude of these changes (minimal), the hepatocellular degeneration/necrosis was considered to be adverse.
In the thyroid glands, minimal follicular hypertrophy correlated with increased weights in males treated at 15 mg/kg/day. In view of the low grade of this change and the association with liver hypertrophy, this was considered to be non-adverse.

Under the conditions of this study, the liver hypertrophy was considered rather an adaptive change (Greaves, 2012). This was consistent with microsomal enzyme induction that resulted in increased turnover of T4 (thyroxine) and secondary thyroid gland hypertrophy due to stimulation of the pituitary-thyroid-endocrine axis (Hall et al, 2012). This was partially confirmed by the TSH increase trend noted in P generation males and in spite no decrease in T4 was observed.
At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and control groups. There were no test item-related macroscopic or microscopic lesions in male or female reproductive organs. This was consistent with the estrous cycle, mating, fertility and delivery results.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: based on adverse degeneration/necrosis in liver of males and females at 15 mg/kg/day

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

other: kidneys

Remarks on result:

other: based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at = 3 mg/kg/day

Target system / organ toxicity (P0)

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

15 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

-Lactating pups (between Days 1 to 21 p.p., before weaning): At 15 mg/kg bw/day and when compared to controls, 1 to 2 litters had pups with dehydration, emaciated appearance, cold to the touch, generalized pallor and/or absence of milk in the stomach (up to 14 pups). A test-item relationship cannot be excluded but these findings were considered to be non-adverse based on absence of any effect on pup body weight and survival during the lactation period). At 3 and 1 mg/kg bw/day, there were no adverse test item-related findings. The remaining findings were recorded in controls, observed with low incidences and no dose relationship and/or are common findings in this species and strain maintained in the experimental conditions of this study. See also table 4 under ''Any other information on results incl. tables''

-Cohort 1A: There were episodes of hypersalivation in both sexes (from 1 mg/kg bw/day in males and from 3 mg/kg bw/day in females). This finding, commonly observed after a gavage procedure, was considered to be test-item related but non-adverse (See also table 5 under under ''Any other information on results incl. tables''). Other findings observed with a low and/or similar incidence across groups (including controls) were those that are commonly observed in this species/strain of animals or in pregnant animals, and they were therefore not considered to be test item treatment-related. See also table 5 under under ''Any other information on results incl. tables''.

-Cohort 1B: There were episodes of hypersalivation in both sexes (from 15 mg/kg bw/day in males and from 3 mg/kg bw/day in females). This finding, commonly observed after a gavage procedure, was considered to be test-item related but non-adverse. Additionally, in females, instances of reflux at dosing, decreased activity and fur erected starting at 3 mg/kg bw/day were observed. Other findings observed with a low and/or similar incidence across the groups (including controls) are those that are commonly observed in this species/strain of animals or in pregnant animals, and they were therefore not considered to be test item treatment-related. See also table 6 under under ''Any other information on results incl. tables''.

-Cohort 1C: There were instances of hypersalivation (after treatment), alopecia and/or thin fur, chromodacryorrhea and/or chromorhynorrhea, scabs (head, dorsal region and/or nose) and tail bent. Other findings observed with a low and/or similar incidence across groups (including controls) are those that are commonly observed in this species/strain of animals or in pregnant animals, and they were therefore not considered to be test item treatment-related. See also table 7 under under ''Any other information on results incl. tables''.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

-Lactating pups (between Days 1 to 21 p.p., before weaning): There were no effects on the pre-culling Day 4 p.p. survival index, lactation index or sex-ratio at weaning. At 15 mg/kg bw/day, sex-ratio was lower than controls (41% vs. 50%, not statistically significant). However, a test item relationship was considered to be unlikely as the percentage was close to the lower values of the Historical Control Data. See also table 4 under ''Any other information on results incl. tables''.

-Cohort 1A: At 1 mg/kg bw/day group, one female was prematurely euthanized on Day 8 on humane grounds. Thin appearance, hunched posture, erected fur and swelling (abdomen) were observed before euthanasia. At necropsy, the ileum, cecum and colon were distended with gas. This death was unrelated to the test item administration since it was an isolated death seen in low-dose females at the beginning of the treatment period with undetermined cause of death. See also table 5 under under ''Any other information on results incl. tables''.

-Cohort 1B: In the control group one male was prematurely euthanized on Day 94 (Week 14). Chromorhynorrhea was observed on Days 48 to 50. Alopecia (forelimbs) was noted from Day 86, scabs (right forelimb) were noted from Day 92 and swelling (right forelimb) was noted on the day of euthanasia. At necropsy, a fracture of right forelimb was noted. This death was not considered to be test item related. A control females was found dead on Day 23 (Week 4). No clinical signs were noted. Perforation of the esophagus was noted at necropsy. A brown thick content was present in the lungs and thoracic cavity. An issue during the gavage procedure was considered to be the cause of death. In the low dose group one male was found dead on Day 40 (Week 6). No clinical signs were noted. No findings were noted at necropsy. The cause of death was not evident. Another low dose male was found dead on Day 52 (Week 8). Reflux at dosing was observed on Day 8. No findings were noted at necropsy. Finally, one high dose female was prematurely euthanized on Day 83 (Week 12). No clinical signs were noted. A fracture of left hindlimb was observed at necropsy. This death was not considered to be test item related. See also table 6 under under ''Any other information on results incl. tables''

-Cohort 1C: One high-dose male was prematurely euthanized after falling from the cage on Day 23 (Week 4). Before euthanasia, loud / abdominal breathing, ventral recumbency and decreased activity were observed. A series of cranial and brain traumas were observed at necropsy. This death was accidental and therefore not test-item related. There were no unscheduled deaths in Cohort 1C females. See also table 7 under under ''Any other information on results incl. tables''.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

-Lactating pups (between Days 1 to 21 p.p., before weaning): No effects (between Days 1 to 21 p.p., before weaning). See table 32 in the attached study report.

-Cohort 1A: There were no adverse effects on mean body weight or mean body weight changes.
When compared to controls, the few statistically significant differences were recorded without any dose relationship and with minimal amplitudes. Therefore, any test item relationship was considered to be unlikely. See table 37 in the attached study report.

-Cohort 1B: There were no effects on mean body weight or mean body weight change. The few statistically significant differences (recorded without a dose relationship and with minimal amplitude when compared to controls) were considered to be fortuitous. See table 45 in the attached study report.

-Cohort 1C: There were no effects on mean body weight or mean body weight changes. When compared to controls, the statistically significant difference was recorded without any dose relationship and with only minimal amplitudes. Therefore, any test item relationship was considered to be unlikely. See table 49 in the attached study report.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

-Cohort 1A: There were no adverse effects on mean food consumption. When compared to controls, the few statistically significant differences were recorded without any dose relationship and/or with minimal amplitudes. Therefore, any test item relationship was considered to be unlikely. See table 38 in the attached study report.

-Cohort 1B: In males, there was no evidence of an effect on mean food consumption. The increased observed at 15 mg/kg bw/day in week 6 (+9.3% vs. controls, p<0.01) was of mild amplitude and isolated; therefore a test-item relationship was considered to be unlikely. In females, from 3 mg/kg bw/day and mainly in weeks 12 to 14, there were dose-related increases in mean food consumption (up to +18.3% vs. controls in Week 14, p<0.001). A test item relationship cannot be excluded, but taking into account the amplitude of the difference, this finding was considered to be non-adverse (less than 20%). See table 46 in the attached study report.

-Cohort 1C: There were no effects on mean food consumption in Cohort 1C animals. See table 50 in the attached study report.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

-Cohort 1A: There were no test item-related effects. A statistically significant difference was recorded in low-dose females (RTC: 0.20 vs. 0.17 T/L in controls, p<0.05) without a similar finding in males or a dose relationship. See table 5 under ''Any other information on results incl. tables''.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

-Cohort 1A: There were no adverse effects on blood biochemistry in Cohort 1A animals.
The statistically significant differences in chloride, calcium, urea, cholesterol, triglycerides, alkaline phosphatase 2C, aspartate aminotransferase, total proteins and albumin were recorded with no dose relationship, were not observed in both sexes and/or remained within the range of Historical Control Data. See table 5 under ''Any other information on results incl. tables''.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

-In Cohort 1A: There were no adverse effects on urinalysis parameters. The statistically significant differences in pH were recorded with no dose relationship, were not observed in both sexes and/or remained within the range of Historical Control Data (see table 5 under ''Any other information on results incl. tables'')

Sexual maturation:

no effects observed

Description (incidence and severity):

In males (all cohorts combined), the mean age at which balanopreputial separation occurred and the mean body weight on the day of occurrence are detailed in table 8 under ''Any other information on results incl. tables''.
There were no effects on mean age at balanopreputial separation or on mean body weight on the day of occurrence.
In females (all cohorts combined), the mean age at which vaginal opening occurred and the mean body weight on the day of occurrence are detailed in table 8 under ''Any other information on results incl. tables''.
There were no effects on mean age at vaginal opening or on mean body weight on the day of occurrence.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

On Day 1 p.p and when compared to controls, there were no effects on mean anogenital distance (AGD) or normalized AGD. See also table 4 under under ''Any other information on results incl. tables''.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no nipples or areolae in male pups examined on Day 12 p.p.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

-Cohort 1A: Increased absolute and/or relative-to-body kidney weights were noted in males at = 1 mg/kg bw/day (up to +43% in relative-to-body weights for males treated at 15 mg/kg bw/day; p<0.01). This correlated with enlargement, tan discoloration and irregular color at gross observation and microscopic degenerative/inflammatory lesions in males. The differences were considered to be related to the test item administration in males. In females, a relationship was considered to be unlikely as the differences were seen only in relative-to-body weights and had no microscopic correlates. Increased absolute and/or relative-to-body liver weights were noted in males and females treated at = 3 mg/kg bw/day (up to +45% in relative-to-body weights for females treated at 15 mg/kg bw/day; p<0.01). This correlated mainly with microscopic hepatocellular hypertrophy in males and females. Increased absolute and/or relative-to-body pituitary gland weights were noted in females treated at = 1 mg/kg bw/day. In view of the poor dose-relationship and absence of microscopic correlates, a relationship with the test item administration was considered to be equivocal. Increased absolute and relative-to-body adrenal gland weights were noted in males treated at = 3 mg/kg bw/day (up to +28% in relative-to-body weights for males treated at 15 mg/kg bw/day; p<0.01). In the absence of microscopic correlates, a relationship with the test item administration was considered to be equivocal. The few other 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 relative-to-body spleen and testes weights in males treated at 15 mg/kg bw/day (respectively +13%; p<0.05 and +10%; p < 0.01). The differences were considered to be unrelated to the test item administration as they had no microscopic correlates and were of low magnitude. See also table 5 under ''Any other information on results incl. tables''.

-Cohort 1B: There were no test item-related organ weight differences. The few organ weight changes were not considered to be test item-related because they were of insufficient magnitude and/or were not dose-related.

-No organ weights were measured in cohort 1C.

-In pups non selected to conform cohorts 1A, 1B or 1C, there were no test item-related organ weight differences. The few organ weight changes were not considered to be test item-related because they were of insufficient magnitude and/or were not dose-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

-There were no test-item related findings at necropsy of pups found dead during weaning.
At 15 mg/kg bw/day, one litter had 4 found dead pups with no milk in the stomach. An absence of maternal care may have led to these deaths. However, finding is common in rats of this strain and was limited to a single litter. Therefore a test item relationship was considered to be unlikely. See also table 4 under under ''Any other information on results incl. tables''

-No test item related macroscopic post-mortem observations in pups euthanized after weaning.
The few findings were observed with low incidences, no dose level relationship and/or are common findings in this species and strain maintained in the experimental conditions of this study.

- Cohort 1A:There were test item-related findings in kidneys of males. Tan discoloration, enlargement and/or irregular color were noted in the kidneys from some males and females treated at = 1 mg/kg bw/day. These changes were considered to be test item-related in males and correlated with increased weights and microscopic degenerative/inflammatory lesions in this sex. In females, the tan discoloration in one female at 1 mg/kg bw/day and in one other female treated at 15 mg/kg bw/day had no microscopic correlates and thus was unrelated to the test item administration. The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in rats of these strain and age. This included the dilated pelvis in kidneys from 4/20 males and 1/20 females treated at 15 mg/kg bw/day, and the tan or white discoloration in the adrenal glands from 2/20 females treated at 15 mg/kg bw/day considered to be incidental. See also table 5 under ''Any other information on results incl. tables''.

-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 abnormal teeth growth in 1/18 males treated at 1 mg/kg bw/day, the dilated renal pelvis or ovarian cyst in 2/20 females treated at 3 mg/kg bw/day and in 2/19 females treated at 15 mg/kg bw/day.

-Cohort 1C: 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 sternum deformation in 1/20 females treated at 15 mg/kg bw/day.

-Non Selected Pups: There were no test item-related gross changes. The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in rats of these strain and age.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

-COHORT 1A: Test item-related changes were noted in the kidneys (males only treated at = 1 mg/kg bw/day) and in liver (males and females treated at = 1 mg/kg bw/day).
. Kidneys
Minimal to marked, dose-related changes were noted in the kidneys of males treated at
= 1 mg/kg bw/day, including tubular degeneration/regeneration, accumulation of hyaline droplets, increased severity/incidence of basophilic tubules, hyaline casts, infiltrate of mononuclear inflammatory cells and tubular/pelvic dilatation. No test item-related changes were noted in females.
. Liver
Minimal to slight hepatocellular hypertrophy in centrilobular zones was noted at = 1 mg/kg bw/day, together with minimal degeneration/necrosis in 1/20 males treated at 15 mg/kg bw/day and microvesicular periportal vacuolation in occasional females treated at = 1 mg/kg bw/day.
See also table 5 under ''Any other information on results incl. tables''
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 mononuclear cell infiltrate in the skeletal muscle from 1/20 males treated at 15 mg/kg bw/day, the vacuolation in the cortex of adrenal glands of 1/20 males treated at 15 mg/kg bw/day, the crust and ulcer in the skin of 1/20 males treated at 3 mg/kg bw/day and in 1/20 males treated at 15 mg/kg bw/day, the ventricular dilatation in the brain of 2/20 females treated at 15 mg/kg bw/day, a malformation (retinal disorganization) in the eyes from 1/20 females treated at 15 mg/kg bw/day, the retrobulbar hemorrhage in one male and one female treated at 1 mg/kg bw/day, one male and one female treated at 3 mg/kg bw/day and in one female treated at 15 mg/kg bw/day, the minimal tingible macrophages in the thymus of 1/20 females treated at 15 mg/kg bw/day, the minimal intrasinusoidal erythrocytes in the mandibular or mesenteric lymph node from 2/20 females treated at 15 mg/kg bw/day, minimal alveolar macrophages in the lungs of 7/20 females treated at 15 mg/kg bw/day, the minimal infiltrate of mononuclear cell infiltrate in the heart of 2/20 females treated at 15 mg/kg bw/day, the minimal tubular dilatation in the kidneys from 1/20 females treated at 15 mg/kg bw/day, the minimal to slight pelvic dilatation in the kidneys from 2/20 females treated at 15 mg/kg bw/day, the slight forestomach hyperplasia from 1/20 females treated at 15 mg/kg bw/day and the slight fracture in the sternum of 1/20 females treated at
3 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.
ENUMERATION OF CORPORA LUTEA IN HE-STAINED SLIDES
At enumeration of corpora lutea in the ovary of high-dose and control groups, there were no statistically significant differences, with a mean number of corpora lutea of 14.35(± 5.97) and 14.75 (± 6.95) per animal, respectively.
ENUMERATION OF THE NUMBER OF PRIMORDIAL FOLLICLES ON PCNA-STAINED SLIDES
At enumeration of primordial follicles in the ovary of high-dose and control groups, there were no statistically significant differences, with a mean number of primordial follicles of 17.77
(± 5.91) and 13.47 (± 6.57) per animal, respectively.

-COHORT 1B, Microscopic examination of macroscopic lesions: There were test item-related findings in kidneys of males treated at = 1 mg/kg bw/day, including accumulation of hyaline droplets, basophilic tubules, hyaline casts, infiltrate of mononuclear inflammatory cells and tubular dilatation. The few other 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.

-COHORT 1C, 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 fracture of sternum seen in one female treated at 15 mg/kg bw/day.

Other effects:

no effects observed

Description (incidence and severity):

-There were no effects on the mean time (number of days) to first estrous after vaginal opening in Cohort 1A females. There were no effects on mean estrous cycle parameters in Cohort 1A females. When compared to controls, a statistically significant difference (see table 5 under ''Any other information on results incl. tables'') was recorded without any dose relationship and with no impact on associated estrous stages. Therefore, any test item relationship was considered to be unlikely. There were no effects on mean estrous cycle parameters in Cohort 1A females. When compared to controls, a statistically significant difference (2.0 vs. 2.4 mean number of days of cycles at 3 mg/kg bw/day, p<0.05) was recorded without any dose relationship and with no impact on associated estrous stages. Therefore, any test item relationship was considered to be unlikely.
- In Cohort 1A animals, when compared to controls, there were no effects on mean plasma TSH or T4 levels.
-No effects on sperm parameters (motility, morphology and sperm cells count) of Cohort 1A animals.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

effects observed, treatment-related

Description (incidence and severity):

LYMPHOCYTE SUBTYPINGIN COHORT 1A:
-Relative counts: A statistically significant difference was observed in terms of NK cell relative counts between the control and high dose group in females. A difference was also observed for groups 3 and 4 versus the male control group. However, while an increase was observed in treated females (+71%) a decrease was observed in males (-35% and -31% in group 3 and 4, respectively) when compared to their respective control groups. In males only, a statistically significant difference was observed in terms of B cell relative counts between group 4 and the control group (+14%).
Absolute counts: In terms of absolute counts, a statistically significant difference was observed in NK cells in female and male animals, but in opposite directions, as for NK cell relative counts.
A dose-related higher mean value for NK cells was observed in females (+40%, +49% and +74% in groups 2, 3 and 4, respectively, compared to control females), while a dose-related lower mean value was observed in males (-21%, -34% and -39% in group 2, 3 and 4, respectively, compared to control males). A test item-related effect on NK cells was found, however, as both relative and absolute counts were found to be increased in females, while they were both decreased in males.
See also table 5 under ''Any other information on results incl. tables''

Details on results (F1)

PATHOLOGY DISCUSSION:
In cohort 1A, similar lesions were noted: No test item-related mortality was observed. Increased kidney weights were noted in males treated at = 1 mg/kg/day. This correlated with enlargement, irregular color and tan discoloration at gross observation and microscopic degenerative/inflammatory lesions in these males (considered as adverse at = 1 mg/kg/day). Increased liver weights were noted in males and females treated at = 3 mg/kg/day. This correlated with microscopic non-adverse hypertrophy and/or vacuolation. Hepatocellular degeneration/necrosis was recorded in males at 15 mg/kg/day and was considered to be adverse, although seen in only 1/20 males at minimal severity. At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and the control groups.
No test item-related changes were noted in the thyroid glands of cohort 1A animals.
At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and control groups. This was consistent with the time to first estrous and estrous cycle results (see main report).
In cohort 1B, no test item-related mortality, no organ weight differences and no gross findings were noted. In the selected organs submitted for microscopic examination, there were test item-related findings in kidneys of males treated at = 1 mg/kg/day, including accumulation of hyaline droplets, basophilic tubules, hyaline casts, infiltrate of mononuclear inflammatory cells and tubular dilatation, similar to P generation and Cohort 1A.
In cohort 1C, no test item-related mortality and no gross findings were noted.
There were no test item-related organ weight differences or gross findings in non-selected pups.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1A)

Effect level:

3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: on adverse degeneration/necrosis in liver of males

2

Key result

Dose descriptor:

LOAEL

Generation:

F1 (cohort 1A)

Effect level:

< 1 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other: based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at = 1 mg/kg/day

Remarks on result:

other: based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at = 1 mg/kg/day

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 mg/kg bw/day (nominal)

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

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 once in the P-premating, P-gestation and P-lactation periods and five times over the F1-postweaning period were all found to be within the acceptance criteria (± 15% of the nominal concentrations). No test item was found in control dose formulations. Dose formulation homogeneity was also demonstrated, as the Relative Standard Deviation (RSD) was found to be ≤ 10% for the group 2 formulation tested in the P-premating period.

The results were as follows:

Group	Theoretical Concentration (mg/mL)	Sampling Location	Measured Concentration (mg/mL)	Bias (%)	RSD (%)
1	0	Middles	Nd	-	-
2	0.2	Top	0.190	-4.9	2.4
			0.200	0.1
		Middle	0.195	-2.6
			0.204	1.9
		Bottom	0.200	-0.2
			0.199	-0.3
		Mean	0.198	-1.0	-
3	0.6	Middle	0.610	1.7	-
4	3	Middle	2.96	-1.3	-

 

 

Table 3: Results P (males until termination after mating, females until weaning F1)

Dose (mg/kg bw/day)		0		1		3		15
Endpoint		M	F	M	F	M	F	M	F
Mortality		0/24	1/24	0/24	0/25	1/24	1/24*	0/24	2/24**
Relevant clinical signs		NTRE
·       Hypersalivation (before treatment)					2/24	1/23		4/24	2/22
·       Hypersalivation (after treatment				3/24	5/24	5/23		8/24	7/22
·       Loud breathing									1/22
·       Fur erected									1/22
Body weight		NTRE
Body weight gain		NTRE
Food consumption		NTRE
Haematology
·       M (G/L)									↑29%  (Mean values within HCR
·       APTT (sec)								↓11% (mean value within HCR)
Clinical chemistry
·       K+ (mmol/L)				↑6%		↑7%		↑4%
·       UREA (mmol/L)									↑18% (mean value within HCR)
·       CHOL (mmol/L)								↑28% (mean value within HCR)
Urinalysis		NTRE
Thyroid hormones -TSH		NTRE
-T4		NTRE
Organ weights	Kidneys			↑9% abs. / ↑10% rel.	↑4% abs. (NTR)	↑16% abs. / ↑16% rel.	↑5% abs. (NTR)	↑25% abs. / ↑29% rel.	↑6% abs. / ↑11% rel. (NTR).
	Liver					↑10% rel.		↑26% abs. / ↑31% rel.	↑14% abs. / ↑20% rel.
	Thyroid glands							↑11% asb.
	Adrenal glands								↑15% abs. / ↑22% rel. (NTR).
Macroscopy	-Liver: accentuated lobular pattern							14/24
	-Kidney: Enlarged, granular and/or discolorated			1/24		1/23		4/24
Microscopy	Kidneys; degeneration/regeneration; tubule			4/24	NA	14/23	NA	24/24
	Kidneys; accumulation; hyaline droplets	1/24		24/24	NA	23/23	NA	24/24
	Kidneys; basophilic tubules	5/24	1/23	20/24	NA	20/23	NA	23/24	2/22
	Kidneys; tubular casts	4/24	1/23	8/24	NA	14/23	NA	21/24	2/22
	Kidneys; infiltrate of mononuclear cells	8/24	5/23	20/24	NA	13/23	NA	18/24	4/22
	Kidneys; dilatation; tubule	1/24		1/24	NA	2/23	NA	6/24	1/22
	Liver; degeneration/necrosis							2/24	1/22
	Liver, hepatocellular hypertrophy			2/24	3/24	16/23	6/23	24/24	22/22
	Liver; vacuolation			5/24		8/23		10/24	1/22
	Thyroid gland; follicular hypertrophy				NA		NA	3/24
Reproductive data
Sperm parameters		NTRE
Estrous circle		NTRE
Enumeration of corpora lutea in HE-stained slides		NTRE
Enumeration of the number of primordial follicles on PCNA-stained slides		NTRE
Pre-coital interval		NTRE
Mating index (% mated)		95.8		100.0		100.0		95.8
Pregnancies		20		22		22		22
Fertility index		87.0		91.7		91.7		95.7
Gestation length		NTRE
Gestation index (%)		95.0		100		95.5		90.9
Live litters born		19		22		21		20
Live birth index (%)		99.0		100.0		99.7		100.0
Implantations		14.9		15.1		14.7		14.4
Mean percent of post-implantation loss (%)		7.8		8.7		10.0		9.7
Number of pups delivered (total)		262		304		276		262
Litter size (before cull)		13.8		13.8		13.1		13.1
Mean number of live pups at birth (before cull)		13.6		13.8		13.1		13.1
Offspring bw (g, on days 1 and 4 p.p before culling)		NTRE
Sex ratio (% M before cull)		49		52		49		43

NTRE= no treatment related effects

M = monocytes

APTT = Activated Partial Thromboplastin Time.

↑/↓= significantly increased/decreased

% compared to controls

* due to delivery difficulties on day 23

** On day 14 p.c. At necropsy, this female had 10 alive concepti and 9 early resorptions. There was red content in vagina that correlated with hemorrhage in lumen.

 

Table 4. Results F1 (between Days 1 to 21 p.p., before weaning)

Dose (mg/kg bw/day)		0		1		3		15
Endpoint		M	F	M	F	M	F	M	F
Mortality
·       Number of litters with found dead pups necropsied, Nb. (%)		4 (21)		2 (9)		2 (10)		1 (5)
·       Number of found dead pups necropsied (%)		4 (1.5)		2 (0.7)		4 (1.4)		6       (2.3)
-        pups with absence of milk in stomach, P/L		0/0		0/0		0/0		4/1
-        pups with autolysis, P/L		4/4		1/1		2/2		2/1
Viability index (%) (day 1-4 , before cull)		99.0		98.6		100.0		97.9
Sex ratio (day 21 % males)		50		50		47		41
Lactation index (%)(Days 4-21 p.p, before cull)		100.0		99.5		98.6		97.4
Body weight day 21		NTRE
Body weight gain		NTRE
Relevant clinical signs
·       - dehydratation, P/L								10/2
·       - cold to the touch, P/L		14/2						14/1
·       - emaciated appearance, P/L								9/2
·       - pallor (generalized), P/L								8/1
·       - absence of milk in stomach, P/L								1/1
AGD (corrected for cube root of BW, mm)		1.82	0.93	1.90	0.99	1.83	0.91	1.80	0.91
Nipple number on day 12	NTRE (0 nipples in males)
Macroscopy	NTRE

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

AGD: Anogenital Distance.

Nb.: Number;

P/L: Nb. of pups / Nb. of litters.

HCR: Historical control range

 

 

 

Table 5: Results F1 until termination COHORT 1A

Dose (mg/kg bw/day)		0		1		3		15
Endpoint		M	F	M	F	M	F	M	F
Mortality					1/20*
Relevant clinical signs: Hypersalivation (after treatment)				1/20		1/20	1/20	3/20	3/20
Body weight day 21		NTRE
Body weight gain		NTRE
Food consumption		NTRE
Time to first estrous		NTRE
Mean Estrous Cycle Parameters
·       Mean number of days of diestrus (± SD)		NTRE
·       Mean number of days of proestrus (± SD)		NTRE
·       Mean number of days of estrus (± SD)		NTRE
·       Mean number of days of metestrus (± SD)		NTRE
·       Mean number of cycles (± SD)			2.5		2.4		↓2.0		2.5
·       Mean duration of cycles (days) (± SD)		NTRE
·       Mean percent of females cycling normally (%)		NTRE
Haematology and coagulation:
·       RTC					↓17%
Clinical Biochemistry
·       Cl-						↑2%	↑2%	↑2%	↑2%
·       Ca++									↑4%
·       UREA (mmol/L)								↑26% (mean value within HCR)
·       CHOL (mmol/L)									↑56% (mean value within HCR)
·       TRIG (mmol/L)									↑500% (mean value within HCR)
·       ALP2c (U/L)					↓21%		↓23%		↓28% (mean value within HCR)
·       ASAT (U/L)									↓23% (mean value within HCR)
·       PROT (g/L)							↑7%	↑6%	↑15% (mean value within HCR)
·       ALB (g/L)							↑5%	↑6%	↑14% (mean value within HCR)
Urinalysis
·       pH						↓6%		↓8%
Splenic lymphocytes						NK cells↓ (34% abs./35% rel.)	NK cells ↑(49% abs.)	NK cells↓ (39% abs./ 31% rel.) B cells ↑(14% rel.)	NK cells ↑(74% abs / 71% rel.)
Thyroid hormones -TSH -T4		NTRE
Organ weights
·       Kidneys				↑16% abs. / ↑16% rel.		↑23% abs. / ↑24% rel.	↑7% rel.	↑39% abs. / ↑43% rel.	↑16% rel.
·       Liver						↑7% rel.	↑11% rel.	↑18% abs. / ↑23% rel.	↑36% abs. / ↑45% rel.
·       Pituitary gland					↑26% abs. / ↑23% rel.		↑24% abs. / ↑24% rel.		↑19% rel.
·       Adrenal glands						↑15% abs. / ↑15% rel.		↑23% abs. / ↑28% rel.
Sperm analysis		NTRE
Macroscopy	-Kidney: Enlarged, granular and/or discolorated			2/20	1/20	2/20		3/20	1/20
Microscopy	Kidneys; degeneration/regeneration; tubule			4/20	NA	16/20	NA	12/20
	Kidneys; accumulation; hyaline droplets			20/20	NA	20/20	NA	20/20
	Kidneys; basophilic tubules	7/20	2/20	15/20	NA	20/20	NA	20/20	3/20
	Kidneys; tubular casts	2/20		4/20	NA	7/20	NA	12/20
	Kidneys; infiltrate of mononuclear cells	4/20	2/20	5/20	NA	6/20	NA	4/20	4/20
	Kidneys; dilatation; tubule	1/20		1/20	NA	2/23	NA	14/20	1/20
	Kidneys; dilatation; pelvis	4/20		5/20	NA	3/20	NA	14/20	2/20
	Liver; degeneration/necrosis							1/20
	Liver, hepatocellular hypertrophy			7/20	1/19	13/20	10/20	20/20	20/20
	Liver; vacuolation				1/19		2/20		1/20
Enumeration of corpora lutea in HE-stained slides		NTRE
Enumeration of the number of primordial follicles on PCNA-stained slides		NTRE

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

% compared to controls

* prematurely euthanized on Day 8 on humane grounds

RTC: Reticulocyte Count.

Cl-: chloride; Ca⁺⁺: calcium; UREA: urea; CHOL: cholesterol; TRIG: triglycerides;  ALP2c: alkaline phosphatase 2C; ASAT: aspartate aminotransferase; PROT: total proteins; ALB: albumin.

HCR: Historical control reange

 

 

Table 6: Results F1 until termination COHORT 1B

Dose (mg/kg bw/day)	0		1		3		15
Endpoint	M	F	M	F	M	F	M	F
Mortality	1/20^(a)	1/20^(b)	2/20^(c)					1/20 ^(d)
Relevant clinical signs
-   Hypersalivation (after treatment)						2/20	1/20
-   Reflux at dosing						2/20		3/19
-   Decreased activity						1/20
-   Fur erected						1/20
-   Hunched posture						1/20
Body weight	NTRE
Food consumption	NTRE

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

% compared to controls

^(a) Prematurely euthanized on Day 94. Chromorhynorrhea was observed on Days 48 to 50. Alopecia (forelimbs) was noted from Day 86, scabs (right forelimb) were noted from Day 92 and swelling (right forelimb) was noted on the day of euthanasia. At necropsy, a fracture of right forelimb was noted.

^(b) Found dead on day 23 . No clinical signs were noted. No findings were noted at necropsy. Perforation of the esophagus was noted at necropsy. A brown thick content was present in the lungs and thoracic cavity. An issue during the gavage procedure was considered to be the cause of death

^(c) Found dead on Days 40 and 52. In one male, no clinical signs were noted and no findings were noted at necropsy, hence the cause of death was not evident. The other male was found dead on Day 52, reflux at dosing was observed on Day 8 and no findings were noted at necropsy.

^(d) Prematurely euthanized on Day 83. No clinical signs were noted. A fracture of left hindlimb was observed at necropsy. This death was not considered to be test item related

 

 

Table 7: Results F1 until termination COHORT 1C

Dose (mg/kg bw/day)	0		1		3		15
Endpoint	M	F	M	F	M	F	M	F
Mortality							1/20*
Relevant clinical signs: hypersalivation (after treatment)					2/20	1/20	3/19	1/20
Body weight	NTRE
Food consumption	NTRE

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

% compared to controls

* euthanized after falling from the cage on Day 23.

 

Table 8: Mean Age of Balanopreputial Separation and mean age at Vaginal Opening with Mean Body Weight on the Day of Occurrence in Males and females from all cohorts (1A, 1B and 1C)

Dose level (mg/kg bw/day)	0	1	3	15
Number of males	60	60	60	59
Number of positive responses	59	59	59	57
Number of negative responses	1	1	1	2
Mean age (days ± SD)	48.7 ± 2.67	49.2 ± 5.47	48.5 ± 3.22	48.7 ± 3.19
Mean body weight (g ± SD)	292.1 ± 32.74	296.4 ± 33.27	289.9 ± 34.53	294.5 ± 29.66
Number of females	60	59	60	60
Number of positive responses	60	59	58	59
Number of negative responses	0	0	2	1
Mean age (days ± SD)	33.3 ± 1.89	33.5 ± 2.40	34.9 ± 11.89	32.7 ± 1.66
Mean body weight (g ± SD)	125.0 ± 14.74	129.9 ± 17.70	131.2 ± 26.02	121.3 ± 13.20

No statistically significant differences vs. controls.

 

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be:
• 1 mg/kg/day in parental (P) generation (based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at = 3 mg/kg/day and on adverse degeneration/necrosis in liver of males and females at 15 mg/kg/day).
• Lower than 1 mg/kg/day in Cohort 1A (based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at = 1 mg/kg/day and on adverse degeneration/necrosis in liver of males at 15 mg/kg/day).

Executive summary:

The objective of this study was to provide general information concerning reproductive and developmental effects of the test item, AZDN, that may occur as a result of pre- and post-natal exposure. Systemic toxicity in pregnant and lactating females and young and adult offspring were also evaluated.

In the assay, sexually mature male and female rats [parental (P) generation] were exposed to the test item from 10 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 (cohorts 1A, 1B and 1C, without extension to mate the Cohort 1B animals to produce an F2 generation).

The test item was also administered to the F1 offspring from weaning to adulthood or euthanasia. Clinical observations and pathology examinations were performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems and the health, growth, development of the offspring.

Methods

Treatment

P generation and F1 lactating offspring:

Three groups of 24 male and 24 female Sprague-Dawley rats (P generation) received the test item, AZDN (batch No. B3J1117), at 1, 3 or 15 mg/kg/day, daily for 10 weeks prior to pairing, during pairing and gestation, and through lactation until weaning of the F1 pups [Day 22
post-partum (p.p.)]. The test item was administered orally (gavage, 5 mL/kg). A control group of 24 males and 24 females received the vehicle alone (corn oil), under the same experimental conditions.

F1 generations:

The test item was administered orally, by gavage (5 mL/kg) to groups of 20 rats/sex (Cohorts 1A, 1B and 1C) receiving 0 (vehicle alone: corn oil), 1, 3 or 15 mg/kg/day. The treatment schedules were the following:

• Cohort 1A: daily from weaning (Day 22 p.) until euthanasia (on Days 90 to 94 p.p.),


• Cohort 1B: daily from weaning (Day 22p.) for at least 10 weeks before euthanasia (after necropsy of cohort 1A, pending no alteration of estrous cycle or sperm parameters, and no macroscopic findings after examination of the reproductive organs ),


• Cohort 1C: daily from weaning (Day 22 p.) until euthanasia (after completion of sexual development testing).

Examination of Parental and F1 generations

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

P generation males and females were paired until mated or until 14 days had elapsed.

P generation females were allowed to deliver normally and rear their progeny. Pregnancy and litter parameters were recorded.

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

Examination of Cohorts

Cohort 1A: animals were selected on Day 22 p.p. 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 14 days before the end of the treatment period.

Cohort 1B: animals were selected on Day 22 p.p. to potentially obtain additional histopathology data.

Cohort 1C: additional animals were selected on Day 22 p.p. for assessment of sexual maturity in F1 animals.

Terminal examinations

A macroscopic post-mortem examination was performed on all P and F1 animals (including F1 pups culled on Day 4 p.p. and F1 pups not selected on Day 22 p.p.). This included examination of the 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 ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated and organs were weighed wet as soon as possible after dissection. A microscopic examination was performed on all macroscopic lesions and tissues (complete list) from animals in all groups or in the control and high-dose groups.

P generation and Cohort 1A: the first 10 surviving animals/sex/group were fasted (food only) for an overnight period of at least 14 hours prior to blood sampling (for hematology, coagulation and blood biochemistry) and urine collection (for urinalysis) at termination. A series of sperm cell evaluations was performed on surviving males from all groups: motility, morphology, sperm cell head count in testicular/epididymal tissues.

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

Thyroid hormone levels (P generation, F1 culled pups and Cohort 1A animals): prior to blood sampling the animals were not fasted (except for animals from which samples were collected for hematology, blood biochemistry and urinalysis). Blood samples were taken on Day 4 p.p. (F1 pups) for measurement of thyroid hormone (T4) levels. Blood samples were also taken on Day 22 p.p. (F1 pups not selected for Cohorts) and at termination [the first ten surviving males/group and the first ten lactating females/group (P generation animals)] for measurement of thyroid hormone (T4) and Thyroid Stimulating Hormone (TSH) levels.

Results

The concentrations in the dose formulations prepared for treatment of the P Generation and Cohorts were all found to be within an acceptable range of variations (measured concentrations at ± 15% of the theoretical concentrations). No test item was found in the control dose formulation.

P generation

Mortality: there were no test-item related deaths.

Clinical signs: there were no test-item related adverse clinical signs

Mean body weight, mean body weight change and mean food consumption: there were no test item-related adverse effects.

Estrous cycle, mating, fertility and delivery: there were no effects

Pre-weaning F1 pups:

Mortality: there were no effects on the repartition of deaths in F1 lactating pups and no test item-related findings at macroscopic post-mortem examination of found dead pups.

F1 pup survival during lactation: there were no effects on the F1 pup viability and lactation indexes.

Clinical observations during lactation: there were no adverse findings.

Body weight and body weight change: there were no effects.

Pup development during lactation: there were no effects on the Day 1 p.p. mean anogenital distance (AGD) or normalized AGD in males or females. There were no nipples or areolae in male pups examined on Day 12 p.p.

Cohorts 1A and 1B

Mortality: there were no test item-related deaths.

Clinical signs: there were no adverse clinical signs.

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

Time to first estrous and estrous cycles: there were no effects in Cohort 1A females.

Sexual development:

Males: there were no effects on the mean age at balanopreputial separation.

Females: there were no effects on the mean age at vaginal opening,

Laboratory investigations:

Hematology, coagulation, blood chemistry and urinalysis in P-generation and/or Cohort 1A animals: there were no adverse findings.

Thyroid hormones: there were no effects on TSH or T4 plasma levels.

Sperm analysis: there were no effects on sperm parameters (motility, morphology, sperm/testicular numerations and daily sperm production rate) in P-generation or Cohort 1A males.

Lymphocyte subtyping: there were no adverse findings in Cohort 1A animals.

 

 

Pathology:

P generation: No test item-related mortality was observed. Test item-related increased kidney weights were noted in males treated at ≥ 1 mg/kg/day. This correlated with enlargement, granular aspect and tan discoloration at gross observation and microscopic degenerative/inflammatory lesions in males (considered as adverse at ≥ 3 mg/kg/day).  Increased liver weights were noted in males treated at ≥ 3 mg/kg/day and in females treated at 15 mg/kg/day. This correlated with increased incidence of accentuated lobular pattern in males at ≥ 3 mg/kg/day and microscopic non-adverse hypertrophy and vacuolation. Hepatocellular degeneration/necrosis was recorded at 15 mg/kg/day and was considered to be adverse, although seen in only 2/20 males and 1/24 females at minimal severity. In addition, non-adverse minimal follicular hypertrophy was seen in the thyroid glands from males treated at 15 mg/kg/day and correlated with increased weights. At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and the control groups.

Cohort 1A: No test item-related mortality was observed. Increased kidney weights were noted in males treated at ≥ 1 mg/kg/day. This correlated with enlargement, irregular color and tan discoloration at gross observation and microscopic degenerative/inflammatory lesions in these males (considered as adverse at ≥ 1 mg/kg/day). Increased liver weights were noted in males and females treated at ≥ 3 mg/kg/day. This correlated with microscopic non-adverse hypertrophy and/or vacuolation. Hepatocellular degeneration/necrosis was recorded in males at 15 mg/kg/day and was considered to be adverse, although seen in only 1/20 males at minimal severity. At quantitative evaluation of primordial follicles or corpora lutea, there were no differences between the high-dose and the control groups.

Cohort 1B: No test item-related mortality and no organ weight differences or gross findings were noted. Test item-related degenerative/inflammatory findings were noted in the kidneys of males treated at ≥ 1 mg/kg/day.

Cohort 1C: There was no test item-related mortality. There were no test item-related gross findings.

Non selected pups from PND22: There were no test item-related organ weight differences or gross findings.

Conclusion

The test item, AZDN, was administered daily by oral gavage, at dose level of 0, 1, 3 or 15 mg/kg/day, to sexually mature male and female rats [parental (P) generation] from 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, the F1 generation was also exposed to the test item and was assigned to Cohorts of animals for reproductive/developmental toxicity.

Systemic toxicity evaluation:

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be:

• 1mg/kg/day in parental (P) generation (based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at ≥ 3 mg/kg/day and on adverse degeneration/necrosis in liver of males and females at 15 mg/kg/day).


• Lower than 1 mg/kg/day in Cohort 1A (based on microscopic degenerative/inflammatory lesions in kidneys of male Sprague-Dawley rats considered as adverse at ≥ 1mg/kg/day and on adverse degeneration/necrosis in liver of males at 15 mg/kg/day).

Reproductive/developmental toxicity evaluation:

The No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be 15 mg/kg/day in the Sprague-Dawley rats, based on the absence of adverse effects at this dose level.