Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Jan. 25, 2011 to March 22, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
see below

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species and strain: Wistar RJHan:WI rats
Source: Laboratoire Elevage Janvier, B.P. 4105, Route des Chênes Secs, 53940 Le Genest-St-Isle CEDEX France
Hygienic level: SPF at arrival; standard laboratory conditions during the study
Justification of species/strain: The rat is regarded as suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.
Number of animals: Main groups: 48 male, 48 female rats, 12 animals/sex/group, 4 groups; a sufficient number of at least 8 pregnant females/group was achieved. Recovery groups: 10 male, 10 female rats, 5 animals/sex/group, 2 groups, Control and High dose. Positive Control MNT group: 12 male and 12 female rats, 1 group. At the completion of the study, the spare animals were returned to LAB Research Ltd. spare colony, as their use was not required (no replacements with spare animals were performed).
Age of animals: Young adult rats, approximately 11-12 weeks old at starting and 13-14 weeks at mating. The age range within the study was kept to the minimum practicable.
Body weight range: Males: 397–463 g, Females: 229-273 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment
Acclimation period: At least 7 d (7 d from animal arrival to pre-treatment ophthalmoscopy examination, 12 d to onset of treatment)

Husbandry

Animal health: Only healthy animals were used for the test, as certified by the veterinarian. Females were nulliparous and non-pregnant.
Room number: 524
Cage type: Type II and/or III polypropylene/polycarbonate
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Details of bedding quality are reported.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.6-23.6°C
Relative humidity: 31 - 62%
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 5 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
The temperature and humidity were measured twice daily; no deviations from the target ranges were noted during the study.

Food and water supply

Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.

Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary).

The food and water are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Animal identification

Each parental animal (P Generation) was identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at LAB Research Ltd. This number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group.
The boxes were arranged in such a way that possible effects due to cage placement were minimized and were identified by cards showing the study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery.

The new-borns (Offspring, F1 Generation) were identified by cutting off digit-tips up to one day after birth.

Randomization

All parental (P) male and female animals were sorted according to body weight by computer and divided to weight ranges. An equal number of animals from each weight group was randomly assigned to each dose group to ensure that test animals were as nearly as practicable of a uniform weight. The grouping was controlled by SPSS/PC software according to the actual body weight, verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Details on exposure:
The test item was formulated in distilled, sterile water for injection at 6.25, 25 and 100 mg/mL concentrations without correction for purity, in the Central Dispensary of LAB Research Ltd. Formulations were prepared and stored refrigerated at 2-8ºC pending use within 7 d.

Rationale for dose selection and route of administration

The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work, including the results of an acute oral toxicity study (LAB study code 10/296-001P) and a repeated dose range finding study in the rat (LAB study code 10/296-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The oral route was selected as it is a possible route of exposure to the test item in humans.

Test item or Control (water)-treated Groups 1-4 Main animals were administered the dosing solutions daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight. Dosing of both sexes began after at least 7 days acclimation (A) and 12 days after the animal arrival; the animals were dosed for 2 weeks before mating, during the mating/post-mating, and were continued up to and including the day of necropsy.

Vehicle:
Name: Distilled, sterile water for injection, PhEUR
Lot No.: 3590210, 7530810, 8490910
Manufacturer: TEVA Pharmaceutical Corporation
Expiry Date: February 2013, August 2013, September 2013 respectively
Storage: Room temperature
Details on mating procedure:
Main and Positive Control MNT animals:

Mating began after the animals have attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 6 d. A vaginal smear were prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were caged individually. Mating pairs were clearly identified in the data, mating of siblings was avoided.

Recovery animals:

Recovery animals were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Top, middle and bottom duplicate samples were taken from test item formulations on 3 occasions, during the first and last weeks and approximately midway during the treatment, one set to analyze (which was collected in replicates as practical) and one set as a back-up for any confirmatory analyses. Similarly, one sample was taken in duplicate from the vehicle Control Group 1 solution for concentration measurements.

The samples were evaluated by UV-HPLC method.

The measured concentrations varied between 95% and 103% of the nominal concentrations (6.25, 25 and 100 mg/mL). No test item was detected in the Control solution samples. These results were considered suitable for the study purposes.
Duration of treatment / exposure:
Males were dosed for at least 28 d (14 d pre-mating, 14 d mating/post-mating period and on the day of necropsy).
Females were dosed for 14 d pre-mating, for up to 6 d mating period, through gestation and up to and including the day of necropsy (at least 4 d post-partum dosing). The day of birth (viz. when parturition was complete) is defined as Day 0 post-partum. Females showing no-evidence of copulation were sacrificed as practical, 26-27 d after the end of the mating period.
Frequency of treatment:
Once daily, 7 d per week
Details on study schedule:
See below for details of experimental schedule under any other information on materials and methods.
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Main groups: 48 male, 48 female rats, 12 animals/sex/group, 4 groups
Recovery groups: 10 male, 10 female rats, 5 animals/sex/group, 2 groups, Control and High dose
Control animals:
yes, concurrent vehicle
Details on study design:
See below under any other information on materials and methods for details of experimental design.
The study design for the in vivo mucronucleus test is detailed in Chapter 7.6.2.
Positive control:
A positive control group was included for the in vivo micronucleus test - see Chapter 7.6.2 for detaills.

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS AND FUNCTIONAL OBSERVATION BATTERY (FOB)

All animals: Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon (pm) as practical during the working day, as no peak period of effects was noted after dosing during the first days of treatment. During Recovery period, the animals were similarly observed daily as practical.

All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including onset, degree and duration of signs as applicable.

More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning (am) or before treatment. These observations were made outside the home cage in a standard arena, at similar times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical signs were observed during the study.

Main animals, 5 males and 5 females/group, “subgroup A”:

Assessment of any potential test item related neurotoxicity was performed during the last exposure week (males, on Day 24 am, females, on PND 3 am). In order to avoid hyperthermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes. Selected animal were subjected to the functional observation battery, including qualitative assessment of the grip strength, and to measurements of the landing foot splay and fore/hind grip strength.

To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rats were dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured.

Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support; results are tabulated with individual and mean data.

Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed. Parameters such as, but not limited to body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.

Recovery animals: Neurotoxicity evaluation was similarly conducted in the Recovery animals towards the end of the Recovery period, 1 d before necropsy (Day 55, for necropsy on Day 56).

BODY WEIGHT MEASUREMENT

All adult Main and Recovery animals were weighed with accuracy of 1 g for randomization purposes, then on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition) and PPD4 (before termination). Body weights of the female animals were additionally weighed on gestational Days GD10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION MEASUREMENT

Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly (see Study Schedule).

OPHTHALMOLOGY

The fundus of eyes of all animals was examined before treatment. Five male and 5 female Control and High dose animals (“subgroup C”) randomly selected from groups 1 and 4, during the last week of treatment prior to necropsy (males, Day 24 pm, females, PND 3 pm). Mydriasis was produced after instillation of a mydriatic agent (eye drops "Mydrum") into the conjunctival sac. The examination was performed using a Gowlland ophthalmoscope. As no ophthalmoscopic alterations were found, no additional examination was performed in other animals.

OBSERVATION OF THE DELIVERY PROCESS, OFFSPRING AND NURSING INSTINCT

Main Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations were recorded as applicable. No evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy. Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded as applicable.

CLINICAL PATHOLOGY

All animals selected for blood sampling were fasted (overnight period of food deprivation).

For terminal blood sampling of Recovery animals, 3 samples were taken from each animal: one for haematology (1.2 mL blood, in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (1.4 mL blood for APTT and PT measurements, in tubes with sodium citrate as anticoagulant) and one to obtain serum (approximately 1 mL blood as practical, in tubes with no anticoagulant) for clinical chemistry.

For Day 14 blood sampling of Main animals selected (subgroup B), 2 samples were taken from each scheduled animal: one for haematology (1.2 mL blood, in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood) and one to obtain serum (approximately 1 mL blood as practical, in tubes with no anticoagulant) for clinical chemistry. Due to technical problems at use of the VITROS 250 equipment, clinical chemistry results collected on Day 14 as available will be archived with the study file but are not reported. Additional blood samples were collected for clinical chemistry evaluation from the subgroup B Main animals prior to the scheduled necropsy by heart puncture under pentobarbital anaesthesia, into empty tubes with no anticoagulant to obtain serum.

For terminal blood sampling of Main animals selected (subgroup B), 2 samples were taken from each scheduled animal: one to obtain serum (approximately 1 mL blood as practical, in tubes with no anticoagulant) for clinical chemistry and one for blood clotting times (1.4 mL blood for APTT and PT measurements, in tubes with sodium citrate as anticoagulant).

For urine collection, the selected animals (Main subgroup B and Recovery) were placed in metabolic cages for approximately 16 hours and food and water deprived, then water were provided at libitum for at least approximately 2 hours prior to necropsy and organ weight measurements.

Main animals, 5 males and 5 females/group, “subgroup B”:

Laboratory examinations for haematology and clinical chemistry evaluation were conducted at the end of pre-mating period, on blood samples collected from the sublingual vein, prior to the start of mating on Day 14 from 5 animals/sex/group randomly selected (“subgroup B”). Coagulation evaluation (APTT and PT) was performed at the completion of the treatment, on blood samples collected by cardiac puncture from subgroup B animals under pentobarbital anaesthesia, immediately prior to scheduled necropsy.

Urine sampling (approximately 16 hours sampling period) was performed prior to necropsy from the same subgroup B (urinalysis on Day 28-males, PND 5-females).

Recovery animals:

Haematology, coagulation and clinical chemistry investigations were conducted at the completion of the Recovery period, 14 days after the first scheduled euthanasia of Main dams. Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.

Urine sampling (approximately 16 hours sampling period) was performed from the Recovery animals prior to necropsy (urinalysis on the day of necropsy, conducted 14 days after the first scheduled euthanasia of the Main dams).

Haematology and blood clotting times

The parameters evaluated are detailed in a table under any other information on materials and methods in Chapter 7.5.1.

Clinical chemistry

The parameters evaluated are detailed in a table under any other information on materials and methods in Chapter 7.5.1.

Urinalysis

The parameters evaluated are detailed in a table under any other information on materials and methods in Chapter 7.5.1.

Oestrous cyclicity (parental animals):
A vaginal smear were prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines).
Sperm parameters (parental animals):
For the adult animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups (Main and Recovery) and all macroscopic findings (abnormalities) from all animals. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Litter observations:
Each litter were examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Observations are reported individually for each adult animal. In addition to the observations on parent animals, the pups (offspring) were monitored for any behavioural changes. Live pups were counted, sexed, weighed individually within 24 h of parturition (ex. Day 0 or 1 post-partum, PND0 or 1) and on PND4, with accuracy of 0.01g. All the litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination in order to identify the possible cause of death. All observed abnormalities were recorded.
Postmortem examinations (parental animals):
Pathology

Gross necropsy was performed on all animals. Terminally, after completion of the treatment or Recovery periods as applicable, animals were sacrificed under pentobarbital anaesthesia followed by exsanguination. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded in the Main females as applicable.

At the time of termination, body weight and weight of the following organs of all parental animals were determined:

- With a precision of 0.01 g: uterus (with and without cervix), vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, brain
- With a precision of 0.001 g: ovaries, pituitary

The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerve were retained in modified Davidson’s fixative. Testes and epididymides were preserved in Bouin’s solution, all other organs in 10% buffered formalin solution.

In addition, for 5 animals/sex/group (subgroup B, Main) and for Recovery animals, the following organs and tissues, or representative samples, were preserved:

Gross findings, Liver, Small intestine (8), Adrenals, Lungs with bronchi (5), Spinal cord (cervical, lumbar, and thoracic levels), Animal identification (1), Lymph nodes (6), Aorta, Mammary gland (inguinal), Spleen, Brain (2), Ovaries with oviduct, Sternum with marrow, Epididymides, Pancreas, Stomach, Eyes with optic nerves (7), Pituitary, Testes, Oesophagus, Prostate, Thymus, Femur with marrow , Salivary gland (mandibular), Thyroid with parathyroids (7), Heart (3), Sciatic nerve, Tongue, Kidneys, Seminal vesicles, Trachea (with main stem bronchi), Large intestine (4), Coagulating glands, Urinary bladder, Lacrimal glands, Skeletal muscle (quadriceps), Uterus (9), Harderian glands, Skin and subcutis (inguinal), Vagina.

1. Fixation and preservation only.
2. Cerebral cortex, midbrain, cerebellum and medulla.
3. Section including both ventricles and atria, septum with papillary muscle.
4. Caecum, colon and rectum.
5. Lungs of euthanized animals were infused with formalin.
6. Mandibular and mesenteric.
7. Parathyroids and optic nerves were examined histologically only if present in routine sections.
8. Duodenum, ileum and jejunum with Peyer’s patches.
9. Horns, body and cervix.


From subgroup B Main animals and Recovery animals, the following organs were weighed in addition to the ones previously mentioned:

- With a precision of 0.01 g: heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals.

For all organs, paired organs were weighed individually. Individual and/or paired absolute organ weight are reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) were calculated and reported.

For the adult animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups (Main and Recovery) and all macroscopic findings (abnormalities) from all animals. As no test item related pathology findings were noted, no additional histopathology evaluation was considered required. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6 µ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.
Postmortem examinations (offspring):
Pups euthanized at PND 4 were carefully examined at least externally for gross abnormalities. Any pups showing abnormalities in structure or behaviour, including the pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination, in order to identify the probable cause of death if possible.
Statistics:
Data were recorded on the appropriate forms from the relevant SOPs of LAB Research Ltd., and then tabulated using the Microsoft Office Word and/or Excel, as appropriate. Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations.

The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.
Reproductive indices:
Formulae for Calculation of Mating and Fertility Indices are included in pdf attached under background information.
Offspring viability indices:
Formulae for Calculation of Pups’ Mortality and Sex Ratio Indices are included in pdf attached under background information.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
Expected staining effect on urine
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY
No test item related mortality occurred during the study.
Low dose female 2504 was found dead on Day 39, due to incidentally difficult parturition. Prior to its death, this female displayed no clinical signs between Days 0 and 37 of treatment. On the day of parturition (treatment Day 38), after delivery of 9 live pups, vaginal bleeding, paleness, piloerection, decreased activity and increased respiration were noted, followed by death on PPD1. At necropsy, one foetus was found dead in the uterus of this female, placed in both uterine horns (transversally), which was considered the cause of the difficult parturition followed by bleeding and incidental death. In addition, dark red, non-collapsed lungs, red fluid in the thoracic cavity and dark, as possible post-mortem changes, and orange discoloration in the stomach due to the coloring effect of the test item were noted.

CLINICAL OBSERVATIONS
In the surviving animals, no clinical signs were noted following daily administration by oral gavage under the conditions of this study, or in the Control animals administered 10 mL/kg vehicle (distilled, sterile water for injection).
In the Main animals, it should be mentioned that dark yellow urine was noted at 250 mg/kg bw/day in 5/5 male and 5/5 female Mid dose animals, and orange, or dark orange at 1,000 mg/kg bw/day in all High dose Main animals, at urinalysis performed prior to necropsy after the animals weof the test substance or its metabolites through urine (urine collection in metabolic cages) and an expected staining effect.

NEUROLOGICAL ASSESSMENT
There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed towards the end of the treatment or Recovery periods.
Increased vocalization was observed on occasion in the animals throughout all the dose groups when subjected to the modified Irwin test (functional observation battery). However, no treatment-related differences to the Control, or dose, or gender related response, were noted, and this sign was considered to be without toxicological significance and within the normal biological variation with respect to behaviour, reactions to different type of stimuli or manipulations.
No test item related effects or statistically significant variations were observed in the landing foot splay test. When compared to Control, there were no statistically or toxicologically significant differences in the mean grip strength values of the forelimbs or hind limbs in the Main animals when evaluated on Day 24 am (males) or PND 3 am (females). In the Recovery 1,000 mg/kg bw/day High dose animals, slightly higher than Control mean grip strength of the forelimbs was observed in the females (24%, p<0.01), however, difference was minor, the mean value was lower than Control in the High dose males, without attaining statistical significance, and these variations were regarded as incidental and not to reflect an adverse or a test item related effect.

OPHTHALMOLOGY
No test item related changes compared to pre-treatment were noted during ophthalmoscopy examination of 5 male and 5 female Control and High dose Main animals during the last week of treatment prior to necropsy (males, Day 24 pm, females, PND 3 pm), thus no additional evaluation was required in other dose groups or Recovery animals.

BODY WEIGHT AND BODY WEIGHT GAIN
No adverse effects considered toxicologically significant were noted on the mean body weight and body weight gain values following daily administration of the test substance at dose levels of up to and including 1000 mg/kg bw/day, either during the treatment or Recovery periods.
In the Main animals, there were no statistically significant differences to Control. In the Recovery animals, lower mean body weight (-5%, p<0.05) and body weight gain (p<0.05) were noted in the High dose males during the second week of treatment; as no similar observations were recorded in the females, or in the Main animals and this variation occurred with isolated incidence, with no associated adverse effects, it was ascribed to individual variability (i.e. male 4017) with no toxicological significance.

FOOD CONSUMPTION
There were no test item-related differences to Control in the mean daily food consumption in any test-item treated Main or Recovery group (62.5, 250, or 1000 mg/kg bw/day) when compared to the Control.
Minor differences to Control were noted or variations within the group, generally associated with changes in the study schedule including mating, delivery, or fasting before blood collection for clinical pathology evaluation, unrelated to treatment and with no statistical or toxicological significance.

CLINICAL PATHOLOGY
- HAEMATOLOGY
No test item related effects, or changes considered toxicologically significant were noted in the haematology parameters evaluated in either Main or Recovery animals.
Variations were noted in a few parameters, on occasion attaining statistical significance, including statistically higher MPV in the Main High dose males, (8%, p<0.05), lower MPV in the Recovery High dose females (-11%, p<0.05), higher MCV (4%, p<0.05) or NE (32%, p<0.05, but -22 % lower in the males) in the Main High dose females. Evaluation of the mean and individual results in comparison with the Control data did not reveal any test-item related cause of the changes noted, and/or no consistent dose or gender-related response was observed. Therefore, these differences observed between the Control and treated groups were considered to be incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range or were with no toxicological significance.

- CLINICAL CHEMISTRY
In the Main animals evaluated at the completion of the treatment prior to necropsy, an apparent total bilirubin T BIL increase was noted in the 1000 mg/kg bw/day Main High dose males and females (74% and 61%, respectively, p<0.01). This was considered to be related to a possible spectral interference with the analytical method caused by the discolouration of the serum by the test item and not to reflect an adverse effect on the liver function. Although T-BIL was 25% higher than Control in the High dose 1,000 mg/kg bw/day Recovery females on Day 56, the difference did not attain statistical significance, was not observed in the males, and was ascribed to individual variability (for example, female 4515) and not a test item related effect.
Other clinical chemistry parameters showed on occasion statistically significant variations, however, there was no dose or gender response or the values were within the physiological ranges. For this reason, these variations were not considered toxicologically significant or related to treatment.

- URINALYSIS
Daily administration by oral gavage at up to and including 1,000 mg/kg bw/day did not result in any test item-related effects considered adverse at urinalysis performed prior to necropsy in either Main or Recovery animals.
In the Main animals, dark yellow urine was noted at 250 mg/kg bw/day in 5/5 male and 5/5 female Mid dose animals, and orange, or dark orange at 1000 mg/kg bw/day in all High dose Main animals (2/5 males and 3/5 females, dark orange, and 3/5 males and 2/5 females, orange), at urinalysis performed prior to necropsy after the animals were placed overnight in metabolic cages for urine collection. These changes were ascribed to elimination of the test substance or its metabolites through urine (urine collection in metabolic cages) and an expected staining effect.
The volume of urine and/or urinary pH showed minor variations, on occasion statistically significant, however, with no dose or gender-dependent, and not considered to be of toxicological importance in correlation with test item administration in the conditions of this study. The few other minor variations observed did not attain statistical significance and/or were regarded as normal background changes.

OESTRUS CYCLE, REPRODUCTIVE ABILITY ASSESSMENT AND INDICES
There were no statistically significant differences between the Control and test item-treated groups with regard to reproductive ability or in the mating or gestation indices, or effects considered adverse or toxicologically significant. The mating indices were 100% in all groups. The fertility indices were 100% in the Control group and 92% in the test item treated groups, due to 1/12 non-pregnant females in each group, values which are comparable with concurrent control data in Wistar rats. Gestation index was 100% in all groups.
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 6 d of pairing (cohabitation).

EVALUATION OF THE GESTATION, PARTURITION AND POST-PARTAL PERIOD
No test item effect on the duration of pregnancy or abnormalities in the gestation outcome ascribed to the treatment were observed.
The mean duration of pregnancy was similar in the control and test item treated groups and varied from 21.08 d (21 to 22 d) in the Controls, 21 d in the Low 62.5 mg/kg bw/day and Mid 250 mg/kg bw/day dose groups, to 21.27 d (21 to 23 d) in the 1,000 mg/kg bw/day high dose group, values comparable with the contemporaneous historical control data (gestation length: 22.40 ± 0.53 d, range: 22 to 23 d, n = 22). All the parturitions were normal.
The number of corpora lutea and implantation sites were comparable in the treated groups up to and including 1,000 mg/kg bw/day with the mean value recorded in the Control group.
There were no statistically significant differences or effects that could be ascribed to treatment on the pre/post-implantation, post-natal or total mortality values (%) at up to and including 1,000 mg/kg bw/day. The pre-implantation and/or total intrauterine mortality values (%) appeared to be slightly higher in the Mid or High dose groups, however, without statistical significance or any dose response, and thus ascribed to biological variability, unrelated to treatment.

PATHOLOGY EVALUATION
- TERMINAL
-- MAIN (DAY 28 or PND5)
--- Parental Generation (including subgroup B)
Macroscopic Findings:
Treatment-related macroscopic findings were observed in the stomach, small intestine, cecum, colon and rectum. Dark discoloration red/orange of the stomach, small intestine, cecum, colon and/or rectum was recorded in 13/23, 24/24 and 24/24 surviving adult rats from the Low, Mid and High Dose groups, respectively.
All other macroscopic findings recorded including dark, red discoloration or pale mottling of the lungs or seminal vesicle, spleen enlargement, dilatation of the renal pelvis, or dark red, bilateral discoloration of the cornea were incidental or terminal procedure-related.

Microscopic Findings:
There were no test item-related findings observed histologically in the stomach, small intestine, colon, cecum and rectum and no correlation with the macroscopic findings noted at necropsy was observed.
No evidence of test substance-related histological findings was recorded in the High and Control animals in the reproductive organs. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females. Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoons were similar in Control and High Dose males.

-- RECOVERY (DAY 56)
Macroscopic Findings:
The sporadic, macroscopic changes observed both in the male and female rats were incidental or terminal procedure-related.

Microscopic Findings:
There was no evidence of the test item-related microscopic findings. The changes in the kidneys, spleen, pituitary, lungs, liver, uterus and prostate were seen unilaterally and/or with low incidence or severity and were considered to be incidental, or were regarded as terminal procedure-related or incidental
In summary, a daily oral (gavage) administration of the test substance to Wistar RJHan:WI rats was associated with treatment-related dark red/orange discoloration of the stomach, small intestines and/or cecum, colon and/or rectum recorded in 13/23, 24/24 and 24/24 surviving adult rats from the 62.5, 250 and 1,000 mg/kg bw/day Mid and High Dose Parental Main rats, respectively. Histologically, no test-item related changes could be observed in the above mentioned organs. Following a 14 d recovery period, no test item-related macroscopic or histological findings were seen at a dose level 1,000 mg/kg bw/day in Parental Generation.

ORGAN WEIGHTS
There were no toxicologically significant changes in organ weight values noted at up to and including 1,000 mg/kg bw/day, evaluated immediately after completion of the treatment, at necropsy on Day 28 (Main males), PND5 (Main females) or after additional 14 d after the first scheduled euthanasia of the dams on Day 41, with necropsy on Day 56 (Recovery male and female animals).
The mean spleen or liver absolute and/or relative weight adjusted for the body or brain weight was slightly higher than control, statistically significant in the Main 250 mg/kg bw/day Mid dose males, or in the Recovery 1,000 mg/kg bw/day High dose females (spleen only). In the absence of any dose or gender response, or of any clinical pathology, macroscopic or microscopic changes, these variations were not considered toxicologically significant or related to treatment.
Other absolute organ weights or relative to the body and/or brain weights were similar in the control and test item treated groups, or showed minor variations, ascribed to biological variability (for example vagina weight in the Recovery 1,000 mg/kg bw/day High dose females).

Results of the in vivo micronucleus test are detailed in Chapter 7.6.3.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Gross pathological findings:
no effects observed

Details on results (F1)

MORTALITY AND CLINICAL OBSERVATIONS

Test substance administration to parental generation at up to 1,000 mg/kg bw/day under the conditions of this study did not lead to any adverse effects considered related to treatment or toxicologically significant in the F1 generation.
The pups found dead and cannibalised were counted and sex determined if possible, but not further examined macroscopically. A few surviving pups were cold to touch, not suckled and/or cyanotic. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. The incidence of these findings was low, within the physiological range expected in the population of Wistar rats and considered without toxicological significance, or to reflect a test item or adverse effect. In the Control group (155 pups examined), 4 pups were cannibalised and 5 were found dead and intact between PND 0 and 4, 2 with positive and 2 with negative lungs flotation test. In the 62.5 mg/kg bw/day Low dose group with 141 pups examined, 4 pups were cannibalized and 3 pups were found dead and intact on PND0, with negative lungs floating test. In the 250 mg/kg bw/day Mid dose group, 1/136 pups was found dead on PND4 without having any previous clinical signs and 3 were cannibalized between PND1 and 3. In the 1,000 mg/kg bw/day High dose group, 4/140 pups were found dead, 1/4 on PND0, with positive lungs flotation test, and 3 on PND4, found autolysed at necropsy, without having any previous symptoms between PND0 and 3; 7 were cannibalized between PND1 and 3.
The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 when evaluated as litter data were comparable to control values at up to and including 1,000 mg/kg bw/day, or showed minor variations ascribed to individual, biological variability.
The sex ratios were slightly lower than control in all treated groups, attaining statistical significance both on PND0 and PND4 in the 1,000 mg/kg bw/day High dose group. Statistically significant differences to Control were also observed in the number of male/females pups on PND0 and/or 4 in the High and/or Mid dose groups. Although a test item related effect cannot be excluded, this variation was considered due to an incidentally higher number of female foetuses in the Control group and individual lower number in the High dose females 4501 and 4503, where the sex ratio is expected to be approximately 50%.

BODY WEIGHT AND BODY WEIGHT GAIN
There were no effects considered adverse on the offspring weight or weight gain following administration of the test substance at 62.5, 250 or 1000 mg/kg bw/day to parental generation under the conditions of this study.
When evaluated per litter basis, the mean litter weights on PND 0 and 4, pups body weight and/or body weight gain evaluated on PND 0 and 4 showed no statistically or toxicologically significant differences compared to controls in the F1 generation.
When evaluated for all pups, statistically significant, slightly higher than Control values were noted in the mean body weight and body weight gain values in the Mid and/or High dose groups, on PND0 and/or PND4. As no test item related clinical signs or macroscopic findings were noted, the higher values observed when the body weight and body weight gain values were calculated for all pups and not per litter basis were ascribed to individual variability and smaller number of pups compared to the Control.

PATHOLOGY EVALUATION
- FOUND DEAD
-- F1 Generation
Thirteen intact pups were found dead between Days 0-4 post-partum. Necropsy was performed on 5, 3, 1 and 4 pups from Control, Low, Mid and High Dose groups, respectively. Positive floating test was observed in 2/5 and 1/4 animals from the Control and High Dose groups, respectively. Negative floating test was seen in 2/5 Control, and 3/3 Low Dose pups. Autolysis of the abdominal and/or thoracic organs was found in four pups (1506/9 and 4512/7, 8, 17). Additionally, colorless, clear liquid in the abdominal cavity was also noted in one Mid Dose animal (3503/11).

- TERMINAL
-- MAIN (DAY 28 or PND5)
--- F1 Generation
Macroscopic Findings:
No macroscopic changes were seen in F1 offspring generation euthanized and examined externally at scheduled termination.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Tabular Summary

 

Dose (mg/kg bw/day)

Control

62.5

250

1000

Pairs started (N)

12

12

12

12

Surviving females showing evidence of copulation (N)

12/12

12/12

12/12

12/12

Females achieving pregnancy (N)

12/12

11/12

11/12

11/12

Conceiving days 1 - 5 (N)

12

12

11

12

Conceiving days 6 - 14 (N)

0

0

1

0

Pregnancy ≤ 21 d (N)

11

11

11

9

Pregnancy = 22 d (N)

1

0

0

1

Pregnancy ≥ 23 d (N)

0

0

0

1

Dams with live young born (N)

12/12

11/11

11/11

11/11

Dams with live young at PN4 (N)

12/12

10/11#

11/11

11/11

Corpora lutea/dam (mean)

20.83

18.64

21.00

19.82

Implantations/dam (mean)

14.08

13.64

13.36

14.27

Live pups/dam at birth (mean)

12.67

12.55

12.36

12.64

Live pups/dam at day 4 (mean)

12.17

12.50#

12.00

11.73

Sex ratio at birth (mean)

61.22

52.30

50.68

40.79**DN

Sex ratio at PN4 (mean)

61.27

52.89

49.51

39.44**DN

Litter weight at birth (mean)

84.9

84.7

84.5

86.4

Litter weight at day 4 (mean)

134.6

138.1

140.6

129.2

Pup weight at birth (litter mean)

6.60

6.63

6.91

6.81

Pup weight at day 4 (litter mean)

11.11

11.18

11.81

11.24

STRUCTURALLY ABNORMAL PUPS

Dams with 0/ Dams with live born

12/12

11/11

11/11

11/11

Dams with 1 or ≥2

0

0

0

0

LOSS OF OFFSPRING#

Pre-implantation (corpora lutea minus implantations)

Females with 0

1/12

2/12$

2/12$

2/12$

Females with 1

1/12

1/12$

1/12$

0/12$

Females with 2

0/12

1/12$

0/12$

1/12$

Females with ≥3

10/12

8/12$

9/12$

9/12$

Pre-natal/post-implantation (implantation's minus live births) (intrauterine)

Females with 0

3/12

7/12$

5/12$

4/12$

Females with 1

6/12

1/12$

4/12$

5/12$

Females with 2

1/12

2/12$

2/12$

2/12$

Females with ≥3

2/12

2/12$

1/12$

1/12$

Post-natal (live births minus alive at post-natal day 4)

Females with 0

6/12

6/11

8/11

7/11

Females with 1

5/12

4/11

2/11

1/11

Females with 2

1/12

0/11

1/11

1/11

Females with ≥3

0/12

1/11

0/11

2/11

Applicant's summary and conclusion

Conclusions:
In conclusion, the NOAEL for parental and F1 effects is considered to be 1,000 mg/kg bw/day.
Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening was conducted according to OECD Guideline 422 in compliance with GLP to obtain information on the possible toxic effects of the test substance following repeated daily administration by oral gavage to Wistar rats. The reproductive/developmental toxicity screening test provided initial information on effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 4 post-partum. The mating indices were 100% in all groups. The fertility indices were 100% in the control group and 92% in the test item treated groups, due to 1/12 non-pregnant females in each group, values which are comparable with concurrent control data in Wistar rats.  Gestation index was 100% in all groups. The sex ratios were slightly lower than control in all treated groups, attaining statistical significance both on post natal day (PND) 0 and PND4 in the 1000 mg/kg bw/day high dose group. Although a test item related effect cannot be excluded, this variation was considered due to an incidentally higher number of female foetuses in the control group and individual lower number in the high dose group. In conclusion, the NOAEL for parental and F1 effects is considered to be 1,000 mg/kg bw/day (Kubaszky, 2011).