1,3,6-Naphthalenetrisulfonic acid, 7-[2-[2-[(aminocarbonyl)amino]-4-[[4-chloro-6-[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]phenyl]diazenyl]-, sodium salt

Toxicity to reproduction

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 April 2011 to 23 June 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

Test item: Structural Analogue 03

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 CiToxLAB Hungary Ltd. spare colony, as their use was not required (no replacements with spare animals were performed)
Age of animals: Young adult rats, approximately 10-11 weeks old at starting and 12-13 weeks at mating. The age range within the study was kept to the minimum practicable.
Body weight range: Males: 366 g – 407 g, Females: 219 g - 249 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment
Acclimation period: At least 7 days (5 days from animal arrival to pre-treatment ophthalmoscopy examination, 7 days from animal arrival to onset of treatment)
Animal health: Only healthy animals were used for the test, as certified by the veterinarian. Females were nulliparous and non-pregnant.
Room number: 524, 505
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: 19.9 – 24.2°C
Relative humidity: 33 - 69%
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/adult 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 CiToxLAB Hungary 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, as indicated in the Experimental design section. 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/adult (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

Details on exposure:

Vehicle
Name: Distilled, sterile water for injection, PhEUR
Lot No.: 8490910, 0110111
Manufacturer: TEVA Pharmaceutical Corporation
Expiry Date: September 2013, January 2014 respectively
Storage: Room temperature

Details on mating procedure:

Main 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 days. 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:

The samples were evaluated by UV-HPLC method

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period).
Females were dosed for 14 days pre-mating, for up to 5 days mating period, through gestation and up to the day of necropsy (at least 4 days post-partum dosing).

Frequency of treatment:

Test item or Control (water)-treated Groups 1-4 Main animals were administered the dosing solutions daily on a 7 days/week basis.

Details on study schedule:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination, as no additional mating was considered required.
Females were dosed for 14 days pre-mating, for up to 5 days mating period, through gestation and up to the day of necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) was defined as Day 0 post-partum. Females showing no-evidence of copulation were sacrificed as practical, 26-27 days after the end of the mating period.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose selection and route of administration: The dose levels and the vehicle were selected by the Sponsor in consultation with the Study Director based on available data, formulation and analytical trials and information from previous experimental work, including the results of an acute oral toxicity study (CiToxLAB Hungary Ltd. study code 11/008-001P) and a repeated dose range finding study in the rat (CiToxLAB Hungary Ltd. study code 11/008-220PE), 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.

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 26 am, females, on PPD 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, on Day 54, 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.

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 26 pm, females, PPD 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.

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.

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.

For terminal blood sampling of Main animals selected (subgroup B), one sample for blood clotting times (1.4 mL blood for APTT and PT measurements, in tubes with sodium citrate as anticoagulant) were taken from scheduled animals.
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).

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.

Litter observations:

Each litter was 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.
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 hours 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.

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.

Histopathological examination was performed on the selected list of specified tissues from the Control and High Dose Main and Recovery rats and all macroscopic lesions from all toxicology 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.

Additionally selected organs/tissues for 5 animals/sex/group (identified as subgroup B Main) were microscopically evaluated from the Control and High dose Main animals, and kidneys and stomach samples from the Subgroup B Low and Mid Dose animals were examined, due to the test item-related microscopic findings noted at the first instance in the animals examined.

The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µm 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 PND4 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 CiToxLAB Hungary 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:

effects observed, treatment-related

Description (incidence and severity):

During the treatment period, slight to moderate dark pink discoloration of the skin was noted in the High dose animals after Day 12 of treatment, until the completion of the study up to Day 55 during the Recovery period. Furthermore, dark red discoloration of the faeces was noted at 1000 mg/kg bw/day. These changes were ascribed to elimination of the test substance or its metabolites through faeces and are neither a toxic nor an adverse effect

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No adverse effects or test item related changes were noted on the mean body weight and body weight gain values at up to and including 250 mg/kg bw/day. At 1000 mg/kg bw/day, slightly lower than control body weight gain values were noted, up to approximately -4% in the males and up to approximately -7% in the females, generally without attaining statistical significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item-related differences to Control in the mean daily food consumption.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No adverse effects related to the test item were seen. The only test-item related effect was an apparent increase in total bilirubin due to the interference of the dye with the photometic test system for this assay.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

No adverse effects were seen. The only test item related effects was pink urine in all animals evaluated from the Main Mid dose 250 mg/kg bw/day group and red urine, in all the High dose 1000 mg/kg bw/day animals examined. These changes were ascribed to elimination of the test item or its metabolites through urine (urine collection in metabolic cages) and an expected staining effect.

Behaviour (functional 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):

In the kidneys, tubular degeneration was associated with eosinophilic intracytoplasmic droplets of variable size and/or vacuolated cytoplasm of the epithelial cells. Regenerating tubular epithelium was characterized by basophilia, surrounded by minimal mononuclear infiltrate and regenerating cells showed lower cuboidal forms. Affected animals included 22/24 from the High Dose groups (10 males, 12 females). In addition, minimal tubular degeneration was also noted in 3/12 Mid Dose males. No tubular degeneration was seen in the Mid Dose females examined or in any rats of the Low Dose animals.

In the stomach, eosinophilic droplets of variable size were present in the cytoplasm of glandular epithelial cells. In addition, these alterations were accompanied with minimal to mild influx of inflammatory cells in the glandular submucosa and mucosa in majority of animals, and sporadically with formation of submucosal lymphoid follicles. The regeneration of the epithelial cells lining glandular mucosa resulted in transition into lower cuboidal epithelial cells with the increase of cytoplasmic basophilia and mitotic figures. Altered animals included 24/24 from the High Dose groups.

At additional histopathological evaluation of the stomach from Low and Mid Dose groups, minimal to mild epithelial degeneration/regeneration of the glandular mucosa was noted in 5/12 Mid Dose males and 3/12 Mid Dose females. This change was accompanied with minimal inflammatory infiltrate in 5/12 Mid Dose males and lymphoid follicle formation in the male 3001. In 2/12 Low Dose males, minimal epithelial degeneration/regeneration was also observed however without influx of inflammatory cells.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

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 in correlation with test substance administration.

Details on results (P0)

Mortality: There was no unscheduled mortality during the study.

Clinical observation: There were no clinical signs at up to and including 250 mg/kg bw/day Structural Analogue 03 or in the Control animals administered 10 mL/kg vehicle (distilled, sterile water for injection).
No systemic adverse clinical signs were noted following administration of Structural Analogue 03 daily by oral gavage at 1000 mg/kg bw/day dose level. During the treatment period, dark red discoloration of the faeces was noted in the High dose Main animals administered 1000 mg/kg bw/day Structural Analogue 03 on Day 1, and of the urine, on Day 2, where the treatment started on Day 0.
The discoloration of the faeces persisted in the 1000 mg/kg bw/Day Recovery animals for up to 3 days after the last dose administration (up to Day 44) and of the urine, until completion of the recovery phase and termination. Slight to moderate dark pink discoloration of the skin was also noted in the High dose animals after Day 12 of treatment, until the completion of the study up to Day 55 during the Recovery period.
These changes were ascribed to elimination of Structural Analogue 03 or its metabolites through faeces or urine 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 toxicologically significant changes, or effects considered adverse or related to test item administration were noted in the landing foot splay or grip strength tests. When compared to Control, there were no statistically or toxicologically significant differences in the mean grip strength (g) or landing foot splay (cm) values of the forelimbs or hind limbs in both the Main and Recovery animals.

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 26 pm, females, PPD 3 pm), thus no additional evaluation was required in other dose groups or Recovery animals.

Body weight and body weight gain: No adverse effects or test item related changes were noted on the mean body weight and body weight gain values following daily administration of Structural Analogue 03 at dose levels of up to and including 250 mg/kg bw/day.
At 1000 mg/kg bw/day, slightly lower than control body weight gain values were noted, up to approximately -4% in the males and up to approximately -7% in the females, generally without attaining statistical significance. The body weight gain however was similarly lower than control in both High dose Main males and females, but attaining statistical significance when evaluated for the whole period of treatment between Days 0 and 28 in the 1000 mg/kg bw/day Main male (96.75 g vs. 115.50 g, p< 0.05), or during the gestation period between GD0 and GD20 in the 1000 mg/kg bw/day Main females (133.50 vs. 161.1 g, p<0.05, mainly based on possible test item related effects during the second half of pregnancy).
In the Recovery animals, the body weights remained lower than controls, with mean values up to approximately -5%, p<0.05 on Days 28 and 35 in the males, and up to approximately -4%, p<0.01, on Day 55 in the females. No statistically significant differences occurred in the body weight gain of the Recovery animals.

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.
The mean values were either higher than control in the test-item treated groups, or, if lower, the differences noted were minor, showed no dose response, and/or were associated with variations within the group, 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 the Main animals at up to and including 250 mg/kg bw/day dose levels.
At 1000 mg/kg bw/day, WBC increases considered possibly related to Structural Analogue 03 administration were noted in the Main animals evaluated on Day 14, up to 45% and 50% higher than control, p<0.01, in the males and females, respectively. The mean values remained 92% higher than control, p<0.01, in the Recovery males evaluated on Day 56, to become comparable with the physiological values in the Recovery females.
Variations were noted in a few parameters, on occasion attaining statistical significance, including for example, statistically higher MPV in the Main males at all dose levels, but without a clear dose response or a similar response in the Main or Recovery females, lower PT up to -6%, especially in the High dose Main males, but remaining within the physiological ranges, or higher PLT in the High dose Main females only. 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 on Day 14, an apparent total bilirubin T BIL increase was noted in the 1000 mg/kg bw/day Main High dose males and females (174% and 166%, p<0.05 and 0.01, respectively). The bile acids were also higher than controls in both males and females at 250 and 1000 mg/kg bw/day, attaining statistical significance in the Mid and Main High dose males (43%, p<0.05 and 105% higher than control, p<0.01, at 250 and 1000 mg/kg bw/day) and in the Main High dose females (180%, p<0.01). After a 14-day recovery period, the T-BIL remained up to 36.2% higher than control in the High dose Recovery males and females. 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.
In addition, lower albumin/globulin ratio, up to -20%, and lower ALT , up to -23%, were recorded in the Main High dose animals, effects considered potentially related to test item administration at 1000 mg/kg bw/day dose level; the ALT mean value remained up to -53% lower than control in the Recovery animals, however, without attaining statistical significance .
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 (including, for example slightly higher than control, but statistically significant Cl- in the Main males at all dose levels, but not in the females, or slightly lower Ca++ in the Main High dose females, but not in the males or in the Recovery animals). For this reason, these variations were not considered toxicologically significant or related to treatment.

Urinalysis: Pink urine was noted in all animals evaluated from the Main Mid dose 250 mg/kg bw/day group (5/5 males and 5/5 females, subgroup B) and red urine, in all the High dose 1000 mg/kg bw/day animals examined (5/5 males and 5/5 females, subgroup B Main and in 5/5 male and 5/5 female Recovery animals), 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 item or its metabolites through urine (urine collection in metabolic cages) and an expected staining effect. The few other minor variations observed did not attain statistical significance and/or were regarded as normal background changes, without toxicological relevance.

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 in correlation with the test item administration.
In all groups, the mating indices were 100%, the fertility indices, 83%, 100%, 92% and 92% due to 2, 0, 1 and 1/12 non-pregnant females, respectively, in Groups 1, 2, 3 and 4, and the gestation indices, 100%, with the exception of the High dose group in which the gestation index was incidentally 91%, due to 1/11 pregnant, but not delivered females in this group, values which are comparable with concurrent control data in Wistar rats.
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 days 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 22.00 days (22 days) in the Controls, 22.17 days (22 to 23 days) in the Low 62.5 mg/kg bw/day group, 22.45 days (p<0.05, 22 to 23 days) in the Mid dose 250 mg/kg bw/day group, to 22.09 days (22 to 23 days) in the 1000 mg/kg bw/day High dose group. These values were comparable with the contemporaneous historical control data (gestation length: 22.40 ± 0.53 days, range: 22 to 23 days, n = 22) and the minor differences to control were not regarded as toxicologically relevant, although the mean duration of pregnancy attained statistical significance in the Mid 250 mg/kg bw/day group only. All the parturitions were normal.
The number of corpora lutea and implantation sites were comparable in the treated groups up to and including 1000 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 250 mg/kg bw/day.
At 1000 mg/kg bw/day, the intrauterine mortality, post-natal mortality and subsequently the total mortality were higher than control, however, only the post-natal mortality attaining statistical significance (intrauterine mortality: 28.19% vs. 11.44%, 146.4% higher than control; post-natal mortality: 16.34%, p<0.05, vs. 0.71%; total mortality: 33.65% vs. 12.03%, 179.6% higher than control in the High dose vs. the control group), effects considered possibly related to Structural Analogue 03 administration at the High dose level, although a role of the individual variability cannot be excluded (females 4501, 4503 and 4506).

Macroscopic Findings: Test item-related pink discoloration was observed in numerous organs/tissues including the stomach, small and large intestine, kidneys, pancreas, testes, seminal vesicles, prostate, epididymides, vagina, uterus and cervix, mandibular salivary gland, mandibular and mesenteric lymph nodes, thymus, tongue, trachea, urinary bladder, eye, lacrimal gland, skin, oral, nasal and conjunctival mucosa, fatty (subcutaneous abdominal and thoracic) and connective tissues (subcutaneous abdominal, thoracic, intramuscular) at necropsy. Affected animals included 24/24 rats from the High Dose group.
Red fluid content (urine) in the urinary bladder was noted in 13/24 High dose Main animals (12/12 males and 1/12 female) and red discolored digestive content was observed in 2/24 animals (2/12 males) in the stomach, small and large intestine also regarded as test item-related.
In the Mid dose animals, pink discoloration of the stomach (1/24), kidneys (1/24) or trachea (5/24) was also seen with low incidence.
These changes were considered due to the expected coloring effect of the test item (dye). All other macroscopic changes were regarded as incidental.

Microscopic Findings: Test item-related findings were microscopically observed in the kidneys and stomach, and were in correlation with the observations noted at necropsy. The changes were characterized as minimal to mild degeneration/regeneration of the cortical tubules in the kidneys and epithelial cells of the glandular mucosa in the stomach.
In the kidneys, tubular degeneration was associated with eosinophilic intracytoplasmic droplets of variable size and/or vacuolated cytoplasm of the epithelial cells. Regenerating tubular epithelium was characterized by basophilia, surrounded by minimal mononuclear infiltrate and regenerating cells showed lower cuboidal forms. Affected animals included 22/24 from the High Dose groups (10 males, 12 females). In addition, minimal tubular degeneration was also noted in 3/12 Mid Dose males. No tubular degeneration was seen in the Mid Dose females examined or in any rats of the Low Dose animals.
In the stomach, eosinophilic droplets of variable size were present in the cytoplasm of glandular epithelial cells. In addition, these alterations were accompanied with minimal to mild influx of inflammatory cells in the glandular submucosa and mucosa in majority of animals, and sporadically with formation of submucosal lymphoid follicles. The regeneration of the epithelial cells lining glandular mucosa resulted in transition into lower cuboidal epithelial cells with the increase of cytoplasmic basophilia and mitotic figures. Altered animals included 24/24 from the High Dose groups.
At additional histopathological evaluation of the stomach from Low and Mid Dose groups, minimal to mild epithelial degeneration/regeneration of the glandular mucosa was noted in 5/12 Mid Dose males and 3/12 Mid Dose females. This change was accompanied with minimal inflammatory infiltrate in 5/12 Mid Dose males and lymphoid follicle formation in the male 3001. In 2/12 Low Dose males, minimal epithelial degeneration/regeneration was also observed however without influx of inflammatory cells.
There was no evidence of Test item-related histopathological findings in the High Dose animals in the reproductive organs. 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. 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.
All other macroscopic changes were regarded as incidental or terminal procedure-related.

RECOVERY (DAY 56)
Macroscopic Findings: Pink discoloration related to the administered test item was macroscopically noted in variety of organs/tissues including the stomach, small and large intestine, adrenal gland, kidneys, pancreas, testes, seminal vesicles, prostate, epididymides, vagina, uterus and cervix, mandibular salivary gland, mandibular and mesenteric lymph nodes, thymus, tongue, trachea, urinary bladder, eye, lacrimal gland, skin, oral, nasal and conjunctival mucosa, fatty (subcutaneous abdominal and thoracic) and connective tissues (subcutaneous abdominal, thoracic, intramuscular).
In addition, red digestive content of the stomach, small and large intestines and red fluid content in the urinary bladder were also considered to be test item-related.
All other macroscopic changes were regarded as incidental or terminal procedure-related.

Microscopic Findings: A residual effect of the test item administration was still present in the kidneys and stomach, and correlated with the macroscopic findings noted at necropsy. Minimal to mild degeneration/regeneration of the cortical tubules in the kidneys was noted in 10/10 High Dose rats. Minimal degree of severity of degeneration/regeneration of the glandular mucosa in the stomach was in 8/10 High Dose animals. The mitotic index was decreased and the influx of the inflammatory cells or formation of the lymphoid follicles were not recorded. All other changes with low incidence and/or severity, observed in control and/or treated animals were considered to be incidental or procedure-related.

In summary, a daily oral (gavage) administration of Structural Analogue 03 to Wistar RJHan:WI rats under the conditions of this study results in pink/red discoloration of numerous organs/tissues observed at necropsy and degeneration/regeneration of the cortical tubules in the kidneys and epithelial cells of the glandular mucosa in the stomach, microscopically present. The severity and/or incidence of these changes indicated a relationship to dose.
In the kidneys, test item-related findings mentioned above were present at dose levels of 250 and 1000 mg/kg bw/day however there was no evidence of degeneration/regeneration in the Mid Dose females (250 mg/kg bw/day).
In the stomach, the findings associated with the administration of the test item were also recorded at dose levels of 250 and 1000 mg/kg bw. There was clear dose responded decrease in the severity when compared High Dose (1000 mg/kg bw/day) and Mid Dose (250 mg/kg bw/day) animals. Also, minimal to mild inflammatory influx in the submucosa was decreased to minimal degree in Mid Dose (250 mg/kg bw/day) animals.
The formation of the submucosal lymphoid follicles was not present in rats dosed at 250 mg/kg bw/day. During additional histopathological examination, minimal epithelial degeneration/regeneration without influx of inflammatory cells was observed in two Low Dose males.
After a 14 day of recovery period, a residual effect of treatment was microscopically seen in the kidneys and stomach in the High Dose groups. In the kidneys, decreased severity of the test item-related microscopic alterations from mild to minimal was more evident in the males compared to the females. Cessation of treatment for 14 days resulted in almost completed regression of the test item-related lesions in the stomach. The decrease of the severity from mild to minimal was observed in all High Dose animals. Also, mitotic index has been decreased and inflammatory influx as well as formation of the submucosal lymphoid follicles, were no more observed.

Organ weights: In the Main animals, evaluated immediately after completion of the treatment, at necropsy on Day 28 (males) or PPD5 (females), there were no toxicologically relevant changes in the organ weight values, or effects considered adverse or related to Structural Analogue 03 administration at up to and including 250 mg/kg bw/day.
At 1000 mg/kg bw/day, slight increases in the kidneys, adrenals or liver weights were noted in both males and females, without attaining statistical significance in the Main animals at evaluation of the absolute mean group values. As the adrenals and liver weights showed no dose response and the mean values were comparable with the physiological values and no microscopic findings were noted at histopathological evaluation, their weight variations were regarded as incidental, not toxicologically significant and unrelated to treatment.
The absolute kidneys weight however was 14% higher than control in the High dose Main males, without attaining statistical significance, but with a relative weight related to the body weight 20% higher than control, p<0.01, and 15% higher, p<0.05, when adjusted for the brain weight. No similar effects were observed in the High dose Main females, however, kidneys weights were higher than control in both male and female Recovery animals. In view of the microscopic findings recorded, these kidneys weight variations were considered to reflect a possible Structural Analogue 03-effect at 1000 mg/kg bw/day administered under the conditions of this study.
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.

Effect levels (P0)

Dose descriptor:

NOAEL

Remarks:

(reproductive toxicity)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicological relevant adverse effects were noted for reproductive toxicity

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, the mean number of viable pups was -26.1% lower than control on PND0 and -30.4% lower on PND4, attaining statistical significance when evaluated for the total number of pups, results correlated with the higher post-natal and total mortality observed in the High dose dams. Survival index was -18.3% lower than control in the High dose group on PND0 and -26.2% on PND4, although without attaining statistical significance. Due to the fact that a higher variability was noted in some animals, a clear substance effects is not given.

Body weight and weight changes:

effects observed, non-treatment-related

Gross pathological findings:

no effects observed

Details on results (F1)

Mortality and clinical observations: Structural Analogue 03 administered to parental generation under the conditions of this study did not lead to any adverse effects considered related to treatment or toxicologically significant in the F1 generation at up to and including 250 mg/kg bw/day dose level.
At 1000 mg/kg bw/day, the mean number of viable pups was -26.1% lower than control on PND0 and -30.4% lower on PND4, attaining statistical significance when evaluated for the total number of pups, results correlated with the higher post-natal and total mortality observed in the High dose dams. Survival index was -18.3% lower than control in the High dose group on PND0 and -26.2% on PND4, although without attaining statistical significance.
The pups found dead and cannibalised were counted and sex determined if possible, but not further examined macroscopically. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. Findings such as cold to touch, occasional cannibalization or lack of suckling were noted with low incidence, 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 (131 pups examined), 3 pups were found dead on PND0, with negative pulmonary floating test (possibly stillborn), and another pup was found dead on PND2 without displaying any clinical signs prior to their death.
In the 62.5 mg/kg bw/day Low dose group with 155 pups examined, 3 pups were found dead on PND0, with negative pulmonary floating test (possibly stillborn), 4 were not suckled on PND0, then survived with no clinical signs until the scheduled necropsy on PND4, and another pup was found dead on PND3 without displaying any clinical signs prior to their death.
In the 250 mg/kg bw/day Mid dose group, with 141 pups examined, 4 pups were found dead on PND0, with negative floating test (possibly stillborn), one was found dead and had positive pulmonary floating test, 5 were cannibalized and 2 cold or not suckled.
In the 1000 mg/kg bw/day High dose group with 113 pups examined, 7 pups were found dead on PND0, with negative floating test (possibly stillborn), 2 were found dead on PND0, with positive pulmonary floating test, 1 was found dead on PND1, and 6 were cold, not suckled and/or cannibalized.
There were no adverse effects, or effects considered related to test item administration on the sex ratios, and no statistical or toxicological significant differences to control were noted.

Body weight and body weight gain: There were no effects considered adverse on the offspring weight or weight gain following administration of Structural Analogue 03 at 62.5 and 250 mg/kg bw/day to parental generation under the conditions of this study. When evaluated per litter basis or for all pups, 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 differences compared to controls in the F1 generation that could be considered toxicologically relevant or related to test item administration.
At 1000 mg/kg bw/day, when evaluated per litter basis, there were no statistically significant differences to control, however, the mean litter weights were lower than control on both PND0 and PND4 (on PND0, 11 litters, 65.7 g, vs. 10 litters, 90.1 g, thus -27.1% lower than controls mean litter weight in the High dose group; on PND4, 9 litters, 112.9 g, vs. 10 litters, 140.1 g, thus -19.4% lower than controls mean litter weight in the High dose group).
When evaluated for all pups, the mean body weights were statistically significant, slightly lower up to -7% than control: on PND0, 6.39 g vs. 6.88 g, and on PND4, 10.36 g vs. 11.03 g. As the differences were minor and the dose response was not clear, a test item related adverse effect on the offspring development following administration of 1000 mg/kg bw/day Structural Analogue 03 to the parental generation in the conditions of this study could not be ascertained, although it cannot be excluded.

Pathology evaluation
FOUND DEAD
F1 Generation: Twenty three pups were found dead between Days 0-3 post partum. Necropsy was performed on 4, 4, 5 and 10 pups from the Control, Low, Mid and High Dose groups, respectively.
Negative floating test was observed in 3/4, 3/4, 4/5 and 7/10 animals from the Control, Low, Mid and High Dose groups. Positive floating test was observed in 1/5 and 2/10 Mid and High dose of the pups found dead on PND 0 (no floating test was required on the remaining pups which died between PND1-3). White liquid/creamy material in the stomach was present in 1/4, 1/4 and 1/10 pups from Control, Low and High Dose. The stomach was empty in 1/5 and 2/10 offsprings from the Mid and High Dose groups. A cause of death of these pups could not be determined.

TERMINAL (MAIN)
F1 Generation: Macroscopic Findings:
No macroscopic changes were seen in F1 offspring generation euthanized and examined externally at scheduled termination on PND 4.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Remarks:

(teratogenicity)

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No teratogenic effects at highest dose tested (1000 mg/kg bw/day)

2

Dose descriptor:

NOAEL

Remarks:

(developmental effects)

Generation:

F1

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Slight effect on pups survival and birth weight at 1000 mg/kg bw/day.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Measured concentrations of the dosing solutions

Analytical occasion	Nominal concentration mg/mL	Measured concentrations with the 95% confidence intervals, mg/mL	Measure concentration in the percentage of the nominal
29 April 2011 (first week of treatment)	6.25	6.18±0.52	99
	25	25.7±0.66	103
	100	102±2.83	102
20 May 2011 (mid term)	6.25	6.09±0.19	97
	25	25.6±1.19	102
	100	107±1.03	107
09 June 2011 (last week of treatment)	6.25	6.07±0.12	97
	25	23.9±1.23	95
	100	99.8±5.38	100

 

Selected Clinical Chemistry Parameters, Main animals

		P (Parent) Male – Main				P (Parent) Female – Main
DOSE GROUP		ALT U/L	T-BILμmol/L	A/G	Bile acidsμmol/L	ALT U/L	T-BILμmol/L	A/G	Bile acidsμmol/L
Control (1)	MEAN	51.40	8.48	1.28	16.68	57	7	2	13
	SD	5.03	4.05	0.08	5.34	8	4	0	7
	n	5	5	5	5	5	5	5	5
62.5 mg/kg bw/day (2)	MEAN	48.00	6.36	1.22	15.96	55	8	1	21
	SD	10.02	2.88	0.11	2.18	7	2	0	4
	n	5	5	5	5	5	5	5	5
	±%	-7	-25	-5	-4	-2	14	-8	61
250 mg/kg bw/day (3)	MEAN	54.00	7.44	1.24	23.78	49	8	2	22
	SD	8.15	0.95	0.09	2.44	7	2	0	9
	n	5	5	5	5	5	5	5	5
	±%	5	-12	-3	43	-14	10	1	71
1000 mg/kg bw/day (4)	MEAN	41.00	23.20	1.06	34.20	44	19	1	36
	SD	3.94	6.47	0.05	9.59	8	3	0	10
	n	5	5	5	5	5	5	5	5
	±%	-20	174	-17	105	-23	166	-20	180
		*	*	**	**	*	**	**	**
		DN	U	DN	U	DN	DN	DN	DN

REMARKS:

±% = Percent Deviation Versus Control

NS = Not Significant

* = p<0.05

** = p<0.01

U = Mann-Whitney U – test Versus Control

DN = Duncan’s multiple range test

 

Selected Clinical Chemistry Parameters, Recovery Main animals

		Male – Recovery				Female – Recovery
DOSE GROUP		AST U/L	T-BILμmol/L	A/G	Bile acidsμmol/L	AST U/L	T-BILμmol/L	A/G	Bile acidsμmol/L
Control (1)	MEAN	142.60	4.92	0.94	9.44	122	6	1	8
	SD	15.13	0.77	0.05	1.97	28	1	0	2
	n	5	5	5	5	5	5	5	5
1000 mg/kg bw/day (4)	MEAN	117.00	6.70	1.10	9.04	98	7	1	8
	SD	21.68	0.35	0.10	2.15	14	0	0	2
	n	5	5	5	5	5	5	5	5
	±%	-18	36.2	17	-4	-20	20	13	6
		NS	**	*	NS	NS	*	NS	NS

REMARKS:

±% = Percent Deviation Versus Control

NS = Not Significant

* = p<0.05

** = p<0.01

T - test versus control

TABULAR SUMMARY REPORT

	Dose (mg/kg bw/day)
	Control	52.5	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)	10/12	12/12	11/12	11/12
Conceiving days 1 – 5 (N)	12	12	11	12
Conceiving days 6 (N)	0	0	1	0
Pregnancy≤21 days (N)	0	0	0	0
Pregnancy = 22 days (N)	10	10	6	10
Pregnancy≥23 days (N)	0	2	5	1
Dams with live young born (N)	10/10	12/12	11/11	10/11
Dams with live young at PN4 (N)	10/10	12/12	11/11	9/11
Corpora lutea/dam (mean)	19.40	20.92	22.36	19.45
Implantations/dam (mean)	14.50	14.17	14.00	13.36
Live pups/dam at birth (mean)	12.80	12.67	12.45	9.64
Live pups/dam at day 4 (mean)	12.80	12.58	12.00	8.91
Sex ratio at birth (mean)	42.87	53.95	50.14	47.05
Sex ratio at PN4 (mean)	42.87	53.72	49.27	34.24
Litter weight at birth (mean)	90.1	85.9	91.9	65.7
Litter weight at day 4 (mean)	140.1	134.2	144.4	112.9
Pup weight at birth (litter mean)	6.90	6.74	7.18	6.54
Pup weight at day 4 (litter mean)	11.12	10.85	12.21	10.12
STRUCTURALLY ABNORMAL PUPS
Dams with 0/ Dams with live born	10/10	12/12	11/11	10/11
Dams with 1 or≥2	0	0	0	0
LOSS OF OFFSPRING
Pre-implantation (corpora lutea minus implantations)
Females with 0	2/11#	1/12	1/11	2/11@
Females with 1	2/11#	1/12	0/11	2/11@
Females with 2	1/11#	0/12	0/11	1/11@
Females with≥3	6/11#	10/12	10/11	6/11@
Pre-natal/post-implantation (implantation’s minus live births) (intrauterine)
Females with 0	1/10#	3/12	4/11	4/11@
Females with 1	4/10#	5/12	3/11	1/11@
Females with 2	3/10#	2/12	2/11	3/11@
Females with≥3	2/10#	2/12	2/11	3/11@
Post-natal (live births minus alive at post-natal day 4)
Females with 0	9/10	11/12	9/11	7/11@
Females with 1	1/10	1/12	1/11	2/11@
Females with 2	0/10	0/12	0/11	0/11@
Females with≥3	0/10	0/12	1/11	2/11@

# females 1502 had incidentally total pre-implantation mortality (14 corpora lutea and no implantation sites), excluded from statistics.

@ female 4501 has 21 corpora lueta, 14 implantation sites and 1 stillborn.

Applicant's summary and conclusion

Conclusions:

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for reproductive toxicity for parental/adult effects is considered to be 1000 mg/kg bw/day. The NOAEL for teratogenic effects is 1000 mg/kg bw/day for the F1 generation and 250 mg/kg bw/day for F1developmental effects based on a possible slight effect on pups survival 1000 mg/kg bw/day. No structural adverse effects were observed in the surviving pups at up to and including 1000 mg/kg bw/day, under the conditions of this study.

Executive summary:

This study has been performed in accordance with the Study Plan, OECD Guidelines for Testing of Chemicals No. 422 (22nd March 1996) and the Principles of Good Laboratory Practice.

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat study was to obtain information on the possible toxic effects of the test item following repeated daily administration by oral gavage to Wistar rats. Reversibility of any treatment-related changes was evaluated following a 14-day Recovery period. The study also comprised a reproductive/developmental toxicity screening test, intended to provide initial information on possible 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. In the Main Groups, male and female Wistar rats were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period and up to and including postpartum/lactation Day PPD5. Additional 5 male and 5 female rats from the Control and High dose Recovery Groups 1 and 4 scheduled for follow-up observations were not mated, but treated up to the first scheduled euthanasia of the Main dams (Day 41), then kept at least for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects, and subjected to necropsy with macroscopic examination on Day 56.Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, neurological and ophthalmoscopic assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. In addition, the reproductive performance and indices, pregnancy, parturition and postpartum/lactation period were monitored in the adult Main animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND4. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult 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), all macroscopic findings (abnormalities) from all animals and kidney and stomach samples from the Mid and Low dose Subgroup B Main groups, based on the results observed at the initial evaluation of the High dose group.

Analysis of formulations (concentration, homogeneity) and assessment of test item stability in this vehicle in the conditions employed on the study was performed at concentrations from approximately 1 to 100 mg/mL in ultrapure water indicated 1 day stability at room temperature and up to 7 days, while stored refrigerated at 2-8ºC in the dark, when the recovery range was 91%-105%, which lies within the acceptance range of 100 ± 10%. Concentration and homogeneity of formulations were evaluated by UV-HPLC method on duplicate samples collected from the top, middle and bottom of test item solutions, and one sample from the control taken and analysed fresh on 3 occasions during the study. The measured concentrations varied between 95% and 107% 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.

No mortality or systemic adverse effects occurred during the study. During the treatment period, slight to moderate dark pink discoloration of the skin was noted in the High dose animals after Day 12 of treatment, until the completion of the study up to Day 55 during the Recovery period. Furthermore, dark red discoloration of the faeces was noted at 1000 mg/kg bw/day from Day 1, and of the urine, from Day 2 onwards. The discoloration of the faeces persisted in the 1000 mg/kg bw/day Recovery animals for up to 3 days after the last dose administration (up to Day 44) and of the urine, until completion of the recovery phase and termination. These changes were ascribed to elimination of the test item or its metabolites through faeces or urine and an expected staining effect. There were no test item-related differences to Control in the mean daily food consumption.

No test item related effects, or changes considered toxicologically significant were noted in the haematology parameters evaluated in the Main animals at up to and including 250 mg/kg bw/day dose levels. At 1000 mg/kg bw/day, WBC increases considered possibly related to the test item administration were noted in the Main animals evaluated on Day 14, up to 45% and 50% higher than control, p<0.01, in the males and females, respectively. The mean values remained 92% higher than control, p<0.01, in the Recovery males evaluated on Day 56, to become comparable with the physiological values in the Recovery females.

In the Main animals evaluated at the completion on Day 14, an apparent total bilirubin T‑BIL increase was noted in the 1000 mg/kg bw/day Main High dose males and females (174% and 166%, p<0.05 and 0.01, respectively). The bile acids were also higher than controls in both males and females at 250 and 1000 mg/kg bw/day, attaining statistical significance in the Mid and Main High dose males (43%, p<0.05 and 105% higher than control, p<0.01, at 250 and 1000 mg/kg bw/day) and in the Main High dose females (180%, p<0.01). After a 14-day recovery period, the T-BIL remained up to 36.2% higher than control in the High dose Recovery males and females. 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. In addition, lower albumin/globulin ratio, up to -20%, and lower ALT, up to -23%, were recorded in the Main High dose animals, effects considered potentially related to test item administration at 1000 mg/kg bw/day dose level; the ALT mean value remained up to -53% lower than control in the Recovery animals, however, without attaining statistical significance.

At urinalysis, pink urine was noted in all animals evaluated from the Main Mid dose 250 mg/kg bw/day group (5/5 males and 5/5 females, subgroup B) and red urine, in all the High dose 1000 mg/kg bw/day animals examined (5/5 males and 5/5 females, subgroup B Main and in 5/5 male and 5/5 female Recovery animals). These changes were ascribed to elimination of the test item or its metabolites through urine (urine collection in metabolic cages) and an expected staining effect.

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 in correlation with the test item administration. In all groups, the mating indices were 100%, the fertility indices, 83%, 100%, 92% and 92% due to 2, 0, 1 and 1/12 non-pregnant females, respectively, in Groups 1, 2, 3 and 4, and the gestation indices, 100%, with the exception of the High dose group in which the gestation index was incidentally 91%, due to 1/11 pregnant, but not delivered females in this group, values which are comparable with concurrent control data in Wistar rats.  

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 days of pairing (cohabitation).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 22.00 days (22 days) in the Controls, 22.17 days (22 to 23 days) in the 62.5 mg/kg bw/day Low dose group, 22.45 days (p<0.05, 22 to 23 days) in the 250 mg/kg bw/day Mid dose group, to 22.09 days (22 to 23 days) in the 1000 mg/kg bw/day High dose group. These values were comparable with the contemporaneous historical control data (gestation length: 22.40 ± 0.53 days, range: 22 to 23 days, n = 22) and the minor differences to control were not regarded as toxicologically relevant, although the mean duration of pregnancy attained statistical significance in the 250 mg/kg bw/day Mid dose group only. All the parturitions were normal. The number of corpora lutea and implantation sites were comparable in the treated groups up to and including 1000 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 250 mg/kg bw/day.At 1000 mg/kg bw/day, the intrauterine mortality, post-natal mortality and subsequently the total mortality were higher than control. However, only the post-natal mortality attained statistical significance (intrauterine mortality: 28.19% vs. 11.44%, i.e. 146.4% higher than control; post-natal mortality: 16.34%, p<0.05, vs. 0.71%; total mortality: 33.65% vs. 12.03%, i.e. 179.6% higher than control in the High dose vs. the control group). These effects were considered to be possibly related to the test item administration at the High dose level, although a role of the individual variability cannot be excluded (females 4501, 4503 and 4506).The test item administered to parental generation under the conditions of this study did not lead to any adverse effects considered related to treatment or toxicologically significant in the F1 generation at up to and including 250 mg/kg bw/day dose level. At 1000 mg/kg bw/day, the mean number of viable pups was -26.1% lower than control on PND0 and -30.4% lower on PND4, attaining statistical significance when evaluated for the total number of pups. These results correlated with the higher post-natal and total mortality observed in the High dose dams. Survival index was -18.3% lower than control in the High dose group on PND0 and -26.2% on PND4, although without attaining statistical significance.

The pups found dead and cannibalised were counted and sex determined if possible, but not further examined macroscopically. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. Findings such as cold to touch, occasional cannibalization or lack of suckling were noted with low incidence, 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. There were no adverse effects, or effects considered related to test item administration on the sex ratios, and no statistical or toxicological significant differences to control were noted.

There were no effects considered adverse on the offspring weight or weight gain at up to and including 250 mg/kg bw/day. At 1000 mg/kg bw/day, when evaluated per litter basis, there were no statistically significant differences to control, however, the mean litter weights were lower than control on both PND0 and PND4 (on PND0,11 litters , 65.7 g, vs. 10 litters, 90.1 g, thus -27.1% lower than controls mean litter weight in the High dose group; on PND4, 9 litters, 112.9 g, vs. 10 litters, 140.1 g, thus -19.4% lower than controls mean litter weight in the High dose group). When evaluated for all pups, the mean body weights were statistically significant, slightly lower up to -7% than control: on PND0, 6.39 g vs. 6.88 g, and onPND4, 10.36 g vs. 11.03 g. As the differences were minor and the dose response was not clear, a test item related adverse effect on the offspring development following administration of 1000 mg/kg bw/day to the parental generation in the conditions of this study could not be ascertained, although it cannot be excluded.

At pathology evaluation, pink/red discoloration of numerous organs/tissues was observed at necropsy in the High dose group. In the Mid dose group, discoloration was only observed in trachea, stomach and kidneys in single animals. Histopathologically, treatment-related changes were observed in the kidney and stomach of High and Mid dose rats. The severity and/or incidence of these changes indicated a relationship to dose. Degeneration/regeneration of the cortical tubules in the kidneys were present in High dose rats and Mid dose males. Degeneration/regeneration of epithelial cells of the glandular mucosa in the stomach accompanied by a minimal to mild inflammatory influx in the submucosa, were recorded in rats at dose levels of 250 and 1000 mg/kg bw/day. There was clear dose responded decrease in the severity when compared High Dose (1000 mg/kg bw/day) and Mid Dose (250 mg/kg bw/day) animals. A minimal epithelial degeneration/regeneration without influx of inflammatory cells was observed in two Low Dose males.

After a 14 day of recovery period, a residual effect of treatment was microscopically seen in the kidneys and stomach in the High Dose groups. In the kidneys, decreased severity of the test item-related microscopic alterations from mild to minimal was more evident in the males compared to the females. Cessation of treatment for 14 days resulted in almost completed regression of the test item-related lesions in the stomach. The decrease of the severity from mild to minimal was observed in all High Dose animals. Also, mitotic index has been decreased and inflammatory influx as well as formation of the submucosal lymphoid follicles, were no more observed. Twenty three pups were found dead, intact and were subjected to necropsy between Days 0-3 post partum (4, 4, 5 and 10 pups from the Control, Low, Mid and High Dose groups). Negative floating test was observed in 3/4, 3/4, 4/5 and 7/10 animals from the Control, Low, Mid and High Dose groups. Positive floating test was observed in 1/5 and 2/10 Mid and High dose of the pups found dead on PND0 (no floating test was required on the remaining pups which died between PND1-3). White liquid/creamy material in the stomach was present in 1/4, 1/4 and 1/10 pups from Control, Low and High Dose. The stomach was empty in 1/5 and 2/10 pups from the Mid and High Dose groups. A cause of death of these pups could not be determined. In the Main animals, there were no toxicologically relevant changes in the organ weight values, or effects considered adverse or test item related at up to and including 250 mg/kg bw/day. At 1000 mg/kg bw/day, the absolute kidneys weight was 14% higher than control in the High dose Main males, without attaining statistical significance. However, the relative kidney weight was 20% higher than control, p<0.01, when adjusted for the body weight, and 15% higher, p<0.05, when adjusted for the brain weight. No similar effects were observed in the High dose Main females, however, kidneys weights were higher than control in both male and female Recovery animals. In view of the microscopic findings recorded, these kidneys weight variations were considered to reflect a possible test item-effect at 1000 mg/kg bw/day administered under the conditions of this study.

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for reproductive toxicity for parental/adult effects is considered to be 1000 mg/kg bw/day. The NOAEL for teratogenic effects is 1000 mg/kg bw/day for the F1 generation and 250  mg/kg bw/day for F1developmental effects based on a possible slight effect on pups survival 1000 mg/kg bw/day. No structural adverse effects were observed in the surviving pups at up to and including 1000 mg/kg bw/day, under the conditions of this study.