Reaction mass of copper complex of [(2,6-difluoroheterocycl-4-yl)amino]hydroxy{[2-hydroxy-3-sulfonato-5-(vinylsulfonyl)phenyl]diazenyl}naphthalene sulfonic acid, dialkali salt and copper complex of [(2,6-difluoroheterocycl-4-yl)amino]-hydroxy{[2-hydroxy-3-sulfonato-5-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl]diazenyl}naphthalene sulfonic acid, trialkali salt

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

30 November 2010 to 25 January 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study with structural analogue conducted to OECD test guidance in compliance with GLP and reported with a valid GLP certificate.

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

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: 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 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: 389 g – 457 g, Females: 230g - 269 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment.
Acclimation period: At least 7 days (7 days from animal arrival to pre-treatment ophthalmoscopy examination, 12 days 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: 525
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: 18.5-24.5°C
Relative humidity: 32 - 66%
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, 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 (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 2.5, 15 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 days.

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 10/252-001P) and a repeated dose range finding study in the rat (LAB study code 10/252-220PE), 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.

Dosing procedure

Main animals

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.
Males were dosed for at least 28 days (14 days pre-mating, 14 days mating/post-mating period and on the day of necropsy), 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 6 days mating period, through gestation and up to and including the day of necropsy (at least 4 days 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, 27-28 days after the end of the mating period.

All F1 offspring were terminated on Day 4 post-partum; in order to allow for overnight fasting of dams prior to urine collection on PND 5, offspring were euthanized on PPD/PND 4, and the dams on PPD/PND 5.

Positive Control MNT animals

Group 5 animals were mated and females allowed to deliver, similarly to the Main animals. All animals were treated with 20 mg/kg bw/day Cyclophosphamide, administered by intraperitoneal injection approximately 24 h prior to scheduled necropsy (males, on Day 27 for necropsy on Day 28; females, on PND4 for necropsy on PND5).

Recovery animals

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.

Details on mating procedure:

Mating procedure

Main and Positive Control micronucleus test (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 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 test item is a multiconstituent composed of 6 different components contained in an amount of ca. 2% to 21%. This is due to the fact that all these 6 components are colouring agents. However, as 2 of these components are only contained in minor amounts (< 5%), the formulation analysis is only performed based on the 4 major components. 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 determination of the concentrations of the dosing solutions was based on the four eluted peaks named Component 1, Component 2, Component 3 and Component 4 peaks, with a limit of quantification of 0.5 µg/mL (Components 1, 2 and 3) and 2 µg/mL (Component 4). Dose formulations were homogenous. The measured concentrations varied between 91% and 103% of the nominal concentrations (2.5, 15 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 days (14 days pre-mating, 14 days mating/post-mating period and on the day of necropsy).
Females were dosed for 14 days pre-mating, for up to 6 days mating period, through gestation and up to and including the day of necropsy (at least 4 days 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 days after the end of the mating period.

Frequency of treatment:

Once daily 7 days per week

Details on study schedule:

See experimental schedule below under any other information on materials and methods

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (control), 25, 150, 1000 mg/kg bw/day
Basis:
actual ingested

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.

Positive control:

A positive control was included for the in vivo micronucleus test

Examinations

Parental animals: Observations and examinations:

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 23 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, 5 days before necropsy (Day 51, 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.

Main animals, 5 males and 5 females/group, groups 1 and 4, “subgroup C”:

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 23 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.


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).

Haematology and blood clotting times

The parameters evaluated are detailed under 'Any other information on materials and methods' in Chapter 7.5.1.

Blood smears (red cell morphology) were prepared for all subgroup B Main and all Recovery animals. As no evaluation is considered required with Sponsor’s approval, based on the haematology results, the slides will be archived at LAB Research Ltd. with study specimens.

Additional blood samples were collected from all Main animals immediately prior to the scheduled necropsy, by heart puncture under pentobarbital anaesthesia, into empty tubes with no anticoagulant to obtain serum. The serum was stored frozen at approximately - 20°C, then will be discarded prior to finalization of the study report with Sponsor’s approval, as no additional investigations are considered required to prove exposure of the animals to the test item, since blue discoloration of the urine collected prior to necropsy from animals placed in metabolic cages was observed.

Clinical chemistry

The parameters evaluated are detailed under 'Any other information on materials and methods' in Chapter 7.5.1.

Urinalysis

The parameters evaluated are detailed under 'Any other information on materials and methods' in Chapter 7.5.1.

Observation of the delivery process 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.

From the Positive Control MNT Group 5 animals, pre-treatment ophthalmoscopy examination was conducted along with all animals prior to their assignment to groups, mortality/morbidity were evaluated twice daily, any positive clinical observations were noted, body weights were measured for dose calculation for Cyclophosphamide administration and the cycle/mating was evaluated by examination of the vaginal smears until the mating was confirmed. No other parameters were monitored in the adults or offspring and the data will be archived but are not reported.

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 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 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.

Postmortem examinations (parental animals):

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. 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 4-6µ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

Postmortem examinations (offspring):

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.

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:

effects observed, treatment-related

Description (incidence and severity):

Discolouration of faeces and urine. See below for details.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Minimal focal reactive hyperplasia of the epithelium in pyloric glands. Minimal to mild foreign basophilic material adherent to the mucosa of non glandular and/or glandular stomach - see below for details.

Other effects:

not specified

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 unscheduled mortality occurred during the study.

Clinical observation

No systemic adverse effects were noted following administration of Reactive Blue F08-0170 daily by oral gavage under the conditions of this study, or in the Control animals administered 10 mL/kg vehicle (distilled, sterile water for injection). During the treatment period, dark blue discoloration of the faeces was noted in the High dose Main animals administered 1000 mg/kg bw/day Reactive Blue F08-0170 after the first treatment on Day 0. The discoloration of the faeces persisted in the 1000 mg/kg bw/Day Recovery animals for 3 days after the last dose administration (on Days 42-44); thereafter no test item-related effects were noted until completion of the 14-Day Recovery period.

In addition, it should be mentioned that dark yellow urine was noted at 150 mg/kg bw/day in 5/5 male and 1/5 female Mid dose Main animals and dark blue urine, at 1000 mg/kg bw/day in all High dose Main 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 Reactive Blue F08-0170 or its metabolites through faeces (cage side observations) or 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, apparently lower grip strength of the forelimbs was noted in the Main animals, statistically significant in the Mid and High dose males when evaluated on Day 23 am and in the High dose females, when evaluated on PND 3 am. Although a dose-related trend was noted, the differences were minor, within -13%, the control values were considered relatively high and a large individual variability was noted; a test item related cause cannot be excluded, however, it cannot be ascertained in the conditions of this study, in the absence of any systemic clinical signs or other adverse effects on the parameters evaluated.

Variations were observed in the grip strength of the hindlimbs, on occasion attaining statistical significance, but the mean values decreased in the males and increased in the females with the increase in the dose level when compared to Control, no histopathological findings were identified at examination of the sciatic nerve and these variations were ascribed to individual variability, unrelated to treatment under the conditions of this study.

The grip strength mean values showed no statistically or toxicologically significant differences to control in the 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 23 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 Reactive Blue F08-0170 at dose levels of up to and including 1000 mg/kg bw/day, either during the treatment or Recovery periods. In the Main animals, minor variations in the mean body weight and body weight gain values occurred in the treated groups compared to Control, attaining statistical significance in the High dose Main males only, when calculated between Days 0 to 7 pre-mating, without a dose or gender consistent response and considered unrelated to treatment, but to reflect expected variations in the population of Wistar rats.

When compared to Control, no statistically or toxicologically significant differences were noted in the High dose Recovery animals in either the mean body weight or body weight gain mean values.

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 (25, 150, or 1000 mg/kg bw/day) when compared to the Control.

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 lower platelets Plt. at 150 and 1000 mg/kg bw/day in the Mid and High dose Main males only (-12% and -14%, p<0.05 and p<0.01, respectively), but not in the females or in the recovery animals, or slightly lower Mean Corpuscular (erythrocyte) Haemoglobin Concentration MCHC, however, with minor differences to Control, of up to -3% in the males and/or 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 after 14 days of treatment, an apparent total bilirubin T -BIL increase was noted at 1000 and 150 mg/kg bw/day in both males and females and at 25 mg/kg bw/day in males only, attaining statistical significance in the High dose males only. 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 slightly higher, statistically significant in the High dose 1000 mg/kg bw/day Recovery males on Day 56, the difference was minor and associated with a relatively low Control mean value.

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

Reactive Blue F08-0170 administration daily by oral gavage at up to and including 1000 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 examined on Day 28 (males) or PND 5 (females), dark yellow urine was noted at 150 mg/kg bw/day in 5/5 male and 1/5 female Mid dose Main animals and dark blue urine, at 1000 mg/kg bw/day in all High dose Main 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 Reactive Blue F08-0170 or its metabolites through urine and an expected staining effect.

The specific gravity (SG) of urine and/or urinary pH showed minor variations in the Main animals, 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.

PATHOLOGY EVALUATION AND ORGAN WEIGHTS

Parental Generation (including subgroup B) (Males, Day 28, Females, PND5)

Macroscopic Findings

Treatment-related macroscopic findings were observed mainly in the stomach, small intestine, cecum, colon and/or rectum. These consisted of dark, diffuse or multifocal, blue or purple, discoloration of the mucosa recorded in 8/24, 24/24 and 24/24 adult rats from the Low, Mid and High Dose groups, respectively. In addition, dark discoloration/focus blue/purple of the thymus (2011), testes (4012) and lungs (2011, 3003, 4008 and 3505) was also considered to be associated with treatment.

All other changes were incidental or terminal procedure-related.

Microscopic Findings

Test item-related findings were histologically observed in the stomach, duodenum and/or cecum and correlated with the gross lesions noted at necropsy.

In the Main animals, minimal focal reactive hyperplasia of the epithelium in pyloric glands was found in 6/12 male and 3/12 female Main High dose 1000 mg/kg bw/day Group 4 animals. Stomach of all Mid dose Group 3 male and female animals was also examined due to macroscopic findings noted at necropsy (dark, purple discoloration of the mucosa), but no hyperplasia was observed (12/12 males and 12/12 females). The 8/24 Low dose Group 2 animals with macroscopic findings of the stomach mucosa did not show any microscopic abnormities, therefore no additional histopathological evaluation of the stomach is considered required in the remaining Low dose Group 2 animals (10/12 males and 6/12 females) that were not evaluated in the absence of the macroscopic findings.

Minimal to mild foreign basophilic material adherent to the mucosa of non glandular and/or glandular stomach was noted in 4/8 Low and 17/24 Mid Dose animals (which showed dark, purple discoloration of the stomach mucosa and were examined histologically) and 23/24 High Dose animals and occasionally to other digestive segments, including duodenum (4003) or cecum (4009), however, this finding is considered without toxicological significance in the absence of associated reactive changes, although additional examination of the relevant organs from the remaining lower dose groups may be conducted if requested by the Sponsor (at additional costs, to be clarified prior to finalization of the study report). In one Low dose male (2011), minimal focal subcapsular foreign basophilic material was seen in the thymus in correlation with macroscopic observation in this rat. It should be noted that dark, purple discoloration of the stomach mucosa was still apparent in 2/5 High dose 1000 mg/kg bw/day Recovery males, but there were no correlated microscopic findings.

There was no evidence of Reactive Blue F08-0170-related histological findings in the High and Control animals, or macroscopic observations from all groups 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.

RECOVERY (DAY 56)
Macroscopic Findings

Dark/purple discoloration of the stomach in 2/10 Recovery High Dose rats was regarded as possibly treatment-related, however, there were no correlated microscopic findings. All other changes were incidental or terminal procedure-related.

Microscopic Findings

There was no evidence of the test item-related microscopic findings. The changes in the testes, epididymis and prostate were seen unilaterally and/or with low incidence or severity and were considered to be incidental. All other changes were regarded as terminal procedure-related or incidental.

In summary, daily oral (gavage) administration of Reactive Blue F08-0170 to Wistar RJHan:WI rats under the conditions of this study was associated, in the Main animals, with minimal focal reactive hyperplasia of the epithelium in the pyloric glands noted in 6/12 male and 3/12 female Main High dose 1000 mg/kg bw/day Group 4, but no similar observations at lower dose levels (24/24 Mid dose and 8/24 Low dose animals with dark, diffuse or multifocal, blue or purple, discoloration of the stomach mucosa, which were further examined histologically) and or in the Recovery animals. Treatment-related foreign basophilic material, adherent mainly to the mucosa of the stomach, duodenum and/or cecum was recorded in 4/8 Low, 17/24 Mid and 23/24 High Dose animals and occasionally to other digestive segments, including duodenum (4003) or cecum (4009), however, this finding is considered without toxicological significance in the absence of associated reactive changes. This microscopic observation was in correlation with dark, diffuse or multifocal, blue or purple, discoloration of the stomach mucosa recorded at these dose levels at necropsy.

Following a 14 day recovery period, no test item-related macroscopic or microscopic findings were seen in the 1000 mg/kg bw/day High dose Recovery animals. No residual microscopic effect of the treatment was noted and the reactive epithelial hyperplasia of the pyloric glands has completely recovered.

Organ weights

There were no toxicologically significant changes in organ weight values noted after Reactive Blue F08-0170 administration at up to and including 1000 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 days after the first scheduled euthanasia of the dams on Day 41, with necropsy on Day 56 (Recovery male and female animals).

In the Main animals, the mean kidney absolute and/or relative weight adjusted for the body weight was slightly higher than control, attaining statistical significance in the High dose group at 1000 mg/kg bw/day. However, the dose response was not clear, all values remained within the historical control range and these variations were not associated with any clinical pathology, macroscopic or microscopic changes and were therefore considered not toxicologically significant and/or unrelated to treatment.

All other examined organ weights (absolute and relative to the body and brain weights) were similar in the control and test item treated groups, or showed minor variations, ascribed to biological variability related to post-partum status or to individual, incidental changes compared to study, or concurrent control data (for example, ovaries weight in the Main females evaluated on PND5, or spleen weight in the High dose Recovery males 4013 or 4016).

No statistically significant differences to Control were noted in the Recovery animals.

Effect levels (P0)

1

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: At 1000 mg/kg bw/day, there were no test item, or effects considered adverse on the reproductive, gestation/parturition or post-partal parameters evaluated.

2

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At 1000 mg/kg bw/day, there were histopathological changes in the glandular stomach.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

MORTALITY AND CLINICAL OBSERVATIONS
The test substance administered to parental generation at up to 1000 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. A few pups were found dead and cannibalised, thus, they were counted and sex determined if possible, but not further examined macroscopically.
In the Control group (135 pups examined), 1 pup was cannibalised and 1 was found dead and intact on PND 1, with positive lungs flotation test. In the 25 mg/kg bw/day Low dose group with 136 pups examined, 2 pups that showed no clinical signs were found dead and intact on PND4, both belonging to the same litter, dam 2507. In the 150 mg/kg bw/day Mid dose group, 1/136 pups was cannibalised on PND1 after being not suckled on PND0; one was found dead and intact on PND 1, with no previous clinical signs and autolysis of abdominal organs observed at necropsy, and one was not suckled, then was found dead and intact on PND 0, showing negative floating test at necropsy. In the 1000 mg/kg bw/day High dose group, 15/107 pups showed no symptoms or were not suckled on PND 0 of birth, then were found dead, with positive floating test (1/15) or cannibalized (14/15) on PND 1 and not further examined; these findings were considered ascribed to individual variability and not a test item related effect, as all pups belonged to litter/dam 4503. One additional pup from litter/female 4508 was not suckled and then found dead on PND 0, with negative floating test.
There were no clinical signs in the pups that survived to scheduled termination on PND4.
No external abnormalities ascribed to test item administration were detected at the clinical or external macroscopic examinations of the pups. The sex ratios were similar in the control and treated groups. No, or minor variations were observed on PN4 compared to PN0.
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 1000 mg/kg bw/day, with a higher than control survival index on PND 4 when evaluated for all pups at 1000 mg/kg bw/day, due to litter/dam 4503 with total post-natal mortality on PND 0-1.

BODY WEIGHT AND BODY GAIN
There were no effects considered adverse on the offspring weight or weight gain following administration of The test substance at 25, 150 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. In addition, the mean litter weights at both PND 0 and PND 4 were higher than control in the treated animals at all dose levels.

When evaluated for all pups, statistically significant variations were noted in the mean body weight and body weight gain values, however, the differences were relatively minor and the values were comparable with the historical control data. As no test item related clinical signs or any adverse effects on other parameters examined, including pups viability, survival index, or sex ratio, these variations 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 large number of pups.

PATHOLOGY EVALUATION
FOUND DEAD
F1 Generation
Seven intact pups were found dead between Days 0-4 post partum. Necropsy was performed on 1, 2, 2 and 2 pups from Control, Low, Mid and High Dose groups, respectively. Positive floating test was observed in 1/1 and 1/2 animals from the Control and High Dose groups. Negative floating test was seen in 1/2 Mid and 1/2 High Dose pups. Autolysis of the abdominal organs was found in two rats (1505/2 and 3512/12). Additionally, dark/red discolored left lobe of the lungs was also noted in one Low Dose animal (2507/13).

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)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 1000 mg/kg bw/day: There were no adverse effects ascribed to test item administration on the F1 offspring viability, clinical signs or development.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Tabular Summary

	Dose (mg/kg bw/day)
	Control	25	150	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	9/12
Conceiving days 1 - 5 (N)	12	12	11	12
Conceiving days 6 - 14 (N)	0	0	1	0
Pregnancy ≤ 21 days (N)	1	3	2	1
Pregnancy = 22 days (N)	8	7	9	7#
Pregnancy ≥ 23 days (N)	3	1	0	0
Dams with live young born (N)	12/12	11/11	11/11	8/9#
Dams with live young at PN4 (N)	12/12	11/11	11/11	7/8$
Corpora lutea/dam (mean)	17.67	17.09	20.18	17.25
Implantations/dam (mean)	12.50	13.55	13.00	14.00
Live pups/dam at birth (mean)	11.25	12.36	12.27	13.25
Live pups/dam at day 4 (mean)	11.08	12.18	12.09	11.38
Sex ratio at birth (mean)	47.32	51.24	47.57	52.39
Sex ratio at PN4 (mean)	46.62	50.68	47.03	43.46
Litter weight at birth (mean)	78.4	83.3	83.6	89.3
Litter weight at day 4 (mean)	130.4	139.0	135.8	145.0
Pup weight at birth (litter mean)	7.05	6.73	6.80	6.68
Pup weight at day 4 (litter mean)	12.02	11.46	11.32	11.15
STRUCTURALLY ABNORMAL PUPS
Dams with 0/ Dams with live born	12/12	11/11	11/11	8/8
Dams with 1 or ≥2	0	0	0	0
LOSS OF OFFSPRING#
Pre-implantation (corpora lutea minus implantations)
Females with 0	5/12	6/12	2/11	4/9
Females with 1	1/12	1/12	1/11	2/9
Females with 2	0/12	1/12	1/11	0/9
Females with ≥3	6/12	4/12@	7/11	3/9
Pre-natal/post-implantation (implantation's minus live births) (intrauterine)
Females with 0	4/12	4/11	6/11	3/9
Females with 1	3/12	4/11	2/11	5/9#
Females with 2	3/12	2/11	3/11	1/9
Females with ≥3	2/12	1/11	0/11	0/9
Post-natal (live births minus alive at post-natal day 4)
Females with 0	10/12	10/11	9/11	7/8
Females with 1	2/12	0/11	2/11	0/8
Females with 2	0/12	1/11	0/11	0/8
Females with ≥3	0/12	0/11	0/11	1/8$

# Due to female 4507, which had one corpora lutea and one implantation site only (pregnant but not delivered, with late embryonic death and 100% intrauterine mortality).

$ Due to female 4503, with 23 corpora lutea and 15 implantation sites/15 pups, which had total post-natal mortality of the whole litter. 

@ Female 2509 had 8 corpora lutea and no implantation site (total pre-implantation mortality).

Applicant's summary and conclusion

Conclusions:

The test substance is considered to be without effect on parental reproduction and F1 offsprings up to 1000 mg/kg bw/day. 

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test the test substance was administered to 12 Wistar rats/sex/dose formulated in water by gavage at dose levels of 0, 25, 150, 1000 mg/kg bw/day.

There were no toxicologically significant changes nor effects ascribed to the test substance, other than blue and reversible discoloration of feces (1000 mg/kg bw/day), dark yellow (150 mg/kg bw/day) or blue (1000 mg/kg bw/day) discoloration of urine, and minimal histopathological changes correlating to focal reactive hyperplasia of the epithelium in the pyloric glands (1000 mg/kg bw/day) and foreign basophilic material adherent to the mucosa of stomach, duodenum and/or cecum (25 mg/kg bw/day). No macroscopic or microscopic findings were seen in the 1000 mg/kg bw/day satellite group after a 14 day recovery period.

The NOAEL for parental toxicity is 150 mg/kg bw/day in males/female rats based on effects in glandular stomach. The NOAEL for parental reproductive and F1 offspring effects is 1000 mg/kg bw/day in males/females rats.

This study is acceptable and satisfies the guideline requirement for a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD 422) in rats.