Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 February 2011 to 21 April 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU, US and OECD test guidance in compliance with GLP and reported with a valid GLP certificate.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2012

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Reactive Red F08-0146

Test animals

Species:
rat
Strain:
other: Wistar RJHan:WI rats
Sex:
male/female
Details on test animals and environmental conditions:
EXPERIMENTAL ANIMALS

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 had to be 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: 359 g – 424 g, Females: 231 g - 267 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)

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 (Appendix 10).
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.1 – 23.8°C
Relative humidity: 30 - 64%
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
Type of inhalation exposure (if applicable):
other: Not applicable
Vehicle:
water
Remarks:
Distilled
Details on exposure:
The test item was formulated in distilled, sterile water for injection at 6.25, 25 and 100 mg/mL concentrations without correction for purity, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared and stored refrigerated at 2-8ºC pending use within 7 days.

Vehicle:
Name: Distilled, sterile water for injection, PhEUR
Lot No.: 3590210, 7530810, 8490910
Manufacturer: TEVA Pharmaceutical Corporation
Expiry Date: February 2013, August 2013, September 2013, respectively
Storage: Room temperature

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 10/285-001P) and a repeated dose range finding study in the rat (CiToxLAB Hungary Ltd. study code 10/285-220PE), 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 in relation to the body weight (10 mL/kg bw) 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 an acclimation period (A) of least 7 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 5 days mating period, through gestation and up to and including the day of necropsy (at least 4 days post-partum). The day of birth (viz. when parturition was complete) is defined as Day 0 post-partum PPD0) for dams or Day 0 post-natal (PND0) for the offspring. Females that proved not to be pregnant were sacrificed as practical, 26 to 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 PPD5, offspring were euthanized on PND4, and the dams on PPD/PND5.

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 PPD4 for necropsy on PPD5).

Recovery animals

Additional 5 male and 5 female rats from the Control (Group 1) and High dose (Group 4) Recovery Groups were treated up to the first scheduled euthanasia of the Main dams (Day 41), but not mated. After cessation of treatment, the recovery animals were kept for at least 14 days without treatment to detect either delayed occurrence, or persistence of, or recovery from toxic effects. All recovery rats were subjected to necropsy with macroscopic examination on Day 56.


Details on mating procedure:
Main and Positive Control MNT animals:

Mating began after the animals have attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 5 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 (GD0) 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:
Test item formulations were analyzed for concentration and homogeneity in the Analytical Laboratory of CiToxLAB Hungary Ltd. 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.

Six samples were taken from each test concentration and two from the control. The samples were diluted and analysed by HPLC using UV detection. Dose formulations were homogenous. 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.
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), 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 and including the day of necropsy (at least 4 days post-partum). The day of birth (viz. when parturition was complete) is defined as Day 0 post-partum PPD0) for dams or Day 0 post-natal (PND0) for the offspring. Females that proved not to be pregnant were sacrificed as practical, 26 to 28 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), 62.5, 250, 1000 mg/kg bw/day
Basis:
nominal in water
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 for details of experimental design.
The study design for the in vivo mucronucleus test is detailed in Chapter 7.6.2.
Positive control:
A positive control was included for the micronucleus test - see Chapter 7.6.2. for details.

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 PPD3 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 post-partal Days PPD0 (within 24 hours after parturition) and PPD4 (before termination). Body weights of the female animals were additionally weighed on gestational Days GD10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

Food consumption measurement

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

Ophthalmology

The fundus of eyes of all animals was examined before treatment. Five male and 5 female Control and High dose animals (“subgroup C”) randomly selected from groups 1 and 4, during the last week of treatment prior to necropsy (males, Day 26 pm, females, PPD3 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, PND5-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 below in a table under any other information on materials and methods.
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 CiToxLAB Hungary 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. As no additional investigations are considered required to prove exposure of the animals to the test item, since orange discoloration of the urine collected prior to necropsy from animals placed in metabolic cages was observed, the samples were discarded prior to finalization of the study report with Sponsor’s approval prior to finalization of the study.

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

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

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. Unless otherwise indicated, all procedures below refer to the Main and/or Recovery animals Groups 1 to 4, as applicable.



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 (with the exception of male 3005, as described in the Deviations section), 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), all macroscopic findings (abnormalities) from all animals and all kidney samples from the Mid and Low dose Main groups, based on the results observed at the initial evaluation of the High dose group. 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 4-6µm 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):
Staining of urine and faeces. See below .
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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See below.
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
See below.

Details on results (P0)

Mortality

There was no unscheduled mortality during the study.

Clinical observation

No systemic adverse effects were noted following administration of REACTIVE RED F08-0146 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, reddish discoloration of the faeces was noted in the High dose Main animals administered 1000 mg/kg bw/day REACTIVE RED F08-0146 from Day 1 (one day after the onset of treatment on Day 0) onwards. The discoloration of the faeces persisted in the 1000 mg/kg bw/Day Recovery animals for up to 2 days after the last dose administration (on Days 42-43); thereafter no test item-related effects were noted until completion of the 14-Day Recovery period. In addition, it should be mentioned that orange urine was noted at 1000 mg/kg bw/day in all High dose subgroup B Main animals (5 males and 5 females) and in High dose Recovery animals, in 5/5 male and 4/5 female (compared to yellow urine in all other dose groups and in 1/10 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 REACTIVE RED F08-0146 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 relevant 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 relevant changes, or effects considered adverse or related to test item administration were noted in the landing foot splay or grip strength tests.

There were no statistically significant differences to control in the landing foot splay of the forelimbs in the Main animals at any of the dose levels tested, or in the Recovery animals, in both the fore- and hind-limbs. Minor increases were observed in the mean values (cm) of the landing foot splay of the hind limbs in the Main animals in both males and females. However, no dose response was observed and the statistically significant variations did not show a consistent response between genders, and in the absence of any other clinical adverse effects, these differences were not considered related to test item administration, but ascribed to biological variability.

When compared to Control, there were no statistically or toxicologically significant differences in the mean grip strength values of the forelimbs or hind limbs in 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, PPD3 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 REACTIVE RED F08-0146 at dose levels of up to and including 1000 mg/kg bw/day, either during the treatment or Recovery periods.

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 and 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 either Main or Recovery animals.

Variations were noted in a few parameters, on occasion attaining statistical significance, including for example, statistically higher MPV in the Main High dose males (10%, p<0.01), lower Plt in the Main Low and Mid dose males (-26%, p<0.01 and -14%, p<0.05, respectively), but higher in the Main High dose females (17%, p<0.01), or higher LUC in the Recovery High dose females (167%, p<0.05, but without statistically significant differences in the males). Evaluation of the mean and individual results in comparison with the Control data did not reveal any test-item related cause of the changes noted, and/or no consistent dose or gender-related response was observed. Therefore, these differences observed between the Control and treated groups were considered to be incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range or were with no toxicological significance.

Clinical chemistry

In the Main animals evaluated at the completion of the treatment prior to necropsy, an apparent total bilirubin T BIL increase was noted in the 1000 mg/kg bw/day Main High dose males and females (171% and 93%, respectively, p<0.01). This was considered to be related to a possible spectral interference with the analytical method caused by the discolouration of the serum by the test item and not to reflect an adverse effect on the liver function. After a 14-day recovery period, the T-BIL remained 25% higher than control in both High dose Recovery males and females and slightly higher than the historical control values, however, without attaining statistical significance.

A few parameters, considered possibly correlated with the REACTIVE RED F08-0146- related histopathological findings noted in the kidneys at 250 and 1000 mg/kg bw/day, showed statistically significant higher values compared to control, mainly in the Main High and/or Mid dose males. These variations included statistically higher up to 4% Na+ and Cl-, up to 22%, total protein, and up to 23% albumin, at 250 and 1000 mg/kg bw/day. In the females, the differences were minor, showing only a trend to similar variations and did not reach statistical significance; therefore, any correlation with the treatment could be as well equivocal, however, in view of the microscopic renal changes, a test item-related cause could not be excluded. In addition, the cholesterol was 31% higher than control in the High dose males (p<0.01) and 16% in the High dose females without attaining statistical significance.

In the 1000 mg/kg bw/day Recovery animals, the Na+ remained statistically significant, very slightly higher than control (1%, p<0.05) in both male and female animals, however, the difference was very minor and the values remained within the physiological ranges and thus were not considered to reflect an adverse effect, or an effect correlated with test item administration.

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 glucose in the Main High dose animals, attaining statistical significance in the females only, 20%, p<0.05, or statistically significant, slightly lower Ca++ in the Main Low and Mid dose males, up to -10%, p<0.05, but without statistical significance in the Main High dose males, or in females at any dose level, and slightly higher than control in the Recovery High dose females, 5%, p<0.01). For this reason, these variations were not considered toxicologically significant or related to treatment.

Urinalysis

REACTIVE RED F08-0146 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.

Orange urine was noted at 1000 mg/kg bw/day in all High dose Main animals examined (5/5 males and 5/5 females, subgroup B) and in 9/10 High dose Recovery animals (5/5 males and 4/5 females), 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 RED F08-0146 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.

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 REACTIVE RED F08-0146 administration. In all groups, the mating indices were 100%, the fertility indices, 92% due to 1/12 non-pregnant females in each group, and the gestation indices, 100%, 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 5 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. They varied from 22.27 days (22 to 23 days) in the Controls, 22.45 days (22 to 23 days) in the Low dose 62.5 mg/kg bw/day group, 22.64 days (22 to 24 days) in the Mid dose 250 mg/kg bw/day group, to 22.36 days (21 to 23 days) in the High dose 1000 mg/kg bw/day group. The values were comparable with the contemporaneous historical control data (gestation length: 22.40 ± 0.53 days, range: 22 to 23 days, n = 22).

All the parturitions were normal, with the exception of one High dose female 4509, which had 15 pups delivered. The seven pups delivered on the first day of delivery were all stillborn or found dead; of the 8 pups delivered on the next day, 1 was cannibalized on PND0 and 7 were surviving to the scheduled termination on PND4. Based on the isolated incidence, this event was considered equivocal to REACTIVE RED F08-0146 administration.

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 statistically higher than control (28.06% vs. 9.73%, 8.12% vs. 0.61% and 32.7% vs. 10.3% in the High dose vs. the control groups), effects considered possibly related to REACTIVE RED F08-0146 administration at the High dose level.

PATHOLOGY EVALUATION AND ORGAN WEIGHTS

Pathology evaluation

TERMINAL

MAIN (DAY 28 or PPD5)

Parental Generation (including subgroup B)

Macroscopic Findings:

Test item-related findings were observed in the stomach, small intestine, caecum, colon, rectum and/or urinary bladder at necropsy. Dark/red discoloration of the stomach and/or intestinal mucosa was observed in 22/24, 24/24 and 24/24 adult rats from the Low, Mid and High Dose groups, respectively. In addition, dark/red discoloration of the urinary bladder was seen in 1/12 Mid Dose and 6/12 High Dose males. This observation was not detected in female parental animals from the Main group. The dark red discolorations are clearly a result of the intended staining effect of the test item (red dye); all other macroscopic changes were regarded as incidental.

Microscopic Findings:

Test item-related findings were histologically observed in the kidneys, stomach, small intestine, caecum, colon and/or rectum. In the stomach and intestines these microscopic alterations correlated with the gross lesions noted at necropsy.

Kidneys

Minimal to mild red-brown cytoplasmic pigment depositions in the tubular cells of the cortex was recorded in 11/11 High Dose animals (6/6 male and 5/5 female High dose animals for which the kidneys were examined histologically).

At the additional histopathology examination conducted on the kidney samples retained from “subgroup B” Mid and Low dose animals, due to the microscopic findings noted in the High dose group, minimal deposition of cytoplasmic red/red-brown cytoplasmic pigment was noted in 1/10 Low Dose (1/5 Low Dose Main females) and 4/10 Mid Dose (1/5 male and 3/5 female Mid Dose Main animals) animals.

Stomach

Minimal to moderate presence of red foreign material in the non glandular/glandular region occurred in 23/24 High Dose rats (11/12 males and 12/12 females for which the stomach was examined histologically). Minimal to moderate red pigment depositions within the cytoplasm of epithelial cells in the glandular stomach was detected in 19/24 animals (11/12 males and 8/12 females for which the stomach was examined histologically) in the High Dose group.

Stomach from 20/24 Low (8 male and 12 female) and 24/24 Mid (12 male and 12 female) Dose group animals was also examined due to dark/red discoloration of the mucosa observed at necropsy. Minimal red foreign material in 4/20 Low Dose (4/12 females) and minimal to mild red foreign material in 12/24 Mid Dose rats (3/12 males and 9/12 females), were seen histologically. Minimal red pigment depositions were only observed in 2/12 Mid Dose females. There was no microscopic evidence of red pigment depositions in any of the Low Dose animals examined.

Intestine

Minimal to moderate luminal/adhered red foreign material in the duodenum, jejunum, ileum, caecum and/or colon in 11/24 (4/12 males and 7/12 females) and minimal to mild red pigment depositions within the cytoplasm of enterocytes in 21/24 rats (11/12 males and 10/12 females) from the High Dose group were noted microscopically.

In the duodenum, jejunum, ileum, caecum and/or colon from the 20 Low (8 male and 12 female) and 24 Mid (12 male and 12 female) Dose groups evaluated due to macroscopic observation (dark/red discoloration), the following incidence and severity were observed: minimal to mild red foreign material in 3/20 Low (3/8 males) and 5/24 Mid Dose rats (3/12 males and 2/12 females), minimal red pigment depositions in 3/24 Mid Dose animals (2/12 males and 1/12 females). No red pigment depositions were observed in animals from the Low Dose group.

Red foreign material was recorded in the urinary bladder in 1/1 Mid dose male 3009 examined due to macroscopic findings noted at necropsy (dark, red discoloration); no similar findings were observed in the urinary bladder in the 24 High dose (12 male and 12 female) animals evaluated.

There was no evidence of Reactive Red F08-0146-related histological findings in the High animals or macroscopic observations from any 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.

All other macroscopic changes were regarded as incidental or procedure-related.

RECOVERY (DAY 56)

Macroscopic Findings:

No test item-related macroscopic findings were noted. Pelvic dilatation of the right kidney or uterus in oestrus were sporadically observed and regarded as incidental.

Microscopic Findings:

A residual effect of the test item administration was still seen in the kidneys, stomach, jejunum, ileum, caecum and/or colon.

Kidneys

Deposition of red-brown pigment, mostly with minimal severity, showing a potential tendency to recovery, was noted in the cortical tubules of the kidneys in 10/10 High Dose animals (5/5 males and 5/5 females).

Stomach and Intestine

Minimal red foreign material was only recorded in 4/10 High dose animals (1/5 High dose Recovery males and 3/5 High Dose Recovery females).

All other changes with low incidence and/or severity, observed in control and/or treated animals were considered to be incidental or procedure-related.

Conclusion

In summary, a daily oral (gavage) administration of Reactive Red F08-0146 to Wistar RJHan:WI rats under the conditions of this study was associated with red-brown pigment depositions in the kidneys, luminal/adhered red foreign material and/or red pigment depositions in the stomach, small intestine, caecum colon and/or rectum observed microscopically, with a clear dose response in terms of severity and/or incidence.


In the kidneys, minimal red-brown cytoplasmic pigment depositions in the tubular cells of the cortex was recorded in 1/10 Low Dose, and minimal to mild, in 4/10 Mid Dose and 11/11 High Dose animals (6/6 High Dose Main male and 5/5 High Dose Main female of the animals which were examined either due to macroscopic findings or as part of the examination of “subgroup B”).

Minimal red foreign material in non glandular/glandular stomach region was noted in 4/20 Low Dose, minimal to mild, in 12/24 Mid Dose rats and minimal to moderate, in 23/24 High Dose rats, of the animals which were examined due to macroscopic findings or as part of the additional examination of “subgroup B”. Minimal to moderate red pigment depositions within the cytoplasm of epithelial cells in the glandular stomach was detected in 19/24 animals in the High Dose group, with minimal severity only, in 2/12 Mid Dose females and no similar findings in the Mid dose males, or in the Low dose animals.

Minimal to moderate luminal/adhered red foreign material in the duodenum, jejunum, ileum, caecum and/or colon in 11/24 and minimal to mild red pigment depositions within the cytoplasm of enterocytes in 21/24 rats from the High Dose group were noted microscopically. Minimal to mild red foreign material was noted in 3/20 Low and 5/24 Mid Dose rats and minimal red pigment depositions in 3/24 Mid Dose animals. No red pigment depositions were observed in animals from the Low Dose group.

A consistent relationship between dose and incidence or severity in treated rats was clearly evident in both the stomach and intestine. The incidence and/or severity of the luminal/adhered red foreign material and/or red cytoplasmic pigment depositions in the epithelial cells of the stomach or in enterocytes of the intestine was higher in the High dose than in the Mid Dose groups. There was no microscopic evidence of red pigment depositions in any of the Low Dose animals.

There was no evidence of Reactive Red F08-0146-related macroscopic or microscopic findings in the High Dose animals in the reproductive organs.

Cessation of treatment followed by observation/recovery period of 14 days resulted in regression for most of the changes in the stomach and intestine, with presence of red material adherent to the stomach and/or intestine mucosa observed in only 4/10 High dose Recovery animals. However resolution of the microscopic alterations in the kidneys was not completed although a decrease of their severity from minimal to mild to predominantly minimal was detected.

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 REACTIVE RED F08-0146 administration at 62.5 mg/kg bw/day.

Statistically 12% higher than control kidneys mean absolute weights were recorded in the Main 250 and 1000 mg/kg bw/day males, p<0.05, without attaining statistical significance when adjusted for the body or brain weights, or in the Main females at any of the dose levels tested. Liver weights were statistically significant, up to 16% higher than control in the High dose Main males at 1000 mg/kg bw/day, p<0.05 for the mean absolute and relative to body weight organ weights, and up to 10% higher than control in the High dose Main females, p<0.05 when adjusted for the brain weight. The differences remained minor, however, in view of the histopathological changes noted in the kidneys and/or possible correlation with minor clinical chemistry variations, a test item related effect cannot be excluded.

The spleen weight was slightly higher than control in both male and female Main animals, with no dose response noted, attaining statistical significance in the males at all dose level when evaluated as absolute values, and in the Mid and High dose groups, when adjusted for the body or brain weight. No statistically significant differences were observed in the females. The values were within the physiological ranges, there was no dose response and no statistically significant differences to control in the females, and in the absence of any clinical pathology, macroscopic or macroscopic adverse effects, these variations were not considered toxicologically significant or related to treatment.

In the Recovery animals evaluated 14 days after completion of the treatment, there were no statistically or toxicologically significant differences to control in the High dose animals that could be considered to reflect a test item related, or an adverse effect.

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.

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

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
(reproductive toxicity)
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Higher intrauterine, postnatal and total foetal mortality at 1000 mg/kg bw/day
Dose descriptor:
NOAEL
Remarks:
(reproductive toxicity)
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No reproductive toxicity was observed at 1000 mg/kg bw/day
Dose descriptor:
NOAEL
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Histopathological changes in the kidneys, stomach and/or intestines were observed at 250 and 1000 mg/kg bw/day, possibly correlated with minor variations in the clinical chemistry and organ weights.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
See below.
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

REACTIVE RED F08-0146 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 number of viable pups was -21.4% lower than control on PND0 and -23.6% 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 -8.3% lower than control in the High dose group on PND0 and -11.7% on PND4, p<0.05.

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 (145 pups examined), 1 pup was cold to touch and not suckled on PND0, but survived with no clinical signs until the scheduled termination on PND4; another pup was cannibalized on PND1.

In the 62.5 mg/kg bw/day Low dose group with 142 pups examined, no clinical signs or unscheduled mortality were noted.

In the 250 mg/kg bw/day Mid dose group (137 pups examined), 2 pups were found dead on PND1, 1 cannibalized and 1 with positive pulmonary test and no macroscopic findings.

In the 1000 mg/kg bw/day High dose group with 126 pups examined, 16 pups were found dead on PND0, 5 cannibalized, and 11 intact and subjected to necropsy, 4 with positive lungs flotation test, and 7 with negative test.

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 REACTIVE RED F08-0146 at 62.5, 250 or 1000 mg/kg bw/day to parental generation under the conditions of this study.

When evaluated per litter basis, the mean litter weights on PND0 and 4, pups body weight and/or body weight gain evaluated on PND0 and 4 showed no statistically or toxicologically significant differences compared to controls in the F1 generation.

Pathology evaluation

FOUND DEAD

F1 Generation

Twelve 12 intact pups were found dead between Days 0-1 post partum. Necropsy was performed on 1 and 11 pups from the Mid and High Dose groups, respectively. Positive floating test was observed in 1/1 and 4/11 animals from the Mid and High Dose groups. No other findings were noted and a specific cause of death could not be determined.


TERMINAL

MAIN (DAY 28 or PPD5)

F1 Generation

Macroscopic Findings:

No macroscopic changes were seen in F1 offspring generation euthanized and examined externally at scheduled termination.

When evaluated for all pups, statistically significant, slightly higher than Control values were noted in the PND0 mean body weight values in the Low and Mid dose groups. As no test item related clinical signs or macroscopic findings were noted, the higher values observed when the mean body weights were calculated for all pups and not per litter basis were ascribed to individual variability.


Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Higher intrauterine, postnatal and total foetal mortality at 1000 mg/kg bw/day

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

TABULAR SUMMARY:

 

Dose (mg/kg bw/day)

Control

62.5

250

1000

Pairs started (N)

12

12

12

12

Surviving females showing evidence of copulation (N)

12/12

12/12

12/12

12/12

Females achieving pregnancy (N)

11/12

11/12

11/12

11/12

Conceiving days 1 - 5 (N)

12

12

12

12

Conceiving days 6 - 14 (N)

0

0

0

0

Pregnancy ≤ 21 days (N)

0

0

0

1

Pregnancy = 22 days (N)

9

6

5

5

Pregnancy ≥ 23 days (N)

3

5

6

5

Dams with live young born (N)

11/11

11/11

11/11

11/11

Dams with live young at PN4 (N)

11/11

11/11

11/11

11/11

Corpora lutea/dam (mean)

22.91

22.73

22.45

23.45

Implantations/dam (mean)

14.55

14.82

13.82

15.18

Live pups/dam at birth (mean)

13.18

12.91

12.45

10.73

Live pups/dam at day 4 (mean)

13.09

12.91

12.27

10.00

Sex ratio at birth (mean)

50.12

46.84

52.72

55.51

Sex ratio at PN4 (mean)

49.82

46.84

53.37

54.22

Litter weight at birth (mean)

87.4

89.3

86.2

76.3

Litter weight at day 4 (mean)

154.2

148.6

140.8

118.8

Pup weight at birth (litter mean)

6.67

7.01

7.05

6.76

Pup weight at day 4 (litter mean)

11.95

11.80

11.94

12.11

STRUCTURALLY ABNORMAL PUPS

Dams with 0/Dams with live born

11/11

11/11

11/11

11/11

Dams with 1 or ≥2

0

0

0

0

LOSS OF OFFSPRING#

Pre-implantation (corpora lutea minus implantations)

Females with 0

0/11

0/12#

1/12#

0/12#

Females with 1

0/11

0/12#

1/12#

0/12#

Females with 2

0/11

0/12#

0/12#

1/12#

Females with ≥3

11/11

12/12#

10/12#

12/12#

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

Females with 0

5/11

1/11

5/11

0/11

Females with 1

4/11

6/11

4/11

1/11

Females with 2

0/11

2/11

0/11

3/11

Females with ≥3

2/11

2/11

2/11

7/11

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

Females with 0

10/11

11/11

10/11

5/11

Females with 1

1/11

0/11

0/11

4/11

Females with 2

0/11

0/11

1/11

2/11

Females with ≥3

0/11

0/11

0/11

0/11

# Due to females 2504, 3501 and 4512, with 14, 7, 7 corpora lutea, respectively, and no implantation sites (total pre-implantation mortality, considered incidental based on the isolated occurrence). 

Applicant's summary and conclusion

Conclusions:
In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for REACTIVE RED F08-0146 for parental/adult systemic toxicity is considered to be 62.5 mg/kg bw/day, based on the histopathological changes in the kidneys, stomach and/or intestines, possibly correlated with minor variations in the clinical chemistry and organ weights at 250 and 1000 mg/kg bw/day only. For reproductive toxicity, the no observed effect level (NOEL) for parental/adult effects is considered to be 1000 mg/kg bw/day for the males and 250 mg/kg bw/day for the females, due to the higher intrauterine, postnatal and total foetal mortality at 1000 mg/kg bw/day, correlated with a NOEL of 250 mg/kg bw/day for F1 offspring, based on a possible REACTIVE RED F08-0146-related effect on pups survival at 1000 mg/kg bw/day. No structural or developmental 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:

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 addition, the test item was evaluated for genotoxic effects by examining the induction of micronuclei in bone marrow erythrocytes of treated and Control animals.

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, according to the following Experimental Design:

 

Gr. No.

Group Designation
Dose Level
(mg/kg bw/day)

Conc. (mg/mL)

Dose volume

(mL/kg bw)

Animal Numbers/ID (Main)

Male

Female

1

Control

0

0

10

1001-1012

1501-1512

2

Low dose

62.5

6.25

2001-2012

2501-2512

3

Mid dose

250

25

3001-3012

3501-3512

4

High dose

1000

100

4001-4012

4501-4512

 

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. 

 

Gr. No.

Group Designation
Dose Level
(mg/kg bw/day)

Conc. (mg/mL)

Dose volume

(mL/kg bw)

Animal Numbers/ID (Recovery)

Male

Female

1

Control

0

0

10

1013-1017

1513-1517

4

High dose

1000

100

4013-4017

4513-4517

 

A Positive Control group for the Mammalian Erythrocyte Micronucleus Test (MNT) was added. The animals were mated and females allowed to deliver, similarly to the Main animals, then treated once with 20 mg/kg bw Cyclophosphamide, administered by intraperitoneal injection approximately 24 h prior to scheduled necropsy, males, on Day 27 for necropsy on Day 28; females, on PPD4 for necropsy on PPD5.

Gr. No.

Group Designation
Cyclophosphamide Dose Level (mg/kg bw)

Cyclophosphamide Conc. (mg/mL)

Dose volume

(mL/kg bw)

Animal Numbers /ID

Male

Female

5

Positive Control MNT

20

10

2

5001-5012

5501-5512

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. Histopathology evaluation was conducted on selected tissues and organs retained in fixative and processed to slides from the Control and High dose 1000 mg/kg bw/day animals scheduled for histopathological evaluation and on kidneys and all organs with macroscopic findings from the Low 62.5 mg/kg bw/day and Mid 250 mg/kg bw/day dose groups. In addition, bone marrow smears were prepared at necropsy and evaluation of induction of micronuclei in bone marrow erythrocytes of treated and Control animals was conducted from the Control and High dose Main animals.

Analysis of formulations (concentration, homogeneity) and assessment of test item stability in this vehicle in the conditions employed on the study was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Stability tests (CiToxLAB Hungary Ltd. study code 10/285-316AN and additional evaluation during the current study) 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 99%-100%, 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, reddish discoloration of the faeces was noted in the High dose Main animals administered 1000 mg/kg bw/day REACTIVE RED F08-0146 from Day 1 onwards, one day after the onset of treatment on Day 0. The discoloration of the faeces persisted in the 1000 mg/kg bw/Day Recovery animals for up to 2 days after the last dose administration (on Days 42-43); thereafter no test item-related effects were noted until completion of the 14-Day Recovery period. Orange urine was noted at 1000 mg/kg bw/day in all High dose subgroup B Main animals (5 males and 5 females) and in High dose Recovery animals, in 5/5 male and 4/5 female (compared to yellow urine in all other dose groups and in 1/10 High dose 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 REACTIVE RED F08-0146 or its metabolites through faeces (cage side observations) or urine (urine collection in metabolic cages) and an expected staining effect. 

 

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. No test item related effects or toxicologically significant changes were observed in the landing foot splay or grip strength tests.

On ophthalmoscopy evaluation, there were no test item related changes compared to pre-treatment during ophthalmoscopy examination of 5 male and 5 female Control and High dose Main animals during the last week of treatment prior to necropsy.

 

No adverse effects considered toxicologically relevant were noted on the mean body weight and body weight gain values, or in the mean daily food consumption in any test-item treatedor Recovery groups.

No test item related adverse effects, or changes considered toxicologically relevant were noted in the haematology, coagulation, or urinalysis parameters evaluated in eitheror Recovery animals.

 

At clinical chemistry evaluation, an apparent total bilirubin T‑BIL increase was noted in the 1000 mg/kg bw/day Main High dose males and females (171% and 93%, respectively,p<0.01). This was considered to be related to a possible spectral interference with the analytical method caused by the discolouration of the serum by the test item and not to reflect an adverse effect on the liver function. After a 14-day recovery period, the T-BIL remained 25% higher than control in both High dose Recovery males and females and slightly higher than the historical control values, however, without attaining statistical significance. 

 

A few parameters, considered possibly correlated with the REACTIVE RED F08‑0146-related histopathological findings noted in the kidneys at 250 and 1000 mg/kg bw/day, showed statistically significant higher values compared to control, mainly in the Main High and/or Mid dose males. These variations included statistically higher up to 4% Na+and Cl-, up to 22%, total protein, and up to 23% albumin, at 250 and 1000 mg/kg bw/day. In the females, the differences were minor, showing only a trend to similar variations and did not reach statistical significance; therefore, any correlation with the treatment could be as well equivocal, however, in view of the microscopic renal changes, a test item-related cause could not be excluded.

 

In addition, the cholesterol was 31% higher than control in the High dose males (p<0.01) and 16% in the High dose females without attaining statistical significance. 

There were no REACTIVE RED F08-0146 adverse effects on the reproductive ability or in the mating or gestation indices. In all groups, the mating indices were 100%, the fertility indices, 92% due to 1/12 non-pregnant females in each group, and the gestation indices, 100%, values within the physiological ranges for Wistar rats.  Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 5 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. They varied from 22.27 days (22 to 23 days) in the Controls, 22.45 days (22 to 23 days) in the Low dose 62.5 mg/kg bw/day group, 22.64 days (22 to 24 days) in the Mid dose 250 mg/kg bw/day group, to 22.36 days (21 to 23 days) in the High dose 1000 mg/kg bw/day group. The values are comparable with the contemporaneous historical control data (gestation length: 22.40 ± 0.53 days, range: 22 to 23 days, n = 22). All the parturitions were normal, with the exception of one Main High dose female 4509, which had 15 pups delivered, 7 on one day but all were stillborn or found dead, and 8 on the next day, of which 1 was cannibalized and 7 surviving to the scheduled termination on PND4. Based on the isolated incidence, this event was considered equivocal to REACTIVE RED F08-0146 administration. 

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 statistically higher than control (28.06% vs. 9.73%, 8.12% vs. 0.61% and 32.7% vs. 10.3% in the High dose vs. the control groups), effects considered possibly related to REACTIVE RED F08‑0146 administration at the High dose level. 

REACTIVE RED F08-0146 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 number of viable pups was -21.4% lower than control on PND0 and -23.6% 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 -8.3% lower than control in the High dose group on PND0 and -11.7% on PND4, p<0.05.

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 effects considered adverse on the offspring weight or weight gain following administration of REACTIVE RED F08-0146 at 62.5, 250 or 1000 mg/kg bw/day to parental generation under the conditions of this study.

On pathology evaluation, a daily oral (gavage) administration of Reactive Red F08‑0146 to Wistar RJHan:WI rats under the conditions of this study was associated with red-brown pigment depositions in the kidneys, luminal/adhered red foreign material and/or red pigment depositions in the stomach, small intestine, caecum, colon and/or rectum observed microscopically, with a clear dose response in terms of severity and/or incidence.

In the kidneys, minimal red-brown cytoplasmic pigment depositions in the tubular cells of the cortex was recorded in 1/10 Low Dose, and minimal to mild, in 4/10 Mid Dose and 11/11 High dose animals (6/6 males and 5/5 femalesof the animals which were examined either due to macroscopic findings or as part of the examination of “subgroup B”).

Minimal red foreign material in non glandular/glandular stomach region was noted in 4/20 Low Dose, minimal to mild, in 12/24 Mid Dose rats and minimal to moderate, in 23/24 High Dose rats, of the animals which were examined due to macroscopic findings or as part of the additional examination of the “subgroup B”. Minimal to moderate red pigment depositions within the cytoplasm of epithelial cells in the glandular stomach was detected in 19/24 animals in the High Dose group, with minimal severity only, in 2/12 Mid Dose females and no similar findings in the Mid dose males, or in the Low dose animals. 

Minimal to moderate luminal/adhered red foreign material in the duodenum, jejunum, ileum, caecum and/or colon in 11/24 and minimal to mild red pigment depositions within the cytoplasm of enterocytes in 21/24 rats from the High Dose group were noted microscopically. Minimal to mild red foreign material was noted in 3/20 Low and 5/24 Mid Dose rats and minimal red pigment depositions in 3/24 Mid Dose animals. No red pigment depositions were observed in animals from the Low Dose group. 

A consistent relationship between dose and incidence or severity in treated rats was clearly evident in both the stomach and intestine. The incidence and/or severity of the luminal/adhered red foreign material and/or red cytoplasmic pigment depositions in the epithelial cells of the stomach or in enterocytes of the intestine was higher in the High dose than in the Mid Dose groups. There was no microscopic evidence of red pigment depositions in any of the Low Dose animals.

There was no evidence of Reactive Red F08-0146-related macroscopic or microscopic findings in the High Dose animals in the reproductive organs.

Twelve intact pups were found dead between Days 0-1 post partum. Necropsy was performed on 1 and 11 pups from the Mid and High Dose groups, respectively. Positive floating test was observed in 1/1 and 4/11 animals from the Mid and High Dose groups. No other findings were noted and a specific cause of death could not be determined.

Cessation of treatment followed by observation/recovery for 14 days resulted in regression for most of the changes in the stomach and intestine, with presence of red material adherent to the stomach and/or intestine mucosa observed in only 4/10 High dose Recovery animals. However resolution of the microscopic alterations in the kidneys was not completed although a decrease of their severity from minimal to mild to predominantly minimal was detected.

Statistically 12% higher than control kidneys mean absolute weights were recorded in the Main 250 and 1000 mg/kg bw/day males, p<0.05, without attaining statistical significance when adjusted for the body or brain weights, or in the Main females at any of the dose levels tested. Liver weights were statistically significant, up to 16% higher than control in the High dose Main males at 1000 mg/kg bw/day, p<0.05 for the mean absolute and relative to body weight organ weights, and up to 10% higher than control in the High dose Main females, p<0.05 when adjusted for the brain weight. The differences remained minor, however, in view of the histopathological changes and possible correlation with minor clinical chemistry variations, a test item related effect cannot be excluded.

In the 62.5 mg/kg bw/day Low dose Main animals and 1000 mg/g bw/day High dose Recovery animals, there were no statistically and/or toxicologically significant differences to control that could be considered to reflect a test item-related or an adverse effect.

No induction of micronuclei in bone marrow erythrocytes was observed following administration of REACTIVE RED F08-0146 to Wistar rats daily by oral gavage to the High dose Main animals at 1000 mg/kg bw/day.

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for REACTIVE RED F08-0146 for parental/adult systemic toxicity is considered to be 62.5 mg/kg bw/day, based on the histopathological changes in the kidneys, stomach and/or intestines, possibly correlated with minor variations in the clinical chemistry and organ weights at 250 and 1000 mg/kg bw/day only. For reproductive toxicity, the no observed effect level (NOEL) for parental/adult effects is considered to be 1000 mg/kg bw/day for the males and 250 mg/kg bw/day for the females, due to the higher intrauterine, postnatal and total foetal mortality at 1000 mg/kg bw/day, correlated with a NOEL of 250 mg/kg bw/day for F1 offspring, based on a possible REACTIVE RED F08-0146-related effect on pups survival at 1000 mg/kg bw/day. No structural or developmental adverse effects were observed in the surviving pups at up to and including 1000 mg/kg bw/day, under the conditions of this study.