Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 October 2011 to 6 November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Substance name: Reactive Yellow F01-0555

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species and strain: Crl:WI rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633
Hygienic level: SPF at the supplier; standard laboratory conditions during the study
Justification of species/strain: The rat is regarded as suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.
Number of animals: Main groups: 48 male, 48 female rats, 12 animals/sex/group, 4 groups; a sufficient number of at least 8 pregnant females/group was achieved.
Recovery groups: 10 male, 10 female rats, 5 animals/sex/group, 2 groups, Control and High dose
Positive Control MNT group: 12 male and 12 female rats, 1 group
At the completion of the study, the spare animals were returned to CiToxLAB Hungary Ltd. spare colony, as their use was not required (no replacements with spare animals were performed)
Age of animals: Young adult rats, approximately 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: 320-404 g, Females: 205 g- 259 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment
Acclimation period: At least 6 days (6 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: 524
Cage type: Type II and/or III polypropylene/polycarbonate
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Details of bedding quality are reported.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.7-23.8°C
Relative humidity: 34 - 55%
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 5 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).

The temperature and humidity were measured twice daily; no deviations from the target ranges were noted during the study.

Food and water supply

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

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

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

Each parental/adult animal (P Generation) was identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB Hungary Ltd. This number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section.

The boxes were arranged in such a way that possible effects due to cage placement were minimized and were identified by cards showing the study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery.

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

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Distilled, sterile
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
Name: Distilled, sterile water for injection, PhEUR
Lot No.: 3590210, 2170511, 2190511
Manufacturer: TEVA Pharmaceutical Corporation
Expiry Date: February 2013, May 2014, respectively
Storage: Room temperature

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The samples were evaluated by UV-HPLC method
Duration of treatment / exposure:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period).
Females were dosed for 14 days pre-mating, for up to 5 days mating period, through gestation and up to the day of necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) was defined as Day 0 post-partum.
Frequency of treatment:
Animals were administered the dosing solutions daily on a 7 days/week basis
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Number of animals: Main groups: 48 male, 48 female rats, 12 animals/sex/group, 4 groups; a sufficient number of at least 8 pregnant females/group was achieved.
Recovery groups: 10 male, 10 female rats, 5 animals/sex/group, 2 groups, Control and High dose
Control animals:
yes, concurrent no treatment
Details on study design:
Formulation and analysis of formulation

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 4 days. Stability tests (CiToxLAB Hungary Ltd. study code 11/174-316AN) at concentrations from approximately 1 to 100 mg/mL in ultrapure water indicated a 1-day stability at room temperature and 4-day stability while stored refrigerated at 2-8ºC, when the recovery range was 100%-103%, which lies within the acceptance range of 100 ± 10%.

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.

For the Positive Control (MNT) Group 5, Cyclophosphamide was dissolved in 0.9% NaCl solution shortly before administration, to obtain a dose conc
entration of 10 mg/mL for a dose volume of 2 mL/kg; no dose formulation analysis was performed.

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 (CiToxLAB Hungary Ltd. study code 11/174-001P) and a repeated dose range finding study in the rat (CiToxLAB Hungary Ltd. study code 11/174-220PE), 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 6 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 5 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.

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 PND 4, and the dams on PPD/PND 5.
Positive control:
A Positive Control group for the Mammalian Erythrocyte Micronucleus Test (MNT) was added.

Examinations

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

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

Main animals, 5 males and 5 females/group, “subgroup A”:
Assessment of any potential test item related neurotoxicity was performed during the last exposure week (males, on Day 26 am, females, on PPD 3 am). In order to avoid hyperthermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes. Selected animal were subjected to the functional observation battery, including qualitative assessment of the grip strength, and to measurements of the landing foot splay and fore/hind grip strength.

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

Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support; results are tabulated with individual and mean data.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed. Parameters such as, but not limited to body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.
Recovery animals: Neurotoxicity evaluation was similarly conducted in the Recovery animals towards the end of the Recovery period, on Day 54, for necropsy on Day 56.

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

Food consumption measurement : Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly.

Ophthalmology: The fundus of eyes of all animals was examined before treatment. Five male and 5 female Control and High dose animals (“subgroup C”) randomly selected from groups 1 and 4, during the last week of treatment prior to necropsy (males, Day 26 pm, females, PPD 3 pm). Mydriasis was produced after instillation of a mydriatic agent (eye drops "Mydrum") into the conjunctival sac. The examination was performed using a Gowlland ophthalmoscope. As no ophthalmoscopic alterations were found, no additional examination was performed in other animals.

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 nec
ropsy, 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 materials and methods.

Clinical Chemistry

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

Urinalysis

The parameters evaluated are detailed below in a table under any other information materials and methods.
Sacrifice and pathology:
Pathology : Gross necropsy was performed on all animals. Terminally, after completion of the treatment or Recovery periods as applicable, animals were sacrificed under pentobarbital anaesthesia (see "Details of Other Materials") followed by exsanguination. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded in the Main females as applicable.

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

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

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

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

- With a precision of 0.01 g: heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals.
For all organs, paired organs were weighed individually. Individual and/or paired absolute organ weight are reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) were calculated and reported.

Histopathological examination was performed on the selected list of specified tissues from the Control and High Dose Main and Recovery rats and all macroscopic lesions from all toxicology animals. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Additionally selected organs/tissues for 5 animals/sex/group (identified as subgroup B Main) were microscopically evaluated from the Control and High dose Main animals, and kidneys and stomach samples from the Subgroup B Low and Mid Dose animals were examined, due to the test item-related microscopic findings noted at the first instance in the animals examined.
The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

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 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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
staining of faeces and urine
Mortality:
mortality observed, treatment-related
Description (incidence):
staining of faeces and urine
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
staining
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See below for details
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See below for details
Histopathological findings: neoplastic:
not examined
Details on results:
Mortality
No test item related mortality occurred during the study.

Clinical observations

No test item-related adverse effects or systemic clinical signs were noted following administration of REACTIVE YELLOW F01-0555 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).

In the Main animals, light orange discoloration of the faeces and dark yellow urine were noted during the treatment period from Day 1 in all animals at 1000 mg/kg bw/day, considered to be due to elimination of REACTIVE YELLOW F01-0555 or its metabolites through urine and an expected staining effect. Low dose female 2501 showed minor decreases in activity, hunched back position, piloerection and paleness between Days 35 and 38, unrelated to test item administration but associated with a total foetal mortality and incidental delivery difficulties.

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, or slightly increased startle response occurred in 2/5 High dose Main males examined, when subjected to the modified Irwin test (functional observation battery). However, there were no treatment-related differences to the Control, or the incidence was considered low, respectively, and no dose, or gender related response were noted, and these variations were considered to be without toxicological significance and within the normal biological variation with respect to behaviour, reactions to different type of stimuli or manipulations.

No test item related effects or statistically significant variations were observed in the landing foot splay test.

When compared to Control, there were no statistically or toxicologically significant differences in the mean grip strength values of the forelimbs or hind limbs in the High dose Main animals when evaluated on Day 24 (males) or PPD 3 (females). In the Low and Mid dose Main animals, slightly higher than Control mean grip strength of the hindlimbs was observed in the males, up to 12%, p<0.01, without a dose or gender response. Slightly lower than Control grip strength of the forelimbs was noted in the High dose Recovery males, -10%, p<0.05, but not in the females. In the absence of a consistent dose or gender response these variations were regarded as incidental and not to reflect an adverse or a test item related effect.

Ophthalmology
No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination.

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 YELLOW F01-0555 at dose levels up to and including 1000 mg/kg bw/day, either during the treatment or Recovery periods.

There were no statistically significant differences to Control in the mean body weights at any of the dose levels tested in both Main and Recovery animals. The body weight gain mean values showed minor increases or decreases in the treated animals, including slightly higher than control mean values in the Low and/or Mid dose Main females during pre-mating period between Days 0 and 14, or slightly lower mean value in the High dose Recovery females, between Days 14 and 21. Based on the isolated incidence and lack of a consistent dose or gender response, these variations were regarded as incidental and without toxicological significance.

Food consumption

There were no test item-related differences to Control in the mean daily food consumption in any test-item treated Main or Recovery group (62.5, 250, or 1000 mg/kg bw/day) when compared to the Control.

Minor differences to Control were noted or variations within the group, generally associated with changes in the study schedule including mating, delivery, or fasting before blood collection for clinical pathology evaluation, unrelated to treatment and with no toxicological significance.

Clinical pathology

Haematology

No test item related effects, or changes considered toxicologically significant were noted in the haematology parameters evaluated in either Main or Recovery animals.

Variations were noted in a few parameters, on occasion attaining statistical significance, including statistically higher eosinophil count up to 128% in the High dose Main males, p<0.05, but up to – 59% lower in the Mid dose Main females, or up to 87% higher than control white blood cell count in the Main females, although associated with a relatively low mean value in the Control group, no dose response and attaining statistical significance at 62.5 and 1000 mg/kg bw/day dose levels, but without similar variations 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.

There were no statistically or toxicologically significant differences to Control in the haematology parameters evaluated in the Recovery animals.

Clinical chemistry

There were no toxicologically significant changes or adverse effects on the animal serum chemistry that could be ascribed to REACTIVE YELLOW F01-0555 administration in the conditions of this study.

In the Main animals evaluated on Day 14, an apparent total bilirubin T BIL increase was noted in the male animals, 16%, 48% and 89% higher than Control in the Low, Mid and High dose groups, respectively, without statistical significance or a similar response in the females. This was considered to be related to a possible spectral interference with the analytical method caused by the discolouration of the serum by the test item and not to reflect an adverse effect on the liver function.

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 YELLOW F01-0555 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.

The urine specific gravity or volume showed minor variations, on occasion statistically significant in the Recovery animals, 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

Pathology evaluation

Parental Generation (including subgroup B)

Macroscopic Findings:

Treatment-related macroscopic findings were observed in the digestive content and mucosa of the stomach, small intestines, cecum, colon and/or rectum. These consisted of dark/orange liquid material in the content and dark/orange discoloration of the mucosa in 15/24, 21/24 and 24/24 adult rats from the Low, Mid and High Dose groups, respectively.

All other changes were incidental or terminal procedure-related.

Microscopic Findings:

Test item-related findings were histologically observed in the stomach, mesenteric lymph node, small intestines, cecum, colon and/or rectum and correlated with the gross lesions noted at necropsy.

Eosinophilic material adhered to the mucosa of the stomach or intestines and/or deposited in the cytoplasm of the glandular epithelial cells in the stomach and enterocytes of the intestines was microscopically observed. In the macrophage cytoplasm of the mesenteric lymph node eosinophilic pigment was microscopically seen. There was clear evidence of the dose relationship to severity and incidence of these changes in both genders.

Stomach

Minimal to moderate severity (mainly minimal/mild) was noted in the 3/16, 18/22 and 21/24 rats from the Low, Mid and High Dose groups. Moderate degree was recorded only in 4/24 High Dose animals.

Small intestine (duodenum, jejunum and/or ileum)

Minimal to moderate grading was present in 1/16 Low, 7/22 Mid and 16/24 High Dose animals. There was similar proportional distribution of the eosinophilic material in particular regions of the small intestine in both males and females.

Caecum, colon and/or rectum

Minimal to mild severity was observed in 4/22 Mid and 11/24 High Dose rats. Rectum was affected only in one High Dose female (4510).

Mesenteric lymph node

Minimal eosinohpilic pigment in the cytoplasm of macrophages was noted in two High Dose females. This observation is consistent with clearance of the test material by lymphatic drainage from the alimentary tract.

All other changes were incidental or regarded as common background.

There was no evidence of REACTIVE YELLOW F01-0555-related histological findings in the High 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:

There was no evidence of test item-related macroscopic findings.

Enlargement of the testes in 1/5 Control male, enlarged spleen or adrenals in 2/10 High Dose, dilatation of the uterus by clear fluid in 2/5 Control and 1/5 High Dose females were incidental, terminal procedure-related or associated with physiological changes during oestrus cycle.

Microscopic Findings:

Following 14 days recovery period, resolution of majority test item-related changes was noted in the stomach, small and large intestines or mesenteric lymph node. Minimal eosinophilic material in the mucosa of the stomach was observed in 4/10 High Dose animals and was regarded as a minor residual effect without toxicological significance in the absence of associated reactive changes.

All other changes were incidental or regarded as common background.

In summary, a daily oral (gavage) administration of REACTIVE YELLOW F01-0555 to Wistar rats under the conditions of this study was associated in the Main animals with eosinophilic material adhered to the mucosa of the stomach, small or large intestines and/or deposited in the cytoplasm of the glandular epithelial cells in the stomach and enterocytes of the intestines, and with eosinohpilic pigment in the cytoplasm of macrophages in the mesenteric lymph node. There was a dose relation to severity and incidence of these findings. These microscopic observations were also in correlation with the dark/orange discoloration noted at necropsy.

Following a 14 day recovery period, almost all residual microscopic effects of the treatment were completely recovered. It occurred that the test item has not been completely cleared following 14 days withdrawal of treatment in the stomach of 3/5 male and 1/5 High Dose female animals, however this finding was regarded as a minor residual effect without toxicological significance in the absence of associated reactive changes.


Organ weights

There were no toxicologically significant changes in organ weight values noted after REACTIVE YELLOW F01-0555 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), PPD5 (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).

Variations were noted in the absolute and/or relative organ weights adjusted for the terminal body or brain weight when compared to Controls, on occasion attaining statistical significance. In the absence of any dose or gender response, or of any clinical pathology, macroscopic or microscopic changes, these variations were not considered toxicologically significant or related to treatment.












Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test item related adverse effects seen at highest dose tested - 1000 mg/kg bw/day
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Measured concentrations of the dosing solutions:

Analytical occasion

Nominal concentration mg/mL

Measured concentrations with the 95% confidence intervals,  mg/mL

Measured concentration in percentage of the nominal

First week

(11 Oct. 2011)

6.25

6.29±0.13

101 %

25

25.5±0.4

102 %

100

103±2

103 %

Midway

(07 Nov. 2011)

6.25

6.23±0.07

100 %

25

25.4±0.3

102 %

100

103±2

103 %

Last week

(23 Nov. 2011)

6.25

6.32±0.11

101 %

25

25.7±0.4

103 %

100

103±3

103 %

Applicant's summary and conclusion

Conclusions:
In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for REACTIVE YELLOW F01-0555 for parental/adult effects is considered to be 1000 mg/kg bw/day.
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 PPD 5, 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 ()

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

 

An 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 were 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 and on 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 was evaluated in bone marrow erythrocytes of the Control and High dose Main animals.

 

Analysis of formulations (concentration, homogeneity) and assessment of test item stability in this vehicle under the conditions employed on the study was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Stability tests (CiToxLAB Hungary Ltd. study code 11/174-316AN) at concentrations from approximately 1 to 100 mg/mL in ultrapure water indicated a 1-day stability at room temperature and a 4‑day stability, while stored refrigerated at 2-8ºC, when the recovery range was 100%-103%, which lies within the acceptance range of 100 ± 10%. 

 

Concentration and homogeneity of formulations were evaluated using a 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 100% and 103% of the nominal concentrations (6.25, 25 and 100 mg/mL). No test item was detected in the Control solution samples. These results were considered suitable for the study purposes.  

 

REACTIVE YELLOW F01-0555 administered daily by oral gavage to Wistar rats did not result in test item related mortality or clinical adverse effects at daily, weekly or neurological assessment, in ophthalmological changes, or changes in the body weight, food consumption, haematology, coagulation, clinical chemistry, or urinalysis parameters at dose levels of 62.5, 250, or 1000 mg/kg bw/Day during either the treatment or after a 14-Day Recovery period under the conditions of this study.In the Main animals, light orange discoloration of the faeces and dark yellow urine was noted during the treatment period as of Day 1, one day after the onset of treatment on Day 0, in all animals at 1000 mg/kg bw/day, due elimination of REACTIVE YELLOW F01-0555 or its metabolites through urine and an expected staining effect. 

 

Treatment-related dark/orange liquid material in the content and dark/orange discoloration of the mucosa of the stomach, small intestines and/or caecum, colon and rectum were observed at 62.5, 250 and 1000 mg/kg bw/day macroscopically in the Main Parental Generation at necropsy.

 

Histologically, eosinophilic material adhered to the mucosa of the stomach, small or large intestines and/or deposited in the cytoplasm of the glandular epithelial cells in the stomach and enterocytes of the intestines, and with eosinohpilic pigment in the cytoplasm of macrophages in the mesenteric lymph node were noted. There was a dose relation to severity and incidence of these findings. These microscopic observations were also in correlation with the dark/orange discoloration noted at necropsy. Following a 14 day recovery period, almost all residual microscopic effects of the treatment were completely recovered. It occurred that the test item has not been completely cleared following 14 days withdrawal of treatment in the stomach of 3/5 male and 1/5 High Dose female animals, however this finding was regarded as a minor residual effect without toxicological significance in the absence of associated reactive changes.

 

Additionally, three pups were found dead and intact and were necropsied between PND 0 and 1. A specific cause of death could not be determined for these animals and based on the low incidence and distribution throughout all the dose groups, their death was considered unrelated to treatment.

No induction of micronuclei in bone marrow erythrocytes was observed following administration of REACTIVE YELLOW F01-0555 to Wistar rats daily by oral gavage to the High dose Main animals at 1000 mg/kg bw/day, thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.

 

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for REACTIVE YELLOW F01-0555 for parental/adult effects is considered to be 1000 mg/kg bw/day.