Octasodium 2,2'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino(1-hydroxy-3,6-disulphonatonaphthalene-2,8-diyl)azo]]bisnaphthalene-1,5-disulphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date (Pre-study chemistry investigations): 19 April 2017 Experimental completion date: 19 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Remarks:

No deviations occured that were considered to have affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Octasodium 2,2'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino(1-hydroxy-3,6-disulphonatonaphthalene-2,8-diyl)azo]]bisnaphthalene-1,5-disulphonate

EC Number:

275-108-6

EC Name:

Octasodium 2,2'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino(1-hydroxy-3,6-disulphonatonaphthalene-2,8-diyl)azo]]bisnaphthalene-1,5-disulphonate

Cas Number:

71002-20-5

Molecular formula:

C52H26Cl2N14Na8O26S8

IUPAC Name:

octasodium 3-({4-chloro-6-[(4-{[4-chloro-6-({8-[(1E)-2-(1,5-disulfonatonaphthalen-2-yl)diazen-1-yl]-1-hydroxy-3,6-disulfonatonaphthalen-2-yl}amino)-1,3,5-triazin-2-yl]amino}phenyl)amino]-1,3,5-triazin-2-yl}amino)-5-[(1E)-2-(1,5-disulfonatonaphthalen-2-yl)diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate

Test material form:

solid

Specific details on test material used for the study:

Test item: Bayscript Magenta BB
Test item identity (including alternative names): 1,5-Naphthalenedisulfonic acid, 2,2’-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino(8-hydroxy-3,6-disulfo-1,7-naphthalenediyl)-2,1-diazenediyl]] bis-,sodium salt (1:8)
Appearance: Solid, brown to black, golden shiny.
Storage conditions: Ambient temperature (15-25ºC) and desiccated.
Expiry date: 17 June 2018

Test animals

Species:

rat

Strain:

other: RccHan™;WIST

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Han Wistar strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier: Envigo RMS (UK).
Number of animals ordered: 44 males and 48 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization
Males: eight days prior to the commencement of treatment.
Females: 22 days prior to the commencement of treatment.
Age of the animals at the start of treatment
Males 86 to 92 days old.
Females 100 to 106 days old.
Weight range of the animals at the start of treatment
Males 298 to 366 g.
Females 192 to 235 g.

Allocation and Identification
Allocation:
On arrival and non-selective allocation to cages.
Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study.
On Day 1 of study all animals were weighed and body weights were reviewed by Study Management before dosing commenced to ensure variations in body weight of animals did not exceed  20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.

Identification of animals:
Each adult animal was assigned a number and identified uniquely within the study using a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

Identification of cages:
Each cage label was colour-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal Replacement
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before allocation:
Irregular estrous cycle - Four females
Body weight range extremes - Two males

Animal Care and Husbandry
Environmental Control
Rodent facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%.
Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
Lighting: Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.

Animal Accommodation
Cages: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Cage distribution: The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Pre-pairing- up to five animals of one sex
Pairing - one male and one female
Males after mating - up to five animals
Gestation - one female
Lactation - one female + litter

Environmental Enrichment
Aspen chew block - A soft white untreated wood block; provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced when necessary.
Plastic shelter Provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced at the same time as the cages.

Diet Supply
Diet: SDS VRF1 Certified pelleted diet.
A sample (100 g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30ºC) until finalization of the report. Samples were discarded after finalization of the report.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability: Non-restricted.

Water Supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at weekly intervals.
Availability: Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen chew blocks.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral gavage route of administration was chosen as the preferred route for human risk assessment.
Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.

Volume dose 10 mL/kg body weight.
Individual dose volume - Calculated from the most recently recorded scheduled body weight.
Control (Group 1) - Vehicle at the same volume dose as treated groups.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

Method of preparation: The test item was ground to a fine powder before weighing, after which requisite amounts were weighed into a suitable container. Working in ascending order of concentration, approximately 50-60% vehicle (as final volume) was added to the powder and magnetically stirred for one hour. Extra vehicle was then added until final volume was obtained. The suspension was then mixed with a high shear homogenizer until homogeneous. The suspension was transferred to final containers, via syringe, whilst magnetically stirring.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation: Weekly.

Storage of formulation:
Refrigerated (2-8ºC).

Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. These investigations demonstrated that formulations in the 1 to 100 mg/mL concentration range are stable following ambient storage (15-25°C) for one day and following refrigerated storage (2-8°C) for 15 days.

Achieved concentration
Samples of each formulation prepared for administration in Weeks 1 and 2 of treatment and on Day 10-12 of lactation were analyzed for achieved concentration of the test item.


The homogeneity and stability of Bayscript Magenta BB in purified water formulations was assessed with respect to the level of concentration at nominal concentrations of 1 mg/mL and 100 mg/mL.
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 day and refrigeration for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 6% of the initial time zero value and the coefficient of variation was less than 4%.
In Week 1 contingency samples were analyzed to confirm the results observed in the original analysis and the results are reported from mean of 6 samples. The results are all above nominal between +11% and +13.9% for Week 1. The coefficient of variation for each group (n=6) is less than 5% confirming the precision of analysis.
In Week 2, the nominal concentrations are above nominal again, with the results observed being +11.8% to +15.0% of nominal concentration. The coefficient of variation for each group (n=3) is less than 2% confirming the precision of analysis.
For Days 10-12 of lactation, the results are within 3% of the nominal concentrations and the coefficient of variation is less than 4% confirming the precision of analysis.

Duration of treatment / exposure:

Males - Two weeks before pairing up to necropsy after minimum of five weeks.
Females - Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

330 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels selected for investigation in this OECD 422 combined repeated dose toxicity study and reproductive/developmental toxicity screening study (0, 100, 330 and 1000 mg/kg/day) were chosen in conjunction with the Sponsor and were based on the results of a 14-day preliminary study conducted at these laboratories (Envigo Study No. XP98DD).
In the preliminary study dose levels of 500, 800 and 1000 mg/kg/day were investigated. There were no test item-related premature deaths, no signs observed in relation to dose administration, no test item-related changes in clinical condition, food and water consumption or spleen weights. Overall body weight gains from Day 1 to 15 of dosing were slightly reduced for males receiving 800 or 1000 mg/kg/day and at all dose levels for females. Mean absolute and body weight relative liver weights were low for males receiving 1000 mg/kg/day and females receiving 800 or 1000 mg/kg/day, kidneys weights were also low for females receiving 800 or 1000 mg/kg/day. Macroscopic findings were limited to abnormal (pink) colouration affecting numerous tissues at all dose levels, increased incidences were observed at higher dose levels (800 or 1000 mg/kg/day).
Therefore, a high dose level of 1000 mg/kg/day was selected for this study as the limit dose for the OECD 422 guideline. The intermediate and low dose levels of 330 and 100 mg/kg/day were chosen to achieve a dose response and/or aid in the determination of a No-Observed-Adverse-Effect-Level (NOAEL).

Examinations

Observations and examinations performed and frequency:

Serial Observations

Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.
Blood samples were collected for hematology and blood chemistry investigations for the group terminated early. A complete necropsy was performed in all cases.

Clinical and Behavioral Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males
Week 1 - daily
Week 2 onwards - once each week
F0 females
Week 1 - daily
Week 2 - once
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12
Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation
One to two hours after completion of dosing
As late as possible in the working day

Detailed physical examination and arena observations
Before treatment commenced and during each week of treatment and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons, therefore observations were made on these occasions without “blinding”.
After removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, male cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. For females, animals were moved into individual cages prior to transport to the testing room. The cage labels on these individual cages showed only the study and animal number. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

The following measurements, reflexes and responses were recorded:
Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores probe/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction

Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response

Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor
Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression

Grip strength
Forelimb and hind limb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.

Motor activity
During Week 5 of treatment for males and at Day 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and the first five lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

Body Weight
The weight of animals was recorded as follows:
F0 males:
Weekly during acclimatization.
Before dosing on the day that treatment commenced (Day 1) and weekly thereafter.
On the day of necropsy.

F0 females:
Weekly during acclimatization.
Before dosing on the day that treatment commenced (Day 1) and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7 and 13 of lactation.
On the day of necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals:
Weekly before pairing, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
For females after mating food consumption was recorded as follows:
Days 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

Estrous cycles
Dry and wet smears were taken as follows:
Dry smears:
For 15 days before pairing using cotton swabs.

Wet smears:
Using pipette lavage during the following phases:
- For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
- After pairing until mating.
- Females showing no evidence of mating - following completion of the pairing period females were separated from the male and vaginal smearing continued for up to five days or until the first estrous smear was seen. If a female showed an estrous smear during this period, she was killed as soon as practically possible and subject to macroscopic examination.
- For four days before scheduled termination.

Sacrifice and pathology:

Method of Kill
All adult animals - Carbon dioxide asphyxiation. Each animal was subsequently exsanguinated.
Offspring - selected for thyroid hormone sampling on Day 4 and 13 of age - Decapitation.
Offspring - all other - Intraperitoneal injection of sodium pentobarbitone.
Sequence - To allow satisfactory inter-group comparison.

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. Examination included a detailed assessment and documentation of any color changes in the internal organs, adipose tissue or skin. If color changes were observed, representative photographs were taken before retained tissues were placed in fixative.

Time of Necropsy
F0 males - After at least five weeks of treatment.
F0 females failing to mate - If an estrous smear was seen following completion of the pairing period animals were terminated as soon as logistically possible.
If no estrous smear was seen, animals were terminated on Day 25 after last day of pairing.
F0 females whose litter died before Day 13 - On or after day the last offspring died.
F0 females - Day 14 of lactation (following terminal blood sampling).
F1 offspring - Selected offspring for thyroid hormone analysis - Day 4 of age.
Scheduled kill - Day 13 of age.
The organs weighed, tissue samples fixed and sections examined microscopically are detailed 'any other information on materials and methods' for F0 animals.

Females
The following were recorded:
Each uterine horn - Number of implantation sites was counted and confirmed if none were visible at visual inspection.
Female whose litter died before Day 13 of lactation - Mammary tissue appearance.

Offspring
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content and particular attention paid to external genitalia was performed. Abnormal tissues were retained.

F1 offspring on Day 4 of age: Blood sampling required.
Externally normal offspring discarded without examination.
Externally abnormal offspring examined externally, and retained pending possible future examination.


F1 offspring on Day 13 of age: Blood sampling required.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Animals observed with external abnormalities were retained pending possible future examination
Thyroid glands were preserved from one male and one female in each litter.


Organ Weights
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals and for the group terminated early.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes - Initially in modified Davidson’s fluid.
Eyes - In Davidson’s fluid.

Histology
Processing - Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List - All F0 animals killed or dying prematurely.
The five lowest numbered surviving F0 males in Groups 1 and 3 and first five lactating F0 females with a surviving litter in Groups 1 and 3 at scheduled termination.
All surviving animals from the group terminated early.
Kidneys and testes - Group 3 remaining F0 animals.
All Group 2 F0 animals.
Abnormalities - All F0 animals.
Routine staining - Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.


Light Microscopy
Tissues preserved for examination were examined as follows:
Scheduled kill:
Premature deaths - F0 males and females in Groups 1 and 4 only - All specified in the table in section 'any othe information on materials and methods'
Group terminated early - All surviving F0 animals - All specified in the table in section 'any othe information on materials and methods'
Scheduled kill
The five lowest numbered surviving F0 males and first five lactating F0 females with a surviving litter in Groups 1 and 3 at scheduled termination - All specified in the table in section 'any othe information on materials and methods'
Scheduled kill
All F0 animals - Abnormalities
Group 3 remaining F0 animals, All Group 2 F0 animals - Males - Males - Kidneys and testes. Females - Kidneys

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

Other examinations:

Hematology, Peripheral Blood
Blood samples were collected after overnight withdrawal of food at the following occasion:

At termination: The five lowest numbered surviving males and the first five lactating females with a surviving litter, in each dose group.
At early termination: All surviving animals.

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

* Derived value calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.


Blood Chemistry
Blood samples were collected after overnight withdrawal of food at the following occasion:

At termination:The five lowest numbered surviving males and the first five surviving lactating females per group.
At early termination: All surviving animals

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acids (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)
Globulin (Glob)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.


Thyroid Hormone Analysis
Blood samples were collected as follows:
At termination: All adults. No samples were collected from animals which failed to mate or with a total litter loss.
Day 4 of age:
F1 offspring, two females per litter (where possible).
- If four female offspring available - one female was selected for T4 (serum)#
- If five or more female offspring available - one female was selected for T4 (serum) and one female for TSH (plasma)
No females were allocated to these procedures if:
- The resultant live litter size fell below eight pups.
- The resultant number of live females fell to less than three (for subsequent allocation to the Day 13 procedures) e.g. three female offspring in the litter – no offspring selected.
# priority was given to serum sample

Day 13 of age:
F1 offspring, two males and two females per litter (where possible)
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female)
# priority given to serum sample


Sequence of blood sampling on each occasion: In order to minimize any potential confounding effect of the time of day of blood sampling, the order of blood sampling was controlled to allow satisfactory inter-group comparisons.
Conditions: No overnight deprivation of food.
Anesthetic: F0 animals: Isoflurane
F1 offspring : None
Blood sample site: F0 adults: Sublingual vein
F1 offspring : Decapitation
Parameter: Thyroid stimulating hormone (TSH)
Anticoagulant: K2 EDTA with no separator gel.
Tubes: Standard Envigo.
Blood volume:
Adults: 0.5 mL.
Offspring: max possible.
Processing: Samples were kept on wet ice prior to centrifugation.
Centrifugation commenced within 30 minutes of sampling.


Parameter: Thyroxine (T4)
Anticoagulant: None
Tubes: Greiner Minicollect - with clot activator.
Blood volume:
Adults: 0.5 mL.
Offspring: max possible.
Processing: Samples were kept at ambient temperature (15 to 25ºC) for a minimum of 30 minutes prior to centrifugation.


Centrifugation conditions: At 2000g for ten minutes at 4°C.
Final storage conditions : Deep frozen (approximately -60°C to -90ºC).
Fate of samples: Dispatched to the Department of Biomarkers, Bioanalysis and Clinical Pathology Envigo.
Thyroid hormone analysis: Performed by the Department of Bioanalysis, Envigo.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs Associated with Dosing and Detailed Physical Examination:
The signs detailed at physical examination for animals receiving 1000 mg/kg/day included staining of the forepaws, hindpaws and tail, these signs were associated with the colour of test item. During gestation, two females receiving 1000 mg/kg/day (4F Nos. 69 and 70) showed reddening of the skin affecting the nose, pinna, lower and upper eyelids.

Administration with Bayscript Magenta BB at doses of 100 and 330 mg/kg/day during the 5 week treatment period for males, and during the 2-week pre-pairing period, gestation and lactation periods for females was well tolerated. There were no test item-related signs observed following dose administration or signs at routine clinical examination that were considered to be associated with treatment, with the exception of staining caused by the colour of the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

Three males (4M Nos. 15, 17 and 18) receiving 1000 mg/kg/day were found dead on Days 11, 8 and 6 of dosing, respectively during the pre-pairing phase of study having shown staining with the test item at recording of clinical signs.
As a result of these deaths and in order to prevent any further suffering, dosing was ceased on Day 11 of the study for all remaining males receiving 1000 mg/kg/day. The remaining animals were subsequently sent to necropsy on Day 12 of the study.
One female (4F 70) receiving 1000 mg/kg/day was found dead on Day 2 of gestation having shown reddening of the skin and staining of the test item at recording of clinical signs.
Given these observations and previous male decedents, all remaining females receiving 1000 mg/kg/day were prematurely terminated to avoid any further suffering to the animals. Dosing for females receiving 1000 mg/kg/day ceased on Day 18 of treatment, and the animals were sent to necropsy two days later on Day 20 of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Prior to the cessation of dosing, males and females given 1000 mg/kg/day showed statistically significantly mean body weight loss during Week 1 for males and Weeks 1 and 2 for females during the pre-pairing treatment period.

During the two-week pre-pairing treatment period, males given 330 mg/kg/day and females given 100 or 330 mg/kg/day showed slightly lower mean body weight gain specifically during the first week of dosing for females.
The mean body weight gain of males in the 330 mg/kg/day group remained lower than Control during the first week of pairing, for the remaining treatment period mean body weight gains were similar to Controls, although overall mean body weight gain from Days 1 to 36 of study was slightly lower than Control.
After mating, the mean body weight of females receiving 330 mg/kg/day was lower than Control, with statistical significance attained for the period of Days 14 to 20 of gestation; overall mean body weight gain from Day 0 to 20 of gestation was 25% lower than Control.
Following parturition, the mean body weight gain for females given 100 or 330 mg/kg/day was essentially similar to Control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption for males given 1000 mg/kg/day group was lower than Control prior to the termination of dosing. Mean food consumption for females given 1000 mg/kg/day was essentially similar to Controls during the pre-pairing treatment period.

Food consumption for males and females receiving 100 and 330 mg/kg/day was similar to that of Controls throughout the pre-pairing treatment period and dosing period for males.
After mating, females receiving 330 mg/kg/day had lower group mean food consumption values when compared to Controls, statistical significance was attained during Days 7 to 13 and Days 14 to 19 of gestation correlating with the reduced mean body weight gain observed during gestation.
Following parturition, mean food consumption for females given 330 mg/kg/day during Days 4 to 6 and Days 7 to 12 of lactation was statistically lower than Controls, however mean body weight gain for this group was essentially similar to Controls for the lactation period.
Mean food consumption for males and females given 100 mg/kg/day was unaffected by Bayscript Magenta BB administration.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The haematological examinations for the animals terminated early revealed changes in mean peripheral white blood cell numbers: these included increased numbers of lymphocytes (with a concomitant increase in total white blood cell counts). Prothrombin times were slightly prolonged in males receiving 1000 mg/kg/day.

The haematological examinations at scheduled termination revealed, when compared with Controls, a dose-dependent reduction in total white blood cell counts in males given 100 or 330 mg/kg/day, which attained statistical significance at the 330 mg/kg/day level. This was a result of lymphocyte, monocyte and neutrophil counts being lower in treated males; however of these only the monocyte count for males receiving 330 mg/kg/day attained statistical significance. White blood cell counts for females on Day 14 of lactation, however, were similar to Control values at the 100 and 330 mg/kg/day dose levels.
All other haematological differences from controls observed were generally small, confined to one sex, or the magnitudes were not dose-related and, consequently, were considered to represent normal biological variation. These included a slight dose-dependent reduction in the number of reticulocytes in males.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination of the blood plasma in animals terminated early revealed: high bilirubin, bile acids and creatinine concentrations in both males and females given 1000 mg/kg/day.

There were no biochemical changes in the plasma at scheduled termination which were attributable to treatment with Bayscript Magenta BB.
Calcium concentrations in males receiving 100 mg/kg/day were statistically significantly lower than Control, however in the absence of a dose relationship it was considered fortuitous and unrelated to Bayscript Magenta BB administration. Sodium concentrations in females given 100 or 330 mg/kg/day were slightly higher than Control, attaining statistical significance at 330 mg/kg/day, however this difference only occurred in one sex. All other biochemical differences from controls were minor, lacked dose relationship or occurred in one sex only and were therefore attributed to normal biological variation.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Bedding Observations
Bedding observation was completed during treatment to document the extent of staining caused by Bayscript Magenta BB, this observation ceased following the termination of Group 4 females. Urine and faeces were observed as an abnormal (pink) colouration which affected all cages in males and females receiving 1000 mg/kg/day Bayscript Magenta BB.

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Following five weeks of treatment, males receiving 330 mg/kg/day showed a body weight adjusted slight decrease in mean testes and epididymides weights when compared to Control. In addition, body weight adjusted kidney weights were slightly higher for males receiving 100 and 330 mg/kg/day compared to Controls.
Assessment of organ weights on Day 14 of lactation revealed slightly high absolute and bodyweight adjusted kidney and liver weights in females at 330 mg/kg/day when compared with Controls. In addition, group mean adjusted ovary, uterus, cervix and oviducts weights were slightly low for females given 100 or 330 mg/kg/day.
All other inter-group differences from control values were minor, not dose-related or was evident in one sex and, consequently, were considered to represent normal biological variation.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Three males (4M Nos. 15, 17 and 18) receiving 1000 mg/kg/day were found dead on Days 11, 8 and 6 of dosing, respectively during the pre-pairing phase of study having shown staining with the test item at recording of clinical signs. Macroscopic examination at necropsy revealed both substantial abnormal staining and/or contents (pink coloration) of multiple organs similar to the colour of Bayscript Magenta BB. In all three males, many tissues were autolytic and were therefore unable to be interpreted on microscopic examination. Significant microscopic findings included hepatocellular apoptosis (minimal or slight severity) in all three males which was spread diffusely throughout the liver, with additional leucocytosis (minimal to slight severity) in male Nos. 15 and 17 and hepatocellular vacuolation (minimal severity) in Male No. 17. In all three males, there was significant autolysis diffusely throughout the mucosa of the stomach, but the possibility of multifocal regions of mucosal necrosis in these animals could not be eliminated.

One female (4F 70) receiving 1000 mg/kg/day was found dead on Day 2 of gestation having shown reddening of the skin and staining of the test item at recording of clinical signs. Macroscopic examination at necropsy revealed dark areas of the lungs, bronchi and oesophagus, in addition to the abnormal staining and/or contents (pink coloration) affecting multiple organs. Microscopic examination revealed inflammation (slight severity), haemorrhage (minimal severity) and epithelial hyperplasia (minimal severity) in the uterus and inflammation (slight severity) and haemorrhage (moderate severity) in the vagina. Hepatocyte apoptosis (moderate severity) and vacuolation (minimal severity) were present in the liver. Hypertrophy (minimal severity) was present in the cortex of the adrenals. The changes identified in the uterus, vagina and liver may have been contributing factors to the death of the animal, but due to autolysis, the precise cause of death could not be determined.

The macroscopic examination conducted for all premature decedents and early terminated animals that received 1000 mg/kg/day revealed abnormal pink colouration affecting multiple tissues. The incidence and distribution of all other findings were considered to be unrelated to treatment.

The macroscopic examination performed after five weeks of treatment for males and on Day 14 of lactation for females revealed, when compared with controls, abnormally pink colouration of the kidneys in all males and females given 100 and 330 mg/kg/day.
Abnormally pink colouration of multiple organs was recorded in the majority of animals given 330 mg/kg/day. Several males and females revealed abnormal colour (pink) of the mesenteric lymph nodes at 100 mg/kg/day. Furthermore, a single female receiving 100 mg/kg/day, and another receiving 330 mg/kg/day had pale areas on the lungs and bronchi.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Animals Receiving 1000 mg/kg/day and Terminated Early
Treatment Related Findings
Changes related to treatment with Bayscript Magenta BB were seen in the kidneys, stomach, spleen and testes.

Kidneys
Degeneration of cortical tubules was present in all animals given 1000 mg/kg/day. Degenerating tubules contained vacuolated epithelial cells with occasional sloughed cells. In addition there was regeneration in the cortical tubules in one male and all females, displaying basophilic tubules and an increase in mitotic epithelial cells. In all males, hyaline droplets were present in the cytoplasm of the epithelial cells lining the cortical tubules. The changes observed in the kidneys were bilateral and spread diffusely throughout the cortex.

Stomach
Eosinophilic globules were present in the cytoplasm of epithelial cells within the mucosa of the glandular region of the stomach in the majority of females and some males given 1000 mg/kg/day. Additionally, in most males and some females there was an accompanying foveolar epithelial hyperplasia. In most males, submucosal inflammation was present in the glandular region of the stomach.

Liver
Apoptosis of hepatocytes and hepatocyte vacuolation were present in three females receiving 1000 mg/kg/day and terminated early. This was accompanied by prominent mitotic activity in hepatocytes in two females.

Testes
An increase in Periodic acid-Schiff (PAS) staining (slight severity) was present in the interstitial macrophages of the testes in all males terminated early that received 1000 mg/kg/day.

Spleen
An increase in cellularity of the white pulp of the spleen was present in one male and four females that received 1000 mg/kg/day.

Incidental Findings
All other findings were considered incidental and not related to administration of Bayscript Magenta BB.
Animals Receiving At Least 5 Weeks of Treatment
Treatment Related Findings

Changes related to treatment with Bayscript Magenta BB were seen in the kidneys and testes.

Kidneys
Hyaline droplets were present in the cytoplasm of the renal tubular epithelium of the cortical tubules in three males given 330 mg/kg/day. In most males and all females given 330 mg/kg/day, degeneration was present in the cortical tubules. Degenerating tubules contained vacuolated epithelial cells with occasional sloughed cells. In addition, in females given 330 mg/kg/day there was regeneration in the cortical tubules, characterised by basophilic tubules and an increase in mitotic epithelial cells. The changes observed in the kidneys were bilateral and spread diffusely throughout the cortex.

Testes
An increase in Periodic acid-Schiff (PAS) staining was present in the interstitial macrophages of males given 330 mg/kg/day. The staining had a granular pattern and was localised to the cytoplasm.

Incidental Findings
All other findings were considered incidental and not related to administration of Bayscript Magenta BB.


Summaries of treatment related findings can be found in section 'any other information on results'.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid Hormone Analysis
There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age. There was therefore no requirement to measure T4 in the samples obtained from offspring on Day 4 of age or from the adult females and none of the TSH (thyroid stimulating hormone) samples required analysis.

Sensory Reactivity and Grip Strength
The sensory reactivity observations conducted during Week 5 of treatment and Days 7 to 9 of lactation revealed no findings which were considered treatment related.

Motor Activity
During Week 5 of treatment group mean high beam (rearing activity) and low beam scores (cage floor activity) for males receiving 330 mg/kg/day were slightly lower than the controls, specifically from the 24 minute interval. As a consequence, the overall high and low beam scores were lower than controls, however, no statistical significances were attained. A similar pattern was observed in females receiving 330 mg/kg/day during Days 7 to 9 of lactation, overall low beam scores were lower than controls with an isolated statistical significance attained at the 54-minute interval. Females receiving 330 mg/kg/day exhibited similar scores to controls for high beam counts, which are generally more sensitive to treatment related effects than the low beam scores.

Estrous Cycles, Pre-Coital Interval, Mating Performance, Fertility and Gestation Length and Gestation Index
All females allocated to the study showed normal 4/5 day estrous cycles during the acclimatization period.
Estrous cyclicity, pre-coital interval, fertility and mating performance were unaffected by treatment with Bayscript Magenta BB. One Control female (1F No. 86) failed to mate.
All females were not cycling before termination (Days 11 to 14 of lactation) and were in diestrous at termination.
A slight shift in gestation length was apparent in comparison with the concurrent Control, the difference for females receiving 330 mg/kg/day attained statistical significance. Two females exhibited a 23.5 day gestation length and a single female also showed a 25 day gestation length, compared to no females in the Control group. The percentage of females showing an extended gestation length is slightly above the Historical Control Data range; however five females receiving 330 mg/kg/day did show a 23 day gestation length (similar to four Control females).

Effect levels

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

1

Critical effects observed:

yes

Lowest effective dose / conc.:

330 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

2

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

3

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

other: lymphatic system

Organ:

spleen

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

4

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Mean serum T4 concentrations (pg/mL)

Group	Treatment	Dose (mg/kg/day)		Adult Male at Termination	Day 13 of age Offspring
					Male	Female
1	Control	0	Mean	47300	45200	55300
			SD	9500	4940	8430
			CV %	20.1	10.9	15.2
			N	10	9	9
2	Bayscript Magenta BB	100	Mean	41600	45300	48800
			SD	3990	6560	5200
			CV %	9.6	14.5	10.7
			N	10	10	10
3	Bayscript Magenta BB	330	Mean	45400	55400	53500
			SD	5720	6380	7410
			CV %	12.6	11.5	13.9
			N	10	9	9

Summary of treatment related findings in the kidneys for animals receiving 1000 mg/kg/day and terminated early.

Group/sex	4M	4F
Dose (mg/kg/day)	1000	1000
Hyaline Droplets, Cortical Tubules
Minimal	3	0
Slight	3	0
Moderate	1	0
Total	7	0
Degeneration, Cortical Tubules
Minimal	5	0
Slight	2	2
Moderate	0	7
Total	7	9
Regeneration, Cortical Tubules
Minimal	1	1
Slight	0	2
Moderate	0	6
Total	1	9
Number of tissues examined	7	9

Summary of treatment related findings in the stomach for animals receiving 1000 mg/kg/day and terminated early

Group/sex	4M	4F
Dose (mg/kg/day)	1000	1000
Eosinophilic Globules, Mucosa, Glandular Region
Minimal	2	7
Slight	3	0
Total	5	7
Foveolar Hyperplasia, Glandular Region
Minimal	3	2
Slight	3	0
Total	6	2
Submucosal Inflammation, Glandular Region
Minimal	5	0
Total	5	0
Number of tissues examined	7	9

Summary of treatment related findings in the liver for animals receiving 1000 mg/kg/day and terminated early

Group/sex	4F
Dose (mg/kg/day)	1000
Hepatocyte Apoptosis
Minimal	2
Slight	1
Total	3
Hepatocyte Vacuolation
Minimal	3
Total	3
Prominent Mitotic Activity
Minimal	2
Total	2
Number of tissues examined	9

Summary of treatment related findings in the testes for animals given 1000 mg/kg/day and terminated early

Group/sex	4M
Dose (mg/kg/day)	1000
PAS Staining Increased, Interstitial Macrophages
Slight	7
Total	7
Number of tissues examined	10

Summary of treatment related findings in the spleen for animals given 1000 mg/kg/day and terminated early

Group/sex	4M	4F
Dose (mg/kg/day)	1000	1000
Increased Cellularity, White Pulp
Minimal	1	4
Total	1	4
Number of tissues examined	9	9

Summary of treatment related findings in the kidneys for animals receiving at least 5 weeks of treatment

Group/sex	1M	2M	3M	1F	2F	3F
Dose (mg/kg/day)	0	100	330	0	100	330
Hyaline Droplets, Cortical Tubules
Minimal	1	1	3	0	0	0
Total	1	1	3	0	0	0
Degeneration, Cortical Tubules
Minimal	0	0	7	0	0	1
Slight	0	0	2	0	0	5
Moderate	0	0	0	0	0	3
Total	0	0	9	0	0	9
Regeneration, Cortical Tubules
Minimal	0	0	0	0	0	3
Slight	0	0	0	0	0	5
Total	0	0	0	0	0	8
Number of tissues examined	5	10	10	5	10	9

 

Summary of treatment related findings in the testes for animals receiving at least 5 weeks of treatment

Group/sex	1M	2M	3M
Dose (mg/kg/day)	0	100	330
PAS Staining Increased, Interstitial Macrophages
Minimal	0	0	6
Slight	0	0	4
Total	0	0	10
Number of tissues examined	10	10	10

Applicant's summary and conclusion

Conclusions:

In conclusion, the oral administration of Bayscript Magenta BB at 1000 mg/kg/day was not tolerated and dosing was terminated early for males and females on Days 11 and 18 of dosing, respectively. The oral administration of Bayscript Magenta BB up to and including 330 mg/kg/day for five weeks in males and for two weeks prior to pairing, throughout gestation and up to Day 13 of lactation in females, was generally well tolerated.

Test item-related histopathological changes were evident in the kidneys, consisted of tubular degeneration in both sexes, tubular regeneration predominately in females and hyaline droplets in males in animals given 330 or 1000 mg/kg/day, associated with increases in adjusted kidney weights in both sexes given 330 mg/kg/day and with gross changes of abnormal coloured kidneys seen at necropsy; these kidney changes were considered to be adverse. Test-item related histopathological changes were also evident in the stomach, spleen and liver of animals given 1000 mg/kg/day and terminated early. These included eosinophilic globules and foveolar hyperplasia in the stomach, with submucosal inflammation observed in males only, increased cellularity of the splenic white pulp and, hepatocellular apoptosis and vacuolation in the liver of females. It was therefore concluded that within the context of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 100 mg/kg/day.

There was no evidence of any adverse effects on mating performance of the adult animals or on the development of the F1 offspring. The reduction in the number of uterine implantations at 330 mg/kg/day (and as a consequence litter size) was associated with the slightly low post implantation survival index. This effect, however, was statistically significant and all parameters were outside of the Historical Control Data values (representing 11 OECD TG 422 studies). This effect is considered to be related to treatment and it is likely that the male and/or female reproductive systems have been affected, but the mechanism is undetermined and potentially adverse. It was therefore concluded that within the context of this study, the NOAEL for reproductive performance was 100 mg/kg/day.
Bayscript Magenta BB showed no evidence of being an endocrine disruptor.

Executive summary:

Summary

The purpose of this study was to assess the general systemic toxic potential in Han Wistar rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of Bayscript Magenta BB (an industrial colourant) by oral gavage administration forat least five weeks. The study was conducted to fulfil the data requirement according to REACH regulation Annex VIII chapter 8.6.1.

Three groups of ten male and ten female rats received Bayscript Magenta BB at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration at a volume dose of 10 mL/kg/day. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, purified water, at the same volume dose as treated groups. Dosing for males receiving 1000 mg/kg/day was suspended on Day 11 of treatment as a result of three male decedents, consequently the remainder of this group were sacrificed early on Day 12 of treatment. Females that received 1000 mg/kg/day were subsequently paired with undosed stock males. One female receiving 1000 mg/kg/day was found dead on Day 18 of study (Day 2 of gestation), resulting in the cessation of dosing Group 4 females and the surviving females in the group were sacrificed early on Day 20 of study.

During the study, for adult animals assessments of clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

Blood samples were collected from all adult animals at termination and from selected offspring on Day 4 and Day 13 of age for thyroid hormone analysis.

Results

Treatment with Bayscript Magenta BB at 1000 mg/kg/day was not tolerated and dosing was suspended early for males and females on Days 11 and 18 of treatment, respectively. There were four premature deaths, three males and one female during the course of the study in the 1000 mg/kg/day group, the precise cause of these deaths were not established due to autolysis however they are considered to be related to treatment. 

Prior to the cessation of dosing at 1000 mg/kg/day the signs observed were related to the colour of the test item, body weight loss was observed in males and females during Week 1 and was still evident in females during Week 2 of dosing (prior to pairing). Mean food consumption was lower for males only. Haematological examinations revealed changes in mean peripheral white blood cell numbers and prothrombin times were slightly prolonged in males only. The biochemical examination of the blood plasma revealed: high bilirubin, bile acids and creatinine concentrations in both males and females given 1000 mg/kg/day. Macroscopic examination of the premature decedents and animals that were terminated early revealed pink colouration affecting numerous tissues. Histopathological evaluation of retained tissues for the animals receiving 1000 mg/kg/day and terminated early revealed treatment related changes in the kidneys, stomach, spleen and testes. In the kidneys, degeneration of cortical tubules was present in all animals and there was regeneration in the cortical tubules in one male and all females given 1000 mg/kg/day. Eosinophilic globules were present in the cytoplasm of epithelial cells in the mucosa of the glandular region of the stomach affecting the majority of animals and this was accompanied by foveolar epithelial hyperplasia in some animals; this was associated with submucosal inflammation in several males. Apoptosis of hepatocytes, hepatocyte vacuolation and prominent mitotic activity in hepatocytes were present in the liver of a few females receiving 1000 mg/kg/day and terminated early. An increase in Periodic acid-Schiff (PAS) staining was present in the interstitial macrophages of the testes in all males given 1000 mg/kg/day. In the spleen, an increase in cellularity of the white pulp was observed in one male and four females that received 1000 mg/kg/day.

Treatment with Bayscript Magenta BB to parental Han Wistar rats at dose levels of 100 and 330 mg/kg/day for two weeks prior to pairing, during pairing and then up to termination of the males after five weeks of treatment and females on Day 14 of lactation was generally well tolerated. There were no premature deaths, no test-item related signs observed during the detailed physical examination and arena observations and no post-dosing observations with the exception of signs related to the colour of the test item. There were no effects on sensory reactivity and grip strength assessments. There was a slight effect observed for motor activity, overall group mean low beam scores were slightly low for both sexes receiving 330 mg/kg/day and overall high beam scores were low for males receiving 330 mg/kg/day.

At the commencement of treatment (prior to pairing), males given 330 mg/kg/day and females given 100 or 330 mg/kg/day showed slightly lower mean body weight gain. Body weight gain of males receiving 330 mg/kg/day for the first week of pairing remained lower than that of Control, subsequent body weight gain for these males were generally similar to Control. After mating, the body weight gain of females receiving 330 mg/kg/day during gestation was lower than that of Control, however following parturition mean body weight gain for females was essentially similar to Control.

Mean food consumption for males receiving 100 and 330 mg/kg/day was generally similar to Controls throughout the dosing period. Mean food consumption for females receiving 330 mg/kg/day was similar to the Control during the pre-pairing period, however lower mean food intake periods were observed during the gestation and lactation periods for these females.  

Estrous cyclicity, pre-coital interval, mating performance and fertility were unaffected by treatment with Bayscript Magenta BB. One Control female (1F No. 86) failed to mate. A slight shift towards longer gestation lengths was apparent for females receiving 330 mg/kg/day in comparison with the concurrent Control.

The haematological examinations at scheduled termination revealed, when compared with Controls, a dose-dependent reduction in total white blood cell count in males given 100 or 330 mg/kg/day. This was a result of lymphocyte, monocyte and neutrophil counts being lower in treated males. White blood cell counts for females on Day 14 of lactation, however, were similar to Control values at the 100 and 330 mg/kg/day dose levels.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 male and female offspring.

Changes in organ weights consisted of slightly lower body weight adjusted epididymides and testes weights in males receiving 330 mg/kg/day. In males, receiving 100 or 330 mg/kg/day slightly high body weight adjusted kidney weights were seen. Slightly high absolute and body weight adjusted kidney and liver weights were evident in females given 330 mg/kg/day. In addition, group mean adjusted ovary and uterus, cervix and oviducts weights were slightly low for females given 100 or 330 mg/kg/day. 

Macroscopic examination of the adult males and females revealed abnormally pink colouration of the kidneys in all males and females given 100 and 330 mg/kg/day. Abnormally pink colouration of multiple organs was recorded in the majority of animals given 330 mg/kg/day. Several males and females at 100 mg/kg/day were noted to have abnormal colour (pink) of the mesenteric lymph nodes. 

Histopathological evaluation of retained tissues revealed degeneration in the cortical tubules of the kidneys in most males and all females given 300 mg/kg/day; this was associated with regeneration in the cortical tubules for females only. An increase in Periodic acid-Schiff (PAS) staining was present in the interstitial macrophages of the testes for males given 330 mg/kg/day.

 

Conclusion

In conclusion, the oral administration of Bayscript Magenta BB at 1000 mg/kg/day was not tolerated and dosing was terminated early for males and females on Days 11 and 18 of dosing, respectively. The oral administration of Bayscript Magenta BB up to and including 330 mg/kg/day for five weeks in males and for two weeks prior to pairing, throughout gestation and up to Day 13 of lactation in females, was generally well tolerated. 

Test item-related histopathological changes were evident in the kidneys, consisted of tubular degeneration in both sexes, tubular regeneration predominately in females and hyaline droplets in males in animals given 330 or 1000 mg/kg/day, associated with increases in adjusted kidney weights in both sexes given 330 mg/kg/day and with gross changes of abnormal coloured kidneys seen at necropsy; these kidney changes were considered to be adverse. Test-item related histopathological changes were also evident in the stomach, spleen and liver of animals given 1000 mg/kg/day and terminated early. These included eosinophilic globules and foveolar hyperplasia in the stomach, with submucosal inflammation observed in males only, increased cellularity of the splenic white pulp and, hepatocellular apoptosis and vacuolation in the liver of females. It was therefore concluded that within the context of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 100 mg/kg/day.

There was no evidence of any adverse effects on mating performance of the adult animals or on the development of the F1 offspring. The reduction in the number of uterine implantations at 330 mg/kg/day (and as a consequence litter size) was associated with the slightly low post implantation survival index. This effect, however, was statistically significant and all parameters were outside of the Historical Control Data values (representing 11 OECD TG 422 studies). This effect is considered to be related to treatment and it is likely that the male and/or female reproductive systems have been affected, but the mechanism is undetermined and potentially adverse. It was therefore concluded that within the context of this study, the NOAEL for reproductive performance was 100 mg/kg/day.

Bayscript Magenta BB showed no evidence of being an endocrine disruptor.