Reaction products of tetrazotized 5-amino-2-[(E)-2-(4-amino-2-sulfophenyl)ethenyl]benzenesulfonic acid with 6-amino-4-hydroxynaphthalene-2-sulfonic acid and 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid in 2-[bis(2-hydroxyethyl)amino]ethanol

Endpoint summary

Description of key information

A subchronic oral toxicity study with administration of Bayscript Blaukomponente TEA to Han Wistar rats for 13 weeks (OECD TG 408) at doses of 100, 300 and 1000 mg/kg bw/day was associated with changes in the kidney which were considered adverse at 1000 mg/kg bw/day. The no-observed-adverse effect level (NOAEL) in this study was considered to be 300 mg/kg bw/day, based on slight to moderate degeneration of the tubular epithelium and moderate to marked tubular basophilia in the kidneys of both sexes given 1000 mg/kg bw/day.

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD TG 422) with oral doses of 100, 330, and 1000 mg/kg bw/day the NOAEL for general systemic toxicity was 330 mg/kg bw based on tubular degeneration in the kidneys of both sexes and with tubular basophilia in the kidney of males.

In the subchronic repeated dose toxicity study (OECD TG 408) and in the Reproductive/Developmental Toxicity Screening Study (OECD TG 422) the body and several organs of animals treated with 1000 mg/kg bw appeared discoloured, due to staining with the test item (dye). Coloured kidneys were observed starting at 100 mg/kg bw indicating the UVCB substance is already absorbed at this dose. This is further evidenced by purple/blue stained urine noticed after acute oral dosing of 300 mg/kg bw and higher in the OECD TG 422.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date (animal arrival) 08 April 2020
Experimental completion date (pathology) 27 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

dark blue to brown crystalline powder

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The RccHan™;WIST strain was used because of the historical control data available at this laboratory and this strain has been used in previous toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals
Strain/Species RccHan:WIST rat.

Supplier Envigo RMS Limited.

Number of animals 45 males and 45 females.

Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization 15 days before commencement of treatment.

Age of the animals at start of treatment 44 to 50 days.

Weight range of the animals at the start of treatment
Males: 146 to 193 g
Females: 120 to 153 g

Allocation and Identification
Allocation Randomly allocated on arrival.
Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.

Identification of animals
Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.

Identification of cages
Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal Replacement
On Day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed 20% of the mean for the appropriate sex. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch. No replacements were necessary.

Animal Care and Husbandry
Environmental Control
Animal 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 20-24C 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 Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
Cage distribution Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed, following a rotation plan, to further minimize possible effects of spatial variations.

Number of animals per cage Five of the same sex.

Bedding Wood based bedding which was changed at appropriate intervals each week.

Environmental Enrichment
Aspen gnawing material Provided to each cage throughout the study and replaced when necessary.
Plastic shelter Provided to each cage throughout the study and replaced when necessary.

Diet Supply
Diet Teklad 2014C Diet.
Availability Non-restricted (removed overnight before blood sampling for hematology or blood chemistry).

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

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

Route of administration:

oral: gavage

Details on route of administration:

The oral gavage route of exposure was selected because ECHA has accepted this as the most appropriate route of administration for the study.

Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at Constant doses in mg/kg bw/day.
Volume dose 10 mL/kg bw/day.

Vehicle:

water

Details on oral exposure:

Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg bw/day.
Volume dose 10 mL/kg bw/day.

Individual dose volume Calculated from the most recently recorded scheduled body weight.

Control (Group 1) Vehicle at the same volume dose as the treated groups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation Analysis
The concentration of Bayscript Blaukomponente TEA in the final solution was quantified by
HPLC using UV detection

High performance liquid chromatograph
(HPLC):
Agilent HP1100 HPLC system and UV/visible
wavelength detector.

Stability and homogeneity The homogeneity and stability of formulations in the concentration range of 0.01 to 50 mg/mL in purified water under ambient storage (15 to 25°C) for 24 hours were confirmed as part of Envigo Study No. CY71SC.

Homogeneity and stability of formulations in the concentration range of 1 and 100 mg/mL in purified water during ambient and refrigerated storage were confirmed as part of Envigo Study No. CD40DP as follows:

• One day stability at ambient temperature (15 to 25°C)
• 15 days refrigerated (2 to 8°C)

Achieved concentration Samples of each formulation prepared for administration in Weeks 1 and 12 of treatment were analyzed for achieved concentration of the test item.

Formulation analysis results:
The mean concentrations of Bayscript Blaukomponente TEA in test formulations analyzed during the study were within ±5% of nominal concentrations confirming the accuracy of formulation.
Bayscript Blaukomponente TEA was not detected in control formulations.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle only

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 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:

10 males
10 females

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection
The doses used in this study (0, 100, 300 and 1000 mg/kg bw/day) were selected in conjunction with the Sponsor and were based on the results of an OECD 422 combined toxicity and reproductive/developmental study in Han Wistar rats (Envigo Study Number CD40DP). In that study, dose levels of 100, 330 and 1000 mg/kg bw/day were well tolerated, with no mortality or clinical signs and no effects on body weight gain or food intake. Minimal to slight grade histopathological changes were seen in the kidney in males and females at 1000 mg/kg bw/day comprising degeneration of the tubular epithelium in both sexes and tubular basophilia in males, accompanied by lipofuscin accumulation. As a consequence of the histopathology changes, the no-observed-adverse-effect level (NOAEL) was considered to be 330 mg/kg bw/day. It was considered that this histopathology finding alone would not preclude further investigation at 1000 mg/kg bw/day in this 90-day study, therefore, a high dose of 1000 mg/kg bw/day was selected for this study as the limit dose for the OECD 408 guideline. The low and intermediate dose levels of 100 and 300 mg/kg bw/day were selected to investigate any dose relationship.

Observations and examinations performed and frequency:

Serial Observations
Clinical and Behavioral Observations
Signs are considered in two parts: detailed observations recorded at times in relation to dose administration, classified as ‘signs associated with dosing’ and extended changes in condition, classified as ‘clinical signs’.

Clinical observations are presented for each animal, providing detail of type of sign, week of occurrence and information on the duration of the sign applicable. There were no signs associated with dosing seen and so no appendix is included in this report.

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 occupants. 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
Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:

• Immediately before dosing (Pre-dose).
• At the end of dosing each group.
• One to two hours after completion of dosing all groups.
• As late as possible in the working day.

Detailed Physical Examination and Arena Observations
Before treatment commenced and during each week of treatment, 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.

After removal from the home cage, animals were assessed for physical condition and behavior 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 all animals during Week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. 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 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/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 hindlimb 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 12 of treatment (before dosing), the motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Covance.

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 necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

Body Weight
The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.

Group mean weight changes were calculated from the weight changes of individual animals.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

Group mean food consumptions and standard deviations for each period were derived from unrounded cage values. Overall mean food consumption values were calculated for each cage and the mean of these cage means were calculated for each group/sex.

Water Consumption
Fluid intake was assessed by daily visual observation. No significant effect was observed and consequently quantitative measurements were not performed.

Ophthalmic Examination
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope as follows:
Occasion Animals
Pretreatment All animals (including spares)
Week 12 Groups 1 and 4

Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

Hematology, Peripheral Blood
Blood samples were collected after overnight withdrawal of food and prior to dosing at the following occasion:
Occasion Animals
Week 13 All 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 K2-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 values 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. A manual count of the differential white blood cell parameters was performed where necessary.

Additional blood samples (nominally 0.5 mL) were taken into tubes containing tri-sodium 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 and prior to dosing at the following occasion:
Occasion Animals
Week 13 All 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 Cobas 6000 Analyzer in respect of:

• Alkaline phosphatase (ALP)
• Alanine aminotransferase (ALT)
• Aspartate aminotransferase (AST)
• Total bilirubin (Bili)
• Urea
• Creatinine (Creat)
• Glucose (Gluc)
• Cholesterol – total (Chol)
• Cholesterol – high density phospholipid (HDL)
• Cholesterol – low density phospholipid (LDL)
• Triglycerides (Trig)
• Sodium (Na)
• Potassium (K)
• Chloride (Cl)
• Calcium (Ca)
• Inorganic phosphorus (Phos)
• Total protein (Total Prot)
• Albumin (Alb)

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

Thyroid Hormone Analysis
Blood samples were collected at necropsy as follows:
Occasion Animals
At termination All animals

Parameters Triiodothyronine (T3)
Thyroxine (T4)
Thyroid stimulating hormone (TSH)
Sequence of blood sampling on each occasion To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was randomized to allow satisfactory inter-group comparisons.

Conditions No overnight deprivation of food.
Blood sample site Sublingual vein.
Anesthetic Isoflurane. Animals were not allowed to recover from the anesthesia.
Anticoagulant None.
Blood tubes Grenier Minicollect tubes with clotting activator.
Blood volume 1.0 mL.
Treatment of samples Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000g for ten minutes at 4°C.

All available serum was transferred to an appropriately labelled polypropylene “cryo” tubes using a plastic disposable pipette, then mixed by gentle ten-fold inversion. Following mixing, each serum sample was divided into two aliquots (see below).

Number of aliquots Aliquot 1 (T3 and T4): 0.2 mL of serum
Aliquot 2 (TSH): all remaining serum
Storage conditions Deep frozen (-60°C to -80C) pending analysis.
T3/T4 The method of analysis and results are presented in Attachment 13.3.
TSH The method analysis and the results are presented in Attachment 13.4.

Additional Hormone Sampling
The sampling schedule was as follows:
Occasion Animals
At termination All animals

Sequence of blood sampling on each occasion To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was randomized to allow satisfactory inter-group comparisons.

Conditions No overnight deprivation of food.
Blood sample site Sublingual vein.
Anesthetic Isoflurane. Animals were not allowed to recover from the anesthesia.
Anticoagulant None.
Blood volume 1.0 mL.
Blood tubes Greiner Minicollect tubes with clotting activator.
Processing Samples were kept at ambient temperature (15 to 25C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000g for ten minutes at 4°C.
All available serum were transferred to an appropriately labelled polypropylene “cryo” tubes using a plastic disposable pipette.
Serum tubes Standard Covance tubes. Serum was transferred to appropriately labelled polypropylene “cryo” tubes using plastic disposable pipettes.

Storage conditions Serum samples were stored at -60 to -80C, pending any future requirement for analysis.

Analysis No examinations were performed. All serum samples were discarded at the time of report finalization.

Vaginal Smears
Wet smears were taken as follows:

Wet smears Using pipette lavage for four days before scheduled termination.
Smears were assessed to establish the stage of estrus (metestrus, diestrus, proestrus and estrus). These observations assisted in the histological evaluation of estrogen sensitive tissues.

Sacrifice and pathology:

Necropsy
All main study 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.

The retained tissues were checked before disposal of the carcass.
Schedule Animals were killed following 13 weeks of treatment.
Sequence To allow satisfactory inter-group comparison.

The organs weighed, are detailed below:
Abnormalities
Adrenals
Aorta
Brain (cerebellum, cerebrum, midbrain)
Bone marrow smear
Cecum
Coagulating glands
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femur (femorotibial joint)
Head
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes - mesenteric
- left axillary
Ovaries
Pancreas
Peyer’s patches
Pituitary
Prostate
Rectum
Salivary glands - submandibular
- sublingual
- parotid
Sciatic nerves
Seminal vesicles
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum (and bone marrow)
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix
Vagina

Bone Marrow
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment period.

Fixation Smears were air dried and subsequently fixed in methanol.

Analysis No examinations were performed, however, the smears were retained for possible future examination.

Retention The smears were transferred to archives and will be retained for the same period as the study raw data.

Organ Weights
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for main study animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:

Testes In modified Davidson’s fluid.
Eyes In Davidson’s fluid.
Bone marrow smears: See Section above.

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 Main study animals of Groups 1 and 4 killed at a scheduled interval.
Abnormalities, kidneys and mesenteric lymph nodes only All main study animals of Groups 2 and 3 killed at a scheduled interval.

Routine staining
Special staining Sections were stained with hematoxylin and eosin.
Perl’s Prussian Blue: all animals from all groups.
Schmorl’s Ferricyanide: all animals from all groups.
Chromotrope Aniline Blue: males only from all groups.

Light Microscopy
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Scheduled kill Main study All animals of Groups 1 and 4. All specified in Section 3.7.

All animals of Groups 2 and 3. Abnormalities only.
The following tissues, which were considered to exhibit a reaction to treatment at the high dose, were examined for all main study animals:
Kidneys
Mesenteric lymph nodes
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.

Statistics:

PLEASE REFER TO "ANY OTHER INFORMATION ON MATERIALS AND METHODS"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Dark coloration of the skin (including ears, limbs, tail and scrotum) was observed from Week 2 of treatment, dark eyes from Week 8 of treatment and dark coloration of the teeth from Week 12 of treatment for males and females receiving 1000 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

No animals died during the treatment period and no signs were observed in association with dose administration.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gain was unaffected by treatment.

PLEASE REFER TO THE ATTACHED TABLE: "BODY WEIGHT - GROUP MEAN VALUES"

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment.

PLEASE REFER TO THE ATTACHED TABLE:"FOOD CONSUMPTION - GROUP MEAN VALUES"

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

The visual assessment of water intake did not reveal any treatment-related effect and, consequently, quantitative measurements were not performed.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related ophthalmic findings.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The hematological investigations in Week 13 of treatment revealed, when compared with the controls, a slight but statistically significant and dose-related reduction in hematocrit for males receiving 300 or 1000 mg/kg bw/day (0.97x and 0.96x control, respectively). These animals also showed slightly low erythrocyte count (0.97x control) though the difference from controls did not attain statistical significance. All individual values were above the lower limit of the background ranges (90-percentile range: 0.424 - 0.535 L/L for hematocrit and 7.68 - 9.08 x1012/L for erythrocyte count). The only change in females was a slightly low reticulocyte count at 1000 mg/kg bw/day (0.86x control); all individual values were within the background range (90-percentile range: 0.109 - 0.207 x1012/L).

Monocyte and large unstained cell counts were slightly low for females receiving 1000 mg/kg bw/day when compared with controls. Total leucocyte counts were slightly low for these animals but the difference did not attain statistical significance and all but one individual value was within the background range (90-percentile range: 2.46 - 7.11 x109/L). Males were unaffected.

Prothrombin time was reduced for males and females receiving 1000 mg/kg bw/day (0.91x and 0.92x control, respectively); all except one female had values within the background range (90-percentile range: 16.9 to 25.3 sec for males and 17.2 – 23.5 sec for females). Conversely, activated partial thromboplastin time was increased, compared to controls, for females receiving 300 or 1000 mg/kg bw/day, without dose-relationship (1.30x and 1.21x controls, respectively). None of the individual values exceeded the upper limit of the background range (24.6 sec).

All other inter-group differences from controls, including those that attained statistical significance, were minor, seen in one sex only or were without dose-relationship and were, therefore considered to represent normal biological variation.

PLEASE REFER TO THE ATTACHED TABLE: "HEAMTOLOGY - GROUP MEAN VALUES DURING WEEK 13 OF TREATMENT"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood chemistry investigations in Week 13 of treatment revealed low alanine amino transferase activities in males and females at 1000 mg/kg bw/day (0.71x and 0.57x control, respectively) with approximately half of individual values being below the background range (90-percentile range: 24 – 71 U/L for males and 23 - 68 U/L for females). Aspartate amino-transferase activities in males at 1000 mg/kg bw/day was higher than controls (1.43x control) though all but one individual value was within the background range (90-percentile range for males: 54 – 120 U/L).

Urea and creatinine concentrations were slightly high (1.13x and 1.16x, respectively) and glucose concentration was low (0.83x control) in males receiving 1000 mg/kg bw/day. Females were unaffected. All individual values for urea and creatinine were within the background range (4.50 – 8.86 mmol/L for urea and 23 – 39 µmol/L for creatinine; 3/10 glucose values were below the background range (6.17 – 11.85 mmol/L).

Cholesterol concentrations (total, high density lipoproteins and low density lipoproteins) were low (0.89x, 0.86x and 0.73x control, respectively) for females receiving 1000 mg/kg bw/day, although only the difference for high density lipids attained statistical significance and with the exception of one animal, all individual values were within the background range (total cholesterol: 1.57 – 2.76 mmol/L; HDL: 1.44 – 2.23 mmol/L; LDL: 0.07 – 0.26 mmol/L). Triglyceride concentrations were high compared to controls for females receiving 300 or 1000 mg/kg bw/day (1.55 and 1.52x control, respectively), with approximately half of individual values above the background range (90-percentile range: 0.28 – 1.00 mmol/L). Males were unaffected.

Sodium concentration was slightly low (0.99x) and potassium concentration was slightly high (1.08x) for males receiving 1000 mg/kg bw/day. Calcium concentrations were slightly low for males at 1000 mg/kg bw/day and for all treated female groups, but without dose-relationship (ranging between 0.96x and 0.98x control). The majority of individual values were within the background range (90-percentile ranges for males: 140 – 146 mmol/L for sodium; 3.80 – 5-19 mmol/L for potassium; 2.57 – 2.85 mmol/L for calcium; 90-precentile range for females: 2.64 – 2.92 mmol/L for calcium).

Total protein concentrations were slightly low for males and females at 1000 mg/kg bw/day (0.96x control for both sexes). With the exception of one animal of each sex receiving 1000 mg/kg bw/day, all individual values were within the background range (90-percentile range: 64-72 g/L for males and 67 – 79 g/L for females).

All other inter-group differences from controls, including those that attained statistical significance, were minor, seen in one sex only or were without dose-relationship and were, therefore considered to represent normal biological variation.

PLEASE REFER TO THE ATTACHED TABLE: "BLOOD CHEMISTRY - GROUP MEAN VALUES DURING WEEK 13 OF TREATMENT"

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis (T3, T4 and TSH)
There was no effect of treatment on thyroid hormones (T3, T4 or TSH).
The mean T3 concentration for females receiving 300 mg/kg bw/day and mean TSH concentration for males receiving 300 mg/kg bw/day were higher than controls, however, considering the lack of dose-relationship (there was no similar effect at 1000 mg/kg bw/day), these differences were considered to reflect normal variation.
Text-table 1: Summary of group mean thyroid hormones
Group 1 2 3 4
Dose (mg/kg bw/day) 0 100 300 1000
Males
T3 (pg/mL) 782 827 845 782
T4 (pg/mL) 42500 40600 42200 36900
TSH (pg/mL) 679 775 1920 738
Females
T3 (pg/mL) 944 1090 1160** 964
T4 (pg/mL) 33800 36900 40400 34200
TSH (pg/mL) 639 903 617 601
Significant when compared with Group 1: ** - p<0.01.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory Reactivity and Grip Strength
Sensory reactivity and grip strength were considered to be unaffected by treatment.

There was a statistically significant reduction in fore limb and hind limb grip strength values for females receiving 1000 mg/kg bw/day, however, the mean value for hind limb grip strength (0.40 kg) was well within the background data range (0.30 to 0.52 kg). The group mean value for forelimb grip strength (0.77 kg) was slightly below the background range (0.80 to 1.14 kg, 33 studies), but this was largely influenced by one animal (No. 72) which was reluctant to grip the fore limb bar. Males were unaffected and this finding, in isolation, was attributed to normal biological variation.

Motor Activity
There was no effect of treatment on motor activity.

There were a few isolated inter-group differences which attained statistical significance, however, in view of the isolated nature of these occurrences, lack of dose-response or consistency and since the total high and low beam breaks during the entire testing period were not statistically different, these findings were attributed to normal biological variation.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Analysis of organ weights for animals killed after 13 weeks of treatment revealed, when compared with the controls, a dose-related increase in absolute and body weight-adjusted kidney weights in males of all treated groups and in females at 300 or 1000 mg/kg bw/day (1.08 to 1.28x control).
Absolute and body weight-adjusted liver weights were slightly, but statistically significantly, increased for males receiving 1000 mg/kg bw/day (1.08x control). Body weight-adjusted liver weights for females given 1000 mg/kg bw/day were also marginally higher than those of the controls, but the difference did not attain statistical significance.

PLEASE SEE TABLE BELOW IN "ANY OTHER INFORMATION ON RESULTS": "SUMMARY OF ORGAN WEIGHT CHANGES RELATED TO TREATMENT"

All other inter-group differences from controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in adjusted to body weight but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small. This included the slightly high body weight-adjusted spleen and thymus weights for females which received 1000 mg/kg bw/day where there was no effect on absolute values and no associated histopathology correlate. The slightly high absolute and body weight-adjusted uterus and cervix weights (1.49x control) observed for females which received 1000 mg/kg bw/day, reflected the stage of estrus at the time of necropsy and no effect of treatment was inferred.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination of animals killed after 13 weeks of treatment revealed abnormal (blue) coloration of many tissues and abnormal (blue) contents in the stomach, intestinal tract and urinary bladder for animals receiving 300 or 1000 mg/kg bw/day. For animals receiving 100 mg/kg bw/day, blue coloration was observed for the kidneys, jejunum and mesenteric lymph nodes.

PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON REULTS": "SUMMARY OF TREATMENT-RELATED MACROPSCOPIC FINDINGS"

The nature and incidence of all other findings were consistent with the commonly seen background of macroscopic changes in Han Wistar rats at these laboratories.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were seen in the kidneys and mesenteric lymph nodes.
Tubular degeneration and an increased incidence of tubular basophilia were seen in the kidneys in both sexes administered 1000 mg/kg bw/day, and accumulation of pigments, staining positively for both Perls’ Prussian Blue and Schmorl’s Ferricyanide, were seen in both sexes administered the test item at all dose levels with a dose-related increased incidence and severity.
Increased incidence of pigmented macrophages in the mesenteric lymph nodes was seen in both sexes administered 300 or 1000 mg/kg bw/day. The presence of the pigments is likely to be related to the nature of the test item.
There were no microscopic findings seen in other tissues reported to have displayed abnormal coloration or abnormal content or any associated microscopic findings in the liver to account for the higher liver weight seen in both sexes given 1000 mg/kg bw/day.

PLEASE SEE THE TABLE IN "ANY OTHER INFORMATION ON RESULTS": "SUMMARY OF TREATMENT RELATED MICROSCOPIC FINDINGS"

All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for this age of Han Wistar rats; therefore, they were considered not test item related.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Stage of Estrous Cycle at Termination
There was no effect of treatment on estrus cycles.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

 

Summary of organ weight changes related to treatment

Sex	Bayscript Blaukomponente TEA
	Males				Females
Dose Level (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
Kidneys
Absolute Weight (g)	2.056	108	108	128	1.398	98	112	123
Body Weight adjusted (%)	2.051	108*	111**	126**	1.395	97	109*	128**
Liver
Absolute Weight (g)	12.007	100	95	110	7.575	103	99	100
Body Weight adjusted (%)	11.971	100	98	108**	7.557	101	95	106
* = p≤0.05; ** = p≤0.01 statistically significant difference (bodyweight-adjusted values) compared with respective control mean value. Note: Values for absolute weight and bodyweight-adjusted organ weights (relative to body) for dosed groups expressed as percentage of the control mean value.

 

: Summary of treatment-related macroscopic findings

Sex	Bayscript Blaukomponente TEA
	Males				Females
Dose Level (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
Number Examined	10	10	10	10	10	10	10	10
General Comments
Many organs abnormal color	0	0	5	10	0	0	2	10
Brain
Dark area(s)	0	0	0	10	0	0	0	7
Cecum
Abnormal color	0	0	1	0	0	0	1	0
Abnormal contents	0	0	0	7	0	0	2	0
Colon
Abnormal color	0	0	0	0	0	0	1	0
Abnormal contents	0	0	0	7	0	0	1	0
Duodenum
Abnormal color	0	0	1	0	0	0	1	0
Abnormal contents	0	0	0	4	0	0	0	1
Ileum
Abnormal color	0	0	2	0	0	0	4	0
Abnormal contents	0	0	1	5	0	0	1	1
Jejunum
Abnormal color	0	3	5	0	0	0	5	0
Abnormal contents	0	0	0	5	0	0	2	1
Kidneys
Abnormal color	0	10	5	0	0	10	8	0
Lymph node Mesenteric
Abnormal color	0	4	5	0	0	4	8	0

 

Text Table 3 (cont): Summary of treatment-related macroscopic findings

Sex	Bayscript Blaukomponente TEA
	Males				Females
Dose Level (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
Number Examined	10	10	10	10	10	10	10	10
Rectum
Abnormal color	0	0	1	0	0	0	0	0
Abnormal contents	0	0	0	6	0	0	0	0
Stomach
Abnormal color	0	0	2	0	0	0	4	0
Abnormal contents	0	0	0	6	0	0	4	4
Testes
Abnormal color	0	0	4	0	-	-	-	-
Urinary Bladder
Abnormal color	0	0	1	0	0	0	1	0
Abnormal contents	0	0	0	7	0	0	0	1

 

Summary of treatment-related microscopic findings

Sex	Bayscript Blaukomponente TEA
	Males				Females
Dose Level (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
Kidneys
Number Examined	10	10	10	10	10	10	10	10
Accumulation, pigments
Minimal	0	2	1	0	0	6	0	0
Slight	0	0	6	0	0	2	9	0
Moderate	0	0	3	10	0	0	1	10
Perls’ Prussian Blue staining, increased
Minimal	0	2	1	0	3	7	0	0
Slight	0	0	8	8	0	1	10	1
Moderate	0	0	1	2	0	0	0	9
Schmorl’s Ferricyanide staining, increased
Minimal	0	1	0	0	7	0	0	0
Slight	0	9	4	2	1	10	6	0
Moderate	0	0	6	8	0	0	4	3
Marked	0	0	0	0	0	0	0	7
Basophilia, tubular
Minimal	2	2	3	0	0	0	5	0
Moderate	0	0	0	8	0	0	0	10
Marked	0	0	0	2	0	0	0	0
Degeneration, tubular
Slight	0	0	0	9	0	0	0	10
Moderate	0	0	0	1	0	0	0	0
Mesenteric lymph node
Number Examined	10	10	10	10	10	10	10	10
Pigmented macrophages, increased
Minimal	0	0	0	2	0	0	3	1
Slight	0	0	8	7	0	0	1	9

Conclusions:

It is concluded that administration of Bayscript Blaukomponente TEA to Han Wistar rats for 13 weeks at doses of 100, 300 and 1000 mg/kg bw/day was associated with changes in the kidney which were considered adverse at 1000 mg/kg bw/day. There were adaptive changes in the liver that were not associated with any histopathological change and considered non-adverse and an increased incidence of pigmented macrophages in the mesenteric lymph nodes at 300 or 1000 mg/kg bw/day related to the nature of the test item. The no-observed-adverse-effect level (NOAEL) in this study was considered to be 300 mg/kg bw/day, based on slight to moderate degeneration of the tubular epithelium and moderate to marked tubular basophilia in the kidneys of both sexes given 1000 mg/kg bw/day.

Executive summary:

SUMMARY

The purpose of this study was to assess the systemic toxic potential of Bayscript Blaukomponente TEA when administered orally by gavage to Han Wistar rats for 13 weeks. This study was requested by the European Chemicals Agency (ECHA).

Three groups, each comprising ten male and ten female RccHan^™;WIST rats, received Bayscript Blaukomponente TEA at doses of 100, 300 or 1000 mg/kg bw/day, at a volume dose of 10 ml/kg bw/day. A similarly constituted control group received the vehicle, purified water, at the same volume dose over the same treatment period.

During the study, thyroid hormone analysis, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, visual water consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

Results

No animals died during the treatment period and no adverse clinical signs were observed.  Dark coloration of the skin, eyes and teeth was observed for animals receiving 1000 mg/kg bw/day, associated with the nature of the test item (a blue dye).  Sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual assessment) and estrus cycles were considered to be unaffected by treatment and there were no treatment-related ophthalmic findings or any effect on thyroid hormones.

The kidney was identified as the principle target organ.  Kidney weights were increased at all doses in males, and in females at 300 or 1000 mg/kg bw/day.  Slightly increased plasma urea and creatinine concentrations seen in males receiving 1000 mg/kg bw/day and alterations in plasma electrolytes (low sodium and high potassium in males at 1000 mg/kg bw/day and low calcium in males at 1000 mg/kg bw/day and all doses in females) indicated an effect on renal function.  At microscopic examination, tubular degeneration at slight to moderate levels and an increased incidence of tubular basophilia at moderate to marked levels were seen in the kidneys in both sexes administered 1000 mg/kg bw/day, and at these severities were considered adverse.  Accumulation of pigments, staining positively for both Perls’ Prussian Blue (suggestive of the presence of ferric iron, likely to be in the form of hemosiderin) and Schmorl’s Ferricyanide (suggestive of the presence of lipofuscin, a “wear and tear” pigment) were seen at all dose levels, in both sexes, with a dose-related increased incidence and severity.

Increased incidence of pigmented macrophages in the mesenteric lymph nodes was seen at histopathology examination in both sexes administered 300 or 1000 mg/kg bw/day. The presence of the pigments is likely to be related to the nature of the test item.

Small increases in liver weight at 1000 mg/kg bw/day in both sexes and a number of changes in the blood plasma were observed suggestive of a slight alteration of liver function.  These included slightly increased aspartate amino transferase activity in males at 1000 mg/kg bw/day, a reduction in alanine amino-transferase activity in males and females at 1000 mg/kg bw/day, a reduction of plasma glucose concentration in males at 1000 mg/kg bw/day, reduced cholesterol in males at 1000 mg/kg bw/day, increased triglyceride concentration in females at 300 or 1000 mg/kg bw/day and low total protein for males and females at 1000 mg/kg bw/day.  There were, however, no associated pathology findings and the majority of individual values were within the background range and consequently, these findings were considered non adverse and may represent responses to the metabolism of the test item after administration of high doses.

The reduction in prothrombin time in both sexes at 1000 mg/kg bw/day and increased activated partial thromboplastin times seen in females at 100 or 300 mg/kg bw/day were minor with the majority of individual values within the background range.  They were not associated with any other findings indicative of an adverse effect on clotting function, consequently, may be a further indicator of an alteration of hepatic metabolism since many clotting factors are biosynthesized in the liver.

 

Small reductions in erythrocyte indices (low hematocrit and erythrocyte count in males receiving 300 or 1000 mg/kg bw/day and slightly low reticulocyte count in females at 1000 mg/kg bw/day) were apparent.  There were also minor alterations of some leucocyte populations (reduced monocyte, large unstained cell counts and total leucocyte counts in females at 1000 mg/kg bw/day).  The differences from controls were minor with the majority of individual values within the background range and there were no histopathological findings that would account for the variations and as such, they were considered non‑adverse.

 

At macroscopic examination, there was abnormal (blue) coloration of many tissues and abnormal (blue) contents in the stomach, intestinal tract and urinary bladder for animals receiving 300 or 1000 mg/kg bw/day.  For animals receiving 100 mg/kg bw/day, blue coloration was observed for the kidneys, jejunum and mesenteric lymph nodes.  With the exception of the kidneys and mesenteric lymph nodes, there were no microscopic findings seen in the tissues which displayed abnormal coloration or abnormal content.

The changes seen at 100 and 300 mg/kg bw/day in this study were minimal and not considered adverse at the severity seen.

 

Conclusion

It is concluded that administration of Bayscript Blaukomponente TEA to Han Wistar rats for 13 weeks at doses of 100, 300 and 1000 mg/kg bw/day was associated with changes in the kidney which were considered adverse at 1000 mg/kg bw/day.  There were adaptive changes in the liver that were not associated with any histopathological change and considered non-adverse and an increased incidence of pigmented macrophages in the mesenteric lymph nodes at 300 or 1000 mg/kg bw/day related to the nature of the test item.  The no-observed-adverse-effect level (NOAEL) in this study was considered to be 300 mg/kg bw/day, based on slight to moderate degeneration of the tubular epithelium and moderate to marked tubular basophilia in the kidneys of both sexes given 1000 mg/kg bw/day.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental start date (Pre-dose trial): 4 April 2017 Experimental completion date (pathology): 30 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

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

Specific details on test material used for the study:

Appearance: Dark blue to black powder
Storage conditions: At ambient temperature (15 to 25ºC) without protection form light.
As the test item is hygroscopic, containers were kept tightly sealed and held under desiccated conditions.
Expiry date: 17 June 2018
Stability under test conditions:The homogeneity and stability of the formulations used were previously analytically confirmed (Envigo No. CY71SC).
The dose formulations were adjusted for purity.

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 RccHan™;WIST (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 (92 in total). Including four spare males and eight spare females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization:
Males: six days prior to the commencement of treatment.
Females: 20 days prior to the commencement of treatment.
Age of the animals at the start of treatment: Males: 84 to 90 days old.
Females: 100 to 106 days old.
Weight range of the animals at the start of the study Males 295 to 370 g
Females 202 to 241 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 that variations in the body weight of animals did not exceed ± 20% of the mean for each sex.
Identification of animals: Each adult animal was assigned a number and identified uniquely within the study by an implanted microchip 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 color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

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. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
The following replacements were made: two females due to irregular estrous cycles, one female due to an abnormality and one female due to body weight range extremes.

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%.
There were no deviations from these ranges.
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 polypropylene 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) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.
Shelters and chew blocks were returned to F0 females after removal of the offspring.

Diet Supply
Diet: SDS VRF1 Certified pelleted diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability - Non-restricted (removed overnight before blood sampling for hematology and blood chemistry investigations). See Section 4.

Water Supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate 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.

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:
A correction factor of 1.073 was used when calculating quantities of test item used during dose preparation.

Method of preparation: The required amount of test material was weighed into a suitable beaker. Approximately 50% of the required volume of vehicle was added to the test item and it was magnetically stirred for a minimum of one hour to ensure accurate mixing. The formulation was transferred to a measuring cylinder which had been wetted with the vehicle. The remaining amount of vehicle was added and it was magnetically stirred for a minimum of 20 minutes. The suspension was transferred to the 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:
Ambient (15 to 25°C) for one day.
Refrigerated (2 to 8°C) for up to 15 days.

A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity
The homogeneity and stability of formulations in the concentration range of 0.01 to 50 mg/mL during ambient storage (15 to 25°C) were previously determined as part of Envigo Study No. CY71SC. In that study, the formulations were confirmed to be stable and homogeneous for up to 24 hours.
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 100 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.
Homogeneity and stability were demonstrated for up to 15 days when stored refrigerated (2 to 8°C) and for one day when stored at ambient temperature (15 to 25°C).

Achieved concentration
Samples of formulations prepared for administration in the first and last weeks of treatment were analyzed for achieved concentration of the test item.
The mean concentrations of Bayscript Blaukomponente TEA in test formulations analyzed for the study were within ±5% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

Males - Two weeks before pairing up to necropsy after a minimum of five weeks of treatment.
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.

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

330 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:

10 males and females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels for investigation were selected based on the results of the 14-day preliminary study (Envigo Study No. VF37VT). In that study, dose levels of 250, 500 or 1000 mg/kg/day were well tolerated, with no effect on body weight gain or food intake and clinical signs were limited to blue coloration of the skin/staining of the fur. At necropsy, there was no effect of treatment upon organ weights and macroscopic findings were limited to dark coloration of the kidneys and blue coloration of various tissues.
Therefore, a high dose level of 1000 mg/kg/day was selected for this study as the limit dose for the OECD422 guideline, and the low and intermediate dose levels of 100 and 330 mg/kg/day were selected to investigate any dose relationship, should effects be observed in the longer duration study.

Observations and examinations performed and frequency:

Serial Observations

Clinical 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 onwards - once each week
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 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 behavior 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, 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 performed to match the body weight recording:
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 daily consumption per animal (g/animal/day) was calculated for each 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.
For four days before scheduled termination (nominally Days 11-14 of lactation).
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 estrus smear was seen. If a female showed an estrus smear during this period, she was killed as soon as practically possible and subject to macroscopic examination.

Sacrifice and pathology:

Method of Kill
All adult animals: Carbon dioxide asphyxiation with subsequent exsanguination.
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.
The macroscopic examination included a detailed assessment and documentation of any color changes in the internal organs, adipose tissue or skin. Representative photographs were taken of observed color changes before retained tissues were placed in fixative.

Time of Necropsy
F0 males - After Week 5 investigations completed.
F0 females failing to mate - Day 25 after last day of pairing.
F0 females failing to produce a viable litter - Day 25 after mating.
F0 females - Day 14 of lactation.
F1 offspring - Selected offspring for thyroid hormone analysis - Day 4 of age.
Day 13 of age.

The organs weighed, tissue samples fixed and sections examined microscopically are detailed in tables in the section 'any other information on materials and methods' for F0 animals.

Females
The following were recorded:
Each uterine horn The number of implantation sites were counted.

Offspring
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
F1 offspring on Day 4 of age: Blood sampling was required.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.
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.
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 all F0 animals killed.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes - 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: The five lowest numbered surviving males and first five lactating females with a surviving litter in Groups 1 and 4.
Abnormalities only: All remaining 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.
Special staining - kidneys From Animal Nos. 1M 21-25, 1F 121, 125, 127,128,147 and 4M 1-5, 4F 103,105,106,107-110: 4-5 m sections were stained with Perl’s Prussian Blue and Schmorl’s Ferricyanide.

Light Microscopy
Tissues preserved for examination were examined as follows:
Premature deaths: F0 males and F0 females in Groups 1 and 4 only. - All tissues listed in tables in section 'any other information on materials and methods'
Terminal sacrifice - The five lowest numbered surviving F0 males and first five lactating F0 females with a surviving litter in Groups 1 and 4. - All tissues listed in tables in section 'any other information on materials and methods'
All remaining F0 animals - Tissue with abnormalities only.
Groups 1 and 4: Five lowest numbered F0 males and female numbers 1F 121, 125, 127, 128, 147 and 4F 103, 105, 106, 107-110.- Kidneys stained with Perl’s Prussian Blue and Schmorl’s Ferricyanide

Gross findings of ‘abnormal color, dark’ or ‘abnormal color, blue’ (with the exception of kidneys and liver) were not examined by light microscopy. A representative selection of tissues with these findings recorded were examined for animals from Group 4 without microscopic correlate.
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.

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 an ACL series 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 lactating females with a surviving litter, in each dose group.

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)

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 F0 adult males and females (no samples were obtained from animals which failed to litter or had a total litter loss).
Day 4 of age : F1 offspring, two females per litter (where possible, ensuring that the number of female offspring did not fall below three).
No pups were allocated to these procedures if the resultant live litter size would fall below eight pups.
- one for T4 (serum)#
- one for TSH (plasma)
# 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 was 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: F0 animals: Following overnight deprivation of food
F1offspring: No overnight deprivation of food
Anesthetic: F0 animals: Isoflurane
F1 offspring : None
Blood sample site: F0 adults: Sublingual vein
F1 offspring : Decapitation
Anticoagulant: Plasma samples: K2EDTA. Microtainers used for collection of samples did not contain separator gel
Serum samples: None (Greiner Minicollect tubes with clotting activators)
Blood volume: F0 animals: 2 x 0.5 mL
F1 offspring: maximum possible
Processing: Plasma samples: Samples were kept on wet ice prior to centrifugation and commenced within 30 minutes of sampling.
Serum samples: Samples were kept at ambient temperature for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions: At 2000g for ten minutes at 4°C.
Number of aliquots per sample: All available plasma/serum was transferred to appropriately labelled polypropylene “cryo” tubes using micropipettes.
Final storage conditions: Deep frozen (-60 to -90ºC).
Fate of samples: Dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences, Envigo.
Thyroid hormone analysis Performed by the Department of Bioanalysis, Envigo.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Blue staining of various parts of the body was observed in males and females receiving 1000 mg/kg/day, principally from the second week of treatment. At the routine weekly observation, blue coloration of the skin was recorded in females receiving 1000 mg/kg/day, occurring from after mating and persisting throughout lactation.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Overall group mean body weight gain (Day 1 to 29) was slightly low in males receiving 330 or 1000 mg/kg/day (91 and 80% of control, respectively) but statistical significance was not attained. For females before pairing, body weight gain was slightly low in those receiving 100, 330 or 1000 mg/kg/day (89, 79 and 61% of control, respectively) with the degree of change demonstrating a dose-relationship but there was no statistical significance. During gestation, the body weight gains of the treated groups were similar to the controls and during lactation females receiving 100, 330 or 1000 mg/kg/day gained slight more weight than the controls (+13, +9 and +12% of control, respectively).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Group mean food intake was slightly low in males receiving 1000 mg/kg/day (92% of control). There was no effect upon food consumption for females before pairing, during gestation and lactation.

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 hematological examination performed after five weeks of treatment for males and on Day 14 of lactation for females revealed, when compared with controls, slightly, but statistically significantly, low reticulocyte count and slightly high mean cell hemoglobin concentration in females receiving 1000 mg/kg/day. There was no effect upon any other erythrocyte parameter and there were no similar findings in the males.
All other inter-group differences from control, including those attaining statistical significance, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the decrease of mean cell volume in males receiving 330 mg/kg/day, where there was no dose-response as the high dose value was similar to control.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination of plasma performed after five weeks of treatment for males and on Day 14 of lactation for females revealed, when compared with controls, statistically significantly high plasma triglyceride (4.45 mmol/L) and cholesterol (3.34 mmol/L) concentrations and low total protein (54 g/L), albumin (30g/L) and globulin (23 g/L) concentrations in females receiving 1000 mg/kg/day. There was a trend towards low plasma creatinine and urea concentrations in females at all dose levels (creatinine: 34, 29 and 29 µmol/L and urea: 9.67, 9.36 and 9.94 µmol/L for females receiving 100, 330 and 1000 mg/kg/day, respectively), with statistical significance attained for the decrease in creatinine.
Plasma phosphorus was slightly, but statistically significantly high in males at all dose levels, however there was no clear dose-relationship. Calcium concentrations were marginally high in high dose males, but conversely, were slightly low females at this dose level.
All other inter-group differences from control, including those attaining statistical significance, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the low bile acid concentration that achieved statistical significance for males given 100 mg/kg/day, where there was no dose-relationship and the individual values were highly variable across the groups.

Urinalysis findings:

not examined

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):

The analysis of organ weights performed after five weeks of treatment for males and on Day 14 of lactation for females revealed, when compared with controls, slightly high absolute and body weight adjusted kidney weights in males and females given 1000 mg/kg/day, with statistical significance attained for the body weigh adjusted value in both sexes. Thymus weights were high, when compared with controls, in males given 1000 mg/kg/day, with statistical significance attained for the body weight adjusted value. Absolute and body weight adjusted liver weights were slightly high in females given 1000 mg/kg/day, but statistical significance was not attained.
All other differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination performed after five weeks of treatment revealed blue or dark coloration of most tissues from all treated groups. Discoloration was seen in the following tissues: the adrenal glands, cecum, colon, duodenum, epididymides, esophagus, eyes, ileum, jejunum, kidneys, liver, lungs, left axillary and mesenteric lymph nodes, ovaries, oviducts, prostate, rectum, stomach, seminal vesicles, testes, thymus, thyroids, thymus, trachea, urinary bladder, skin and subcutis, uterus, uterine cervix, vagina, esophagus and head.
All other macroscopic findings were considered to be unrelated to the administration of the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment Related Findings
Changes related to treatment with Bayscript Blaukomponente TEA were seen in the kidneys of animals treated with 330 mg/kg/day or 1000 mg/kg/day.

Kidneys
Dark brown to black pigment accumulation in renal tubules was seen in all males and females given 330 or 1000 mg/kg/day. The severity of pigment accumulation was dose dependent. In most males and females given 1000 mg/kg/day, pigment accumulation was associated with tubular degeneration and a greater increase in tubular basophilia in males.
In males and females given 1000 mg/kg/day there was an increase in positive staining in the kidneys which were stained with Perl’s Prussian Blue and Schmorl’s Ferricyanide.
Summary of treatment related findings for the kidneys is given in the table in section 'any other information on results'

Incidental Findings
All other microscopic findings were considered to be incidental and unrelated to the test item. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatment related cell or stage specific abnormalities were noted.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis
Plasma thyroxine (T4) concentrations were slightly low, when compared with controls, in adult males receiving 1000 mg/kg/day, but there was no dose-relationship.
There was considered to be no effect of treatment upon T4 levels in offspring on Day 13 of age.

Sensory Reactivity and Grip Strength
There were no effects on grip strength, approach response, pinna reflex, auditory startle reflex or tail pinch response.
Forelimb grip strength was slightly, but statistically significantly low, when compared with controls, in males receiving 1000 mg/kg/day, but there was no dose-response and females were unaffected. The group mean value for the affected animals was, however, below the background range (range 1.09 to 1.23 kg; six studies) but the group mean value for the males receiving 100 mg/kg/day was also below this range.

Motor Activity
Motor activity was considered to be unaffected by treatment with Bayscript Blaukomponente TEA.
The assessment of motor activity revealed, when compared with controls, a statistically significant increase in the total number of high (rearing) beam breaks in males receiving 1000 mg/kg/day, which was principally attributed an increase of beam breaks during the initial 30 minutes of the assessment, with the value at the 12-minute interval achieving statistical significance. The majority of values were, however, within the background range and several of the individual control values were below the background range (12 minute interval range: 44.0 to 65.6; total range: 173.6 to 383.2; six studies). The total number of low (cage-floor) beam breaks was slightly high in males receiving 1000 mg/kg/day, but statistical significance was not attained for the overall value, but the increase at the 12-minute interval was statistically significant. There was no dose-relationship for the changes in high or low beam breaks.
The total number of low (cage-floor) beam breaks slightly low, when compared with controls, in females at all dose levels at the 6-minute interval, with statistical significance attained at 330 and 1000 mg/kg/day. The total number of beam breaks was slightly low in all dose groups, however statistical significance was not attained, there was no dose-response and the majority of values were within the background range (6-minute interval range: 132.8 to 187.2; total range: 486.6 to 846.2; six studies).

Estrous Cycles, Pre-Coital Interval, Mating Performance, Fertility and Gestation Length
All females allocated to study showed normal 4/5 day estrous cycles during the acclimatization period.
Estrous cycles, pre-coital interval, gestation length and index, mating performance and fertility were considered unaffected by treatment with Bayscript Blaukomponente TEA. All females showed diestrus at termination.
One female receiving 1000 mg/kg/day (Animal No. 102) was noted to have an irregular estrous cycle during treatment; the animal was subsequently found to be not pregnant. One female receiving 330 mg/kg/day had a total litter loss.

Dose descriptor:

NOAEL

Effect level:

330 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Organ:

kidney

Thyroid Hormone Analysis

Group	Treatment	Dose (mg/kg/day)		Adult males (pg/mL)	Offspring at Day 13 of age (pg/mL)
					Male	Female
1	Vehicle (purified water)	0	Mean	56600	43500	46500
			SD	22800	4900	3890
			CV	40.3	11.3	8.4
			N	10	10	10
2	Bayscript Blaukomponente TEA	100	Mean	39700	45800	44200
			SD	6200	7310	7210
			CV	15.6	16.0	16.3
			N	10	10	10
3	Bayscript Blaukomponente TEA	330	Mean	46200	52900	47900
			SD	8430	11300	7030
			CV	18.2	21.4	14.7
			N	10	9	9
4	Bayscript Blaukomponente TEA	1000	Mean	30400	43400	48700
			SD	7070	4660	5340
			CV	23.3	10.7	11.0
			N	10	9	9

Summary of treatment related findings in the kidneys for animals killed after five weeks of treatment.

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	330	1000	0	100	330	1000
Accumulation, Pigment, Tubular
Minimal	0	0	10	6	0	0	8	2
Slight	0	0	0	4	0	0	1	5
Total	0	0	10	10	0	0	9	7
Basophilia, Tubular
Minimal	0	0	0	3	0	0	0	1
Total	0	0	0	3	0	0	0	1
Degeneration, Tubular
Minimal	0	0	0	2	0	0	0	5
Slight	0	0	0	3	0	0	0	1
Total	0	0	0	5	0	0	0	6
Number of tissues examined	5	10	10	10	5	10	9	7
Perl’s staining positive
Slight	0	0	0	2	0	0	0	0
Moderate	0	0	0	3	0	0	0	5
Total	0	0	0	5	0	0	0	5
Schmorl’s staining positive, increase
Moderate	0	0	0	5	0	0	0	0
Marked	0	0	0	0	0	0	0	5
Total	0	0	0	5	0	0	0	5
Number of tissues examined	5	0	0	5	5	0	0	5

 

Conclusions:

It was concluded that the oral administration of Bayscript Blaukomponete TEA to Han Wistar rats at dose levels of 100, 330 or 1000 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 14 of lactation in females was generally well-tolerated in the adult animals. The kidney was identified as the primary target organ with adverse effects reported in males and females given 1000 mg/kg/day (degeneration of the tubular epithelium in both sexes and tubular basophilia in males). These changes were accompanied by lipofuscin accumulation (confirmed by Schmorl’s Ferricyanide staining).
Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment and, in the context of this study, Bayscript Blaukomponente TEA showed no evidence of being an endocrine disruptor.
Due to the adverse findings histopathological findings at 1000 mg/kg/day, the NOAEL of Bayscript Blaukomponente TEA for systemic toxicity was considered to be 330 mg/kg/day. The NOAEL for reproductive/developmental toxicity was 1000 mg/kg/day.

Executive 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 endpoints potentially relevant for endocrine active compounds, with administration of Bayscript Blaukomponente TEA by oral gavage for at least five weeks.

Four groups of 10 male and 10 female rats received Bayscript Blaukomponente TEA at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration. 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.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, 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 onmale offspring on Day 13 of age.

Results - with respect to general toxicity

No animals died during the treatment period and no signs were observed in association with dose administration. There was no adverse effect of treatment upon the appearance or behavior of the animals and sensory reactivity, grip strength and motor activity were considered to be unaffected by treatment.

Body weight gain was slightly low in males receiving 330 or 1000 mg/kg/day and in females at receiving 100, 330 or 1000 mg/kg/day before pairing, with a corresponding decrease in food consumption of males receiving 1000 mg/kg/day. During gestation and lactation, the body weight gain of treated females was similar to or slightly above control.

Estrous cycles, pre-coital interval, gestation length and index, mating performance and fertility were considered unaffected by treatment.

The hematological examination revealed slightly low reticulocyte count and slightly high mean cell hemoglobin concentration in females receiving 1000 mg/kg/day. 

The blood chemistry investigations revealed high plasma triglyceride and cholesterol concentrations and low creatinine, urea, total protein, albumin and globulin concentration in females receiving 1000 mg/kg/day. Phosphorus and calcium concentrations were slightly high in males at all dose levels and calcium was slightly low in females receiving 1000 mg/kg/day. There was considered to be no effect of treatment upon circulating levels of thyroxine (T4) in males.

The evaluation of organ weights revealed high kidneys weights in both sexes given 1000 mg/kg/day, high thymus weights in high dose males and slightly high liver weights in high dose females. The macroscopic examination revealed blue or dark coloration of most tissues from all treated groups, which was considered to be due to the dark blue to black color of the test substance. Microscopically, there was dark brown to black pigment accumulation in the renal tubules of males and females given 330 or 1000 mg/kg/day which associated, at the high dose, with tubular degeneration in both sexes and also with tubular basophilia in males. Staining of the kidneys with Schmorl’s Ferricyanide confirmed the presence of lipofuscin in both sexes given 1000 mg/kg/day.

 

Conclusion

It was concluded that the oral administration of Bayscript Blaukomponete TEA to Han Wistar rats at dose levels of 100, 330 or 1000 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 14 of lactation in females was generally well-tolerated in the adult animals. The kidney was identified as the primary target organ with adverse effects reported in males and females given 1000 mg/kg/day (degeneration of the tubular epithelium in both sexes and tubular basophilia in males). These changes were accompanied by lipofuscin accumulation (confirmed bySchmorl’s Ferricyanide staining).

Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment and, in the context of this study, Bayscript Blaukomponente TEA showed no evidence of being an endocrine disruptor. 

Due to the adverse findings histopathological findings at 1000 mg/kg/day, the no-observed-adverse-effect level (NOAEL) of Bayscript Blaukomponente TEA for systemic toxicity was considered to be 330 mg/kg/day. The NOAEL for reproductive/developmental toxicity was 1000 mg/kg/day. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

High qualitiy database with GLP-compliant Guideline studies according to OECD TG 408 and OECD TG 422.

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The available studies with repeated oral exposure consistently show histopathological effects in the kidneys of rats at the limit dose of 1000 mg/kg bw/day only.

OECD TG 408:

The registered substance was administered orally for 13 weeks (OECD TG 408) to groups of 10 Wistar rats/sex in doses of 100, 300 or 1000 mg/kg bw/day formulated in water. 

No animals died during the treatment period and no adverse clinical signs were observed.  Dark coloration of the skin, eyes and teeth was observed for animals receiving 1000 mg/kg bw/day, associated with the nature of the test item (a blue dye).  Sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual assessment) and estrus cycles were considered to be unaffected by treatment and there were no treatment-related ophthalmic findings or any effect on thyroid hormones.

The kidney was identified as the principle target organ.  Kidney weights were increased at all doses in males, and in females at 300 or 1000 mg/kg bw/day.  Slightly increased plasma urea and creatinine concentrations seen in males receiving 1000 mg/kg bw/day and alterations in plasma electrolytes (low sodium and high potassium in males at 1000 mg/kg bw/day and low calcium in males at 1000 mg/kg bw/day and all doses in females) indicated an effect on renal function.  At microscopic examination, tubular degeneration at slight to moderate levels and an increased incidence of tubular basophilia at moderate to marked levels were seen in the kidneys in both sexes administered 1000 mg/kg bw/day. 

Small increases in liver weight at 1000 mg/kg bw/day in both sexes and a number of changes in the blood plasma were observed suggestive of a slight alteration of liver function. There were, however, no associated pathology findings and the majority of individual values were within the background range and consequently, these findings were considered non adverse and may represent responses to the metabolism of the test item after administration of high doses. An increased incidence of pigmented macrophages in the mesenteric lymph nodes at 300 or 1000 mg/kg bw/day is related to the coloured nature of the test item, seen in many tissued and organs.

The no-observed-adverse-effect level (NOAEL) in this study was considered to be 300 mg/kg bw/day, based on slight to moderate degeneration of the tubular epithelium and moderate to marked tubular basophilia in the kidneys of both sexes given 1000 mg/kg bw/day.

 

OECD TG 422:

In a study in rats following OECD TG 422 groups of each 10 male and female rats received the registered substance in the vehicle water at doses of 100, 330 or 1000 mg/kg bw by oral gavage administration. 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.

The oral administration of the substance was generally well-tolerated in this study in the adult animals. No animals died during the treatment period and no signs were observed in association with dose administration. There was no adverse effect of treatment upon the appearance or behavior of the animals and sensory reactivity, grip strength and motor activity were considered to be unaffected by treatment.

Body weight gain was slightly low in males receiving 330 or 1000 mg/kg bw and in females at receiving 100, 330 or 1000 mg/kg bw before pairing, with a corresponding decrease in food consumption of males receiving 1000 mg/kg bw. During gestation and lactation, the body weight gain of treated females was similar to or slightly above control.

The hematological examination revealed slightly low reticulocyte count and slightly high mean cell hemoglobin concentration in females receiving 1000 mg/kg bw. The blood chemistry investigations revealed high plasma triglyceride and cholesterol concentrations and low creatinine, urea, total protein, albumin and globulin concentration in females receiving 1000 mg/kg bw. Phosphorus and calcium concentrations were slightly high in males at all dose levels and calcium was slightly low in females receiving 1000 mg/kg bw. There was considered to be no effect of treatment upon circulating levels of thyroxine (T4) in males. The substance showed no evidence of being an endocrine disruptor.

The evaluation of organ weights revealed high kidneys weights in both sexes given 1000 mg/kg bw, high thymus weights in high dose males and slightly high liver weights in high dose females. The macroscopic examination revealed blue or dark coloration of most tissues from all treated groups, which was considered to be due to the dark blue to black color of the test substance. Microscopically, there was dark brown to black pigment accumulation in the renal tubules of males and females given 330 or 1000 mg/kg bw which associated, at the high dose, with tubular degeneration in both sexes and also with tubular basophilia in males. 

Overall, the kidney was identified as the primary target organ with adverse effects reported in males and females given 1000 mg/kg/day (degeneration of the tubular epithelium in both sexes and tubular basophilia in males). The NOAEL for systemic toxicity was thus considered to be 330 mg/kg bw.

Justification for classification or non-classification

No classification concluded for Repeated dose Toxicity according to Regulation (EC) No 1272/2008, Annex I.