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

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

[Reaction mass of 2,7-Naphthalenedisulfonic acid, 5-amino-3-[[4-[2-[4-[(7-amino-1-hydroxy-3-sulfo-2-naphthalenyl)azo]-2-sulfophenyl]ethenyl]-3-sulfophenyl]azo]-4-hydroxy-, compd. with 2,2',2''-nitrilotris[ethanol] (1:5) and 3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:6) and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:4)]

EC Number:

916-899-6

Molecular formula:

not available

IUPAC Name:

[Reaction mass of 2,7-Naphthalenedisulfonic acid, 5-amino-3-[[4-[2-[4-[(7-amino-1-hydroxy-3-sulfo-2-naphthalenyl)azo]-2-sulfophenyl]ethenyl]-3-sulfophenyl]azo]-4-hydroxy-, compd. with 2,2',2''-nitrilotris[ethanol] (1:5) and 3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:6) and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:4)]

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

dark blue to brown crystalline powder

Test animals

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.

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Vehicle only

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

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.

Examinations

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"

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

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

Any other information on results incl. tables

 

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

Applicant's summary and conclusion

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.