Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key 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
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Remarks:
No deviations occured that were considered to have affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
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
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.

Test animals

Species:
rat
Strain:
other: RccHan;WIST
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The 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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on 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.



Details on mating procedure:
Pairing commenced After a minimum of two weeks of treatment.
Male/female ratio 1:1 from within the same treatment groups.
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.

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.
Details on study schedule:
Age of the animals at the start of treatment:
Males 86 to 92 days old.
Females 100 to 106 days old.
Doses / concentrationsopen allclose all
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.

Examinations

Parental animals: Observations and examinations:
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, See Section 4), 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.

Parturition Observations and Gestation Length
Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

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).

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
Anesthetic: F0 animals: Isoflurane
Blood sample site: F0 adults: Sublingual vein
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
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.



Oestrous cyclicity (parental animals):
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.

Sperm parameters (parental animals):
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.
Litter observations:
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter: Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights: Days 1, 4, 7 and 13 of age.
Ano-genital distance: Day 1 - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring.


Thyroid Hormone Analysis
Blood samples were collected as follows:
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: F1offspring: No overnight deprivation of food
Anesthetic: F1 offspring : None
Blood sample site: 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: 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.
Postmortem examinations (parental animals):
Method of Kill
All adult animals: Carbon dioxide asphyxiation with subsequent exsanguination.

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.

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.


Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for 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.

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'
Scheduled kill - 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 ovaries a qualitative evaluation of one section from each ovary was made.





Postmortem examinations (offspring):
Method of Kill
Offspring - selected for thyroid hormone sampling on Day 4 and 13 of age: Decapitation.
Offspring - all other: Intraperitoneal injection of sodium pentobarbitone.
.
Time of Necropsy
F1 offspring - Selected offspring for thyroid hormone analysis - Day 4 of age.
Day 13 of age.

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.
Reproductive indices:
Mating Performance and Fertility
Group values were calculated for males and females separately for the following:
Percentage mating (%) = Number of animals mating / Animals paired x 100

Conception rate (%) = Number of animals achieving pregnancy / Animals mated x 100

Fertility index (%) = Number of animals achieving pregnancy / Animals pairing x 100


Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:

Gestation index (%) = Number of live litters born / Number pregnant x 100


Offspring viability indices:
Survival Indices
The following were calculated for each litter:

Post-implantation survival index (%) = Total number of offspring born / Total number of uterine implantation sites x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = Number of live offspring on Day 1 after littering / Total number of offspring born x 100

Viability index (%) = Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering x 100

Lactation index (%) = Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling) x 100

Group mean values were calculated from individual litter values.
Sex Ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after culling) and 13 of age.

Percentage males = Number of males in litter / Total number of offspring in litter x 100

Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.
Dermal irritation (if dermal study):
not examined
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
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males Killed After Five Weeks of Treatment and Females on Day 14 of Lactation
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.
A summary of treatment related findings is given 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
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).

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
All females allocated to study showed normal 4/5 day estrous cycles during the acclimatization period.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
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.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
330 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Systemic toxicity
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
other: Reproductive toxicity

Target system / organ toxicity (P0)

Critical effects observed:
not specified
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Organ:
kidney

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs considered to be related to parental treatment with Bayscript Blaukomponete TEA.
All offspring from the litter of 3F 134 were noted to have abnormally blue coloration of the skin on Day 1 of age, which persisted in isolated individuals until Day 3 of age but was not apparent thereafter.

Offspring ano-genital distances were considered unaffected by treatment. Nipples were recorded in a few isolated male offspring, including controls, but there was no relationship to treatment.
Statistical significance was attained for the marginally greater ano-gential distances of female offspring derived from parental animals receiving 330 or 1000 mg/kg/day, but the individual values were generally similar to controls and there was no effect in the male offspring.

Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Offspring survival to Day 13 of age was unaffected by parental treatment with Bayscript Blaukomponete TEA.
Animal No. 137 (330 mg/kg/day) had a total litter loss; only one pup was observed to be born and at the macroscopic examination, two implantations were visible in the uterus and the mammary tissue was noted to be inactive.
Animal No. 102 (1000 mg/kg/day) was not pregnant.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was considered to be no effect of parental treatment upon offspring group mean body weights or body weight gain.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic findings that were considered to be related to parental treatment.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis
There was considered to be no effect of treatment upon T4 levels in offspring on Day 13 of age.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
mortality
clinical biochemistry
gross pathology

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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 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.
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 forat 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

F0 responses

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.

F1 responses

The clinical condition, litter size, sex ratio, survival indices and body weight gain of offspring were unaffected by parental treatment.

There was no effect of parental treatment upon circulating levels of thyroxine (T4) in offspring on Day 13 of age.

The ano-gential distances of offspring were unaffected by parental treatment and there was no treatment-related occurrence of nipples in male offspring on Day 13 of age.

No macroscopic findings considered to be related to parental treatment were recorded.

 

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.