Tetrasodium 3,3'-[[6-[(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(2-methyl-4,1-phenylene)azo]]bisnaphthalene-1,5-disulphonate

Toxicity to reproduction

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 May 2017 Experimental completion date (pathology): 3 November 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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium 3,3'-[[6-[(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(2-methyl-4,1-phenylene)azo]]bisnaphthalene-1,5-disulphonate

EC Number:

256-851-5

EC Name:

Tetrasodium 3,3'-[[6-[(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(2-methyl-4,1-phenylene)azo]]bisnaphthalene-1,5-disulphonate

Cas Number:

50925-42-3

Molecular formula:

C39H30N10Na4O13S4

IUPAC Name:

tetrasodium 3,3'-({6-[(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl}bis[imino(2-methyl-4,1-phenylene)diazene-2,1-diyl])dinaphthalene-1,5-disulfonate

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

solid

Specific details on test material used for the study:

Test item: Bayscript Gelb BR.
Test item identity: Bayscript Yellow BR.
Chemical name: 1,5-Naphthalenedisulfonic acid, 3,3’-[[6-[(2-hydroxyethyl)amino]- 1,3,5-triazine-2,4-diyl]bis[imino (2-methyl-4,1-phenylene)-2,1-diazenediyl]] bis-, sodium salt (1:4)
Appearance: Yellow odourless liquid.
Storage conditions: Room temperature (15-25ºC).
Expiry date: 11 July 2018

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

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 Crl:CD(SD) 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: Charles River (UK) Ltd.
Number of animals ordered: 44 males and 48 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 approximately 71 days old.
Females approximately 85 days old.
Weight range of the animals at the start of treatment
Males 317 to 371 g
Females 239 to 281 g

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

Identification of animals: Each adult animal was assigned a number and identified uniquely within the study using a 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 occupant(s).

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

Animal Care and Husbandry
Environmental Control
Rodent facility: Limited barrier - 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 cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.

Cage distribution: The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

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

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

Plastic shelter: Provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced at the same time as the cages.

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

Availability: Non-restricted.

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

Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Volume dose -10 mL/kg body weight.
Individual dose volume - Calculated from the most recently recorded scheduled body weight.
Control (Group 1) - Vehicle at the same volume dose as treated groups.

PREPARATION OF DOSING SOLUTIONS:
A correction factor (including purity) of 1.165 was used when calculating quantities of test item to be used during dose preparation.
Method of preparation: The required amount of test item was weighed and approximately 50 to 60 % of the final volume of vehicle was added. It was magnetically stirred for a minimum of one hour to ensure accurate mixing. It was made up to the required volume with vehicle and mixed using a high shear homogenizer until homogenous. 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:
Refrigerated (2-8ºC).
Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

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
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. These investigations demonstrated that formulations in the 1 to 100 mg
/mL concentration range are stable following ambient storage (15-25°C) for one day and following refrigerated storage (2-8°C) for 15 days.
The homogeneity and stability of Bayscript Gelb BR in purified water formulations was assessed with respect to the level of concentration at nominal concentrations of 1 mg/mL and 100 mg/mL.
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 day and refrigeration for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 4% of the initial time zero value and the coefficient of variation was less than 2%. Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method.

Achieved concentration
Samples of each formulation prepared for administration in Week1 of treatment and on Day 10-12 of lactation were analyzed for achieved concentration of the test item.
The mean concentrations of Bayscript Gelb BR in test formulations analyzed during the study were within 5%, confirming the accuracy of formulation, with the exception of Week 1, Group 4 (+12.0 %), this result was confirmed by the analysis of contingency samples and reported as the mean of
four values (two original and two contingency). The difference from mean was within 4%, confirming precise analysis.

Duration of treatment / exposure:

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

Frequency of treatment:

Once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels selected for investigation in this OECD 422 combined repeated dose toxicity study and reproductive/developmental toxicity screening study (0, 100, 300 and 1000 mg/kg/day) were chosen in conjunction with the Sponsor and were based on the results of a 14-day preliminary study conducted at these laboratories (Envigo Study No. DG28JR).
In the preliminary study dose levels of 250, 500 and 1000 mg/kg/day were investigated. There were no test item-related premature deaths, no signs observed in relation to dose administration, no test item-related changes in clinical condition, no inter-group differences in body weight performance,
food consumption or weights of the kidneys, liver or spleen, and no test item-related macroscopic abnormalities at any dose level investigated.
The high dose level for the current OECD 422 study was set at 1000 mg/kg/day. The intermediate and low dose levels of 300 and 100 mg/kg/day were chosen to achieve a dose response and/or aid in the determination of a No-Observed-Adverse-Effect-Level (NOAEL).

Examinations

Parental animals: Observations and examinations:

Serial Observations

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

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


Detailed physical examination and arena observations
Before treatment commenced and during each week of treatment and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons, therefore observations were made on these occasions without “blinding”.
After removal from the home cage, animals were assessed for physical condition and 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 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 5 of treatment for males and at Day 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and the first five lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing

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

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

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals:
Weekly before pairing, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Day 15-22), but recommenced for males in Week 4.
For females after mating food consumption was recorded as follows:
Days 0-6, 7-13 and 14-19 after mating.
Days 1-3, 4-6 and 7-12 of lactation.
From these records the mean 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.

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.

For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

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:

Records Made During Littering Phase
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 maternal 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.

Postmortem examinations (parental animals):

Method of Kill
All adult animals: Carbon dioxide asphyxiation with subsequent exsanguination.
Sequence: To allow satisfactory inter-group comparison.

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

Time of Necropsy
F0 males - After at least five weeks of treatment.
F0 females failing to produce a viable litter - Day 25 after mating.
F0 females - Day 14 of lactation (following terminal blood sampling).

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 Number of implantation sites was counted and confirmed if none were visible at visual inspection.

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

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

Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: The five lowest numbered surviving F0 males and first five lactating F0 females with a surviving litter in Groups 1 and 4 at scheduled termination.
Abnormalities only: All F0 animals.
Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Light Microscopy
Tissues preserved for examination were examined as follows:
Scheduled kill - 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 F0 animals - Abnormalities.

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.

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 a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.


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

At termination: The five lowest numbered surviving males and the first five 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)
Globulin (Glob)

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

Thyroid Hormone Analysis
Blood samples were collected as follows:
At termination: All adults.

Sequence of blood sampling on each occasion: In order to minimize any potential confounding effect of the time of day of blood sampling, the order of blood sampling was controlled to allow satisfactory inter-group comparisons.
Conditions: No overnight deprivation of food.
Anesthetic: F0 animals: Isoflurane

Blood sample site: F0 adults: Sublingual vein

Anticoagulant:
Thyroid stimulating hormone (TSH): K2 EDTA with no separator gel.
Thyroxine (T4): None
Tubes:
Thyroid stimulating hormone (TSH): Standard Envigo
Thyroxine (T4): Greiner Minicollect - with clot activator.
Blood volume (x 2): Adults: 0.5 mL

Processing:
Thyroid stimulating hormone (TSH): Samples were kept on wet ice prior to centrifugation. Centrifugation commenced within 30 minutes of sampling.
Thyroxine (T4): Samples were kept at ambient temperature (15 to 25ºC) for a minimum of 30 minutes prior to centrifugation.

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

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.
Sequence: To allow satisfactory inter-group comparison.

Time of Necropsy
F1 offspring - Selected offspring for thyroid hormone analysis - Day 4 of age.
- Day 13 of age.


Offspring
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content and particular attention paid to external genitalia was performed. Abnormal tissues were retained.
F1 offspring on Day 4 of age: Blood sampling was required.
Externally normal offspring discarded without examination. Externally abnormal offspring identified on dispatch to necropsy, an external macroscopic examination was performed and offspring retained pending possible future examination.
F1 offspring on Day 13 of age: Blood sampling required.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Animals observed with external abnormalities were retained pending possible future examination. Thyroid glands were preserved from one male and one female in each litter.


Thyroid Hormone Analysis
Blood samples were collected as follows:
Day 4 of age: FF1 offspring, two females per litter (where possible).
No offspring were allocated to these procedures if the resultant live litter size would fall below ten offspring or if it would leave less than three females.
- 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 given to serum sample

Sequence of blood sampling on each occasion: In order to minimize any potential confounding effect of the time of day of blood sampling, the order of blood sampling was controlled to allow satisfactory inter-group comparisons.
Conditions: No overnight deprivation of food.
Anesthetic: F1 offspring : None
Blood sample site: F1 offspring : Decapitation
Anticoagulant:
Thyroid stimulating hormone (TSH): K2 EDTA with no separator gel.
Thyroxine (T4): None
Tubes:
Thyroid stimulating hormone (TSH): Standard Envigo
Thyroxine (T4): Greiner Minicollect - with clot activator.
Blood volume (x 2): F1 offspring: maximum possible

Processing:
Thyroid stimulating hormone (TSH): Samples were kept on wet ice prior to centrifugation. Centrifugation commenced within 30 minutes of sampling.
Thyroxine (T4): Samples were kept at ambient temperature (15 to 25ºC) for a minimum of 30 minutes prior to centrifugation.

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

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:

no effects observed

Description (incidence and severity):

Administration with Bayscript Gelb BR at doses of 100, 300 and 1000 mg/kg/day during the 5-week treatment period for males, and during the 2-week pre-pairing period, gestation and lactation periods for females was well tolerated. There were no test item-related signs observed following dose administration or signs at routine clinical examination that were considered to be associated with treatment,
and no premature deaths occurred.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight performance of animals receiving Bayscript Gelb BR at doses up to and including 1000 mg/kg/day was considered to be unaffected by treatment throughout the 5 week dosing period for males, and throughout the 2-week pre-pairing period, gestation period and lactation period for
females.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Mean food consumption for all groups of animals receiving Bayscript Gelb BR was similar to that of Controls and no effect of treatment was inferred.
During Days 0-7 and Days 14-20 of gestation, the mean food intake of females given 300 or 1000 mg/kg/day was marginally higher than Control, with differences attaining statistical significance. There was no dose response apparent and the magnitude of the differences from Control was minor, therefore they were considered to be fortuitous.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Assessment of haematological parameters at the end of the treatment period did not reveal any adverse effects of treatment with Bayscript Gelb BR.
All groups of treated males showed a slight but statistically significantly higher than Control mean cell haemoglobin concentration, however there was no dose relationship apparent. Among females given 1000 mg/kg/day mean cell haemoglobin was slightly higher than Control, with statistical significance attained; this difference was attributable to a single female (No. 66) with an atypically high mean cell haemoglobin value and no effect of treatment was inferred. All other haematological differences from controls observed were minor, lacked dose relationship or occurred in one sex only and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no biochemical changes in the plasma at scheduled termination which were attributable to treatment with Bayscript Gelb BR.
Triglyceride concentrations in males receiving 300 mg/kg/day were higher than Control and sodium concentrations in males given 300 or 1000 mg/kg/day were slightly lower than Control; these differences attained statistical significance, however in the absence of a dose relationship were considered fortuitous and unrelated to Bayscript Gelb BR administration. All other biochemical differences from controls were minor, lacked dose relationship or occurred in one sex only and were therefore attributed to normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The microscopic examination performed after 5 weeks of treatment for males and on Day 14 of lactation for females revealed no test item related lesions.
All findings were considered to be incidental and unrelated to treatment.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males. There was therefore no requirement to measure T4 in the samples obtained from the adult females and none of the TSH (thyroid stimulating hormone) samples required analysis.

Sensory Reactivity and Grip Strength
The sensory reactivity observations conducted during Week 5 of treatment for males and Days 7-9 of lactation for females revealed no findings which were considered treatment related.
Females in the 100 mg/kg/day group showed slightly increased grip strength compared to Controls, with statistical significance attained for hindlimb grip strength. In the absence of similar differences in the 300 or 1000 mg/kg/day groups, these differences were considered incidental and unrelated to treatment.

Motor Activity
Motor activity scores for males and females given Bayscript Gelb BR were considered to be unaffected by treatment.
Some minor differences from Control were evident in the high and low beam scores, in terms of the individual 6-minute recording periods, however there was no consistent pattern of change and therefore these minor differences were considered to be attributable to natural variation and were unrelated to treatment with Bayscript Gelb BR.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females allocated to the study showed normal 4/5 day estrous cycles during the acclimatisation period.
Estrous cyclicity, pre-coital interval, mating performance, fertility, gestation length and index were unaffected by treatment.
All females were not cycling before termination (Days 11 to 14 of lactation) and were in diestrous at termination.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

With the exception of Group 2F No. 59 all females were pregnant, successfully gave birth to live young and reared their offspring to Day 13 of age.
There was no effect of parental treatment with Bayscript Gelb BR on the mean numbers of implantation sites or litter size.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: Systemic toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

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:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed among the offspring which were considered to be related to parental treatment with Bayscript Gelb BR.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no effect of parental treatment with Bayscript Gelb BR on offspring survival to Day 13 of age.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean offspring body weights on Day 1 of age and subsequent body weight gain up to Day 13 of age was similar in all groups and no effect of parental treatment with Bayscript Gelb BR was inferred.

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 findings in the decedent offspring, or offspring at termination on Day 13 of age that were considered to be related to parental treatment.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

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

There was no effect of parental treatment with Bayscript Gelb BR on the sex ratio of offspring.

The mean ano-genital distance of male and female offspring was essentially similar to Control in all treated groups and no effect of parental treatment with Bayscript Gelb BR was inferred.

The single incidence of nipples observed among male offspring on Day 13 of age occurred in the Control group, therefore was unrelated to parental treatment with Bayscript Gelb BR.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

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

Mean serum T4 concentrations (pg/mL)

Group	Treatment	Dose		Adult Male at Termination	Day 13 of age Offspring
		(mg/kg/day)			Male	Female
1	Vehicle (purified water)	0	Mean	40700	38500	39400
			SD	6570	6440	4970
			% CV	16.1	16.7	12.6
			N	10	10	10
2	Bayscript Gelb BR	100	Mean	41200	41100	40700
			SD	9820	6180	14800
			% CV	23.8	15.0	36.4
			N	10	9	9
3	Bayscript Gelb BR	300	Mean	41000	38000	42000
			SD	5400	7310	5250
			% CV	13.2	19.2	12.5
			N	10	10	10
4	Bayscript Gelb BR	1000	Mean	41600	37400	40600
			SD	10100	4390	5090
			% CV	24.3	11.7	12.5
			N	10	10	10

 

Applicant's summary and conclusion

Conclusions:

In conclusion, within the context of this combined repeated dose toxicity and reproductive/ developmental toxicity screening studyit was concluded that a dose level of 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity and reproductive/developmental toxicity in the CD rat. Bayscript Gelb BR showed no evidence of being an endocrine disruptor.

Executive summary:

Summary

The purpose of this study was to assess the general systemic toxic potential in Crl:CD(SD) rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, following administration of Bayscript Gelb BR by oral gavage administration forat least five weeks. The study was conducted according to OECD TG 422 guideline: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.

Three groups of ten male and ten female rats received Bayscript Gelb BR at doses of 100, 300 or 1000 mg/kg/day by oral gavage administration at a volume dose of 10 mL/kg/day. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, purified water, at the same volume dose as treated groups.

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

For the offspring,clinical condition, litter size and survival, sex ratio, body weight, ano‑genital distance and nipple counts (males only), and macropathology were also assessed.

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

Results

F0 responses

Oral administration of Bayscript Gelb BR to parental Sprague Dawley (Crl:CD(SD)) rats at dose levels of 100, 300 or 1000 mg/kg/day for two weeks prior to pairing, during pairing and then up to termination of the males after 5 weeks of treatment and females on Day 14 of lactation was well tolerated. There were no premature deaths,no test article-related signs observed during the detailed physical examination and arena observations, no post-dosing observations, no effects on sensory reactivity, grip strength or motor activity and no effects on body weight performance or food consumption. Organ weights at scheduled termination were essentially similar in all groups, and macroscopic and microscopic examination conducted after five weeks of treatment for males and on Day 14 of lactation for females did not reveal any test item-related lesions.

Estrous cyclicity, pre-coital interval, mating performance, gestation length and index were unaffected by treatment with Bayscript Gelb BR.

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

F1 responses

The clinical condition, litter size, sex ratio, body weight, survival and ano-genital distances of the F1 offspring was unaffected by parental treatment and at scheduled termination there were no findings associated with treatment.

 

Conclusion

In conclusion, within the context of this combined repeated dose toxicity and reproductive/ developmental toxicity screening studyit was concluded that a dose level of 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity and reproductive/developmental toxicity in the CD rat. Bayscript Gelb BR showed no evidence of being an endocrine disruptor.