Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 November 2018 to 16 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Natural white powder
- Storage conditions: Room temperature (15-25 °C), protected from light and humidity

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar RccHan™;WIST rat.
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Males approximately 12 weeks old. Females approximately 14 weeks old.
- Weight at study initiation: Males 317 to 355 g. Females 206 to 246 g.
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation, 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. The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Pre-pairing: Up to four animals of one sex
Pairing: One male and one female
Males after mating: Up to four animals
Gestation: One female
Lactation: One female + litter
- Diet: Non-restricted (removed overnight for urinalysis investigations).
- Water: Non-restricted (removed overnight for urinalysis investigations).
- Acclimation period:
Males: Six days before commencement of treatment.
Females: 22 days before commencement of treatment.
Oestrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4 - 5 day 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.
Before the commencement of treatment, any individuals rejected during the acclimatisation period were replaced with spare animals of suitable weight from the same batch:
Body weight range extremes: Two males and one female.
Abnormal estrous cycles: Four females.

DETAILS OF FOOD AND WATER QUALITY: The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20 - 24 °C.
- Humidity (%): Monitored and maintained within the range of 40 - 70 %.
- Air changes (per hr): Filtered fresh air was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
Details on oral exposure
- PREPARATION OF DOSING SOLUTIONS:
- Treatment of test material prior to testing: The required amount of test material was weighed. Approximately 40 % of the final volume of vehicle was added and stirred by hand, crushing any large particles. Stirring continued using a magnetic stirrer followed by a high shear homogeniser to fully disperse the test material. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer. The suspension was transferred to the final containers, via syringe whilst magnetically stirring. A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test material in ascending order.
- Frequency of preparation: Weekly.
- Storage of formulation: Refrigerated (2 - 8 °C).
- Formulated concentrations: 0, 20, 60 and 200 mg/L.
- Volume dose: 5 mL/kg
- Treated at: Constant doses in mg/kg/day.
- Individual dose volume: Calculated from the most recently recorded scheduled body weight.
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

- VEHICLE: Purified water.
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 at 10 and 200 mg/mL were analysed to assess the stability and homogeneity of the test material in the liquid matrix.
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 3 and the final week of treatment were analysed for achieved concentration of the test material.

Preparation of Test Samples
A representative sample of test formulation (3 mL, accurately weighed) was sampled into a pre-weighed glass centrifuge tube. The centrifuge tubes were transferred to a drying oven and allowed to stand overnight at 100 °C. The samples were removed and equilibrated to ambient temperature. The samples were re-weighed to determine the mass of the test material within each formulation sample.

Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (3 mL) of control matrix (purified water) with known amounts of the test material. The prepared procedural recoveries were analysed in a ccordance with the analytical procedure. Procedural recoveries were used to correct analytical values.

Calculations
The concentration of the test material was determined using the following equation:

Analysed concentration (mg/mL) = (((W4 - W2) - W3) / W1) × D

Procedural recovery values were determined using the following equation:

Procedural recovery, mg/mL = ((W4 - W2) - W3) / V

Sample concentrations were corrected for procedural recoveries using the following equation:

Corrected concentration, mg/mL = Analysed concentration, mg/mL × 100 / R

Where
W1 = Weight of sample taken (g)
W2 = Original mass of centrifuge tube (mg)
W3 = Mean weight of residue from control sample (mg)
W4 = Final mass of centrifuge tube and solid residue (mg)
V = Nominal weight of recovery sample
D = Density of sample (g/mL)
R = Appropriate mean procedural recovery value at analysis

Validation of the Analytical Procedure
The analytical procedure was validated by determining the following parameters:
The specificity of the analysis (test material analysed weight should be < 0.01g) in control samples. The method accuracy and precision, by determining five procedural recoveries at nominal concentrations of 10 mg/mL and 200 mg/mL during the method validation.

Homogeneity in Purified Water Formulations
The homogeneity of the test material in purified water formulations was assessed at nominal concentrations of 10 mg/mL and 200 mg/mL, during ambient and refrigerated storage. Freshly prepared specimen formulations (400 mL) were equally sub divided into four amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Ambient Temperature Storage (15 to 25 °C)
On receipt, the contents of one bottle of each formulation were mixed by 20-fold inversion followed by magnetic stirring. After stirring for a minimum of 20 minutes (representing 0 hour) and 4 hours, single samples (nominally 3 mL) were removed for analysis from the top, middle and bottom of the continuously stirred formulation. The remainder of the bottle was stored at ambient temperature and after storage for 1 day, the contents were remixed and sampled as detailed above.

Refrigerated Storage (2 to 8 °C)
The remaining bottles were refrigerated on receipt and on Day 1, Day 8 and Day 15 the appropriate bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottle were mixed by 20-fold inversion followed by magnetic stirring for a minimum of 20 minutes and single samples (nominally 3 mL) were removed for analysis from the top, middle and bottom of the stirred formulation.

Concentration of Dose Formulations
For Week 1, Week 3 and the Final week of treatment, freshly prepared test formulations were sample d (4 × 3 mL, accurately weighed) by Pharmacy personnel and submitted for analysis. Two samples were analysed in accordance with the analytical procedure, and the remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved. Additional samples were taken for Week 3 and analysed due to out of specification results obtained for Week 1.
Procedural recoveries were prepared as a quality control measure and were used to correct for the formulation samples.

RESULTS
Method Validation
The analytical procedure was successfully validated for the test material in purified water with respect to the specificity of the analysis, method accuracy and precision.
The specificity of the assay was demonstrated by the absence of the test material in the control sample (< 0.01g).
Method accuracy and precision were confirmed and results are: A mean procedural recovery value of 70.3 % (CV=0.76 %, n=5) was obtained for 10 mg/mL and 69.0 % (CV=0.94 %, n=5) was obtained for 200 mg/mL. Mean procedural recovery values were outside of the acceptance criteria as the test material was partially soluble in the vehicle, which confirmed that gravimetric analysis was the only suitable assay for this formulation. Trial and routine formulations samples followed the same trend, therefore, each set of samples were corrected for procedural recoveries. It is considered to have no impact on the integrity of the study.

Homogeneity of Dose Formulations
The homogeneity of the test material in purified water formulations was assessed with respect to the level of concentration at nominal concentrations of 10 mg/mL and 200 mg/mL.
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for up to 4 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 analysed concentration for the three samples remained within 9 % of the initial time zero value and the coefficient of variation was less than 5 %. Recovery results during the trial remained within ± 7.5 % of the mean recovery found during validation showing the continued accuracy of the method, with the exception of one procedural recovery prepared at 10 mg/mL on Trial 1 Day 15, which was excluded as an outlier as per SOP.

Concentration of Dose Formulations
The mean concentrations were within the applied limits of +10/-15 % of the nominal concentration, confirming the accuracy of formulation, with the exception of Week 1 Groups 2 to 4. The difference from mean remained within 4 %, confirming precise analysis, with the exception of Week 1 Groups 2 to 4. The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure.
For Week 1, treated formulation samples were outside of the acceptance criteria. Samples were supplied in glass scintillation vials instead of pre weighed glass centrifuge tubes in error. As the weight of the scintillation vials was not known, the 3 mL samples supplied from Pharmacy were stirred for a minimum of 20 minutes and 1 mL samples (accurately weighed) were taken and sampled into the pre-weighed centrifuge tubes. Results showed that the formulation samples were -30.0 % to -40.5 % from nominal for groups 2 to 4. The losses seen were attributed to the sampling of the formulation samples as 1 mL was not a representative sample size for accurate gravimetric analysis.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of the analysis, method accuracy and precision.
The homogeneity was confirmed for the test material in purified water formulations at nominal concentrations of 10 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for up to 4 hours, ambient temperature storage (15 to 25 °C) for 1 day and refrigerated storage (2 to 8 °C) for up to 15 days.
The mean concentrations of the test material in test formulations analysed for the study were within +10/-15 % of nominal concentrations, confirming accurate formulation, with the exception of Week 1 Groups 2 to 4. The difference from mean remained within 4 %, confirming precise analysis, with theexception of Week 1 Groups 2 to 4. The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical 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.
Duration of treatment / exposure:
Males: Two weeks pre-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.
Duration of test:
Males: Two weeks pre-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.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals per sex per dose.
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: The doses used in this study (0, 100, 300 and 1 000 mg/kg/day) were selected in conjunction with the Sponsor and were based on the results of a 14-day preliminary study conducted at the test facility (Study No. CM06WP).
In the preliminary study dose levels of 250, 500 and 1 000 mg/kg/day were investigated in male and female Han Wistar rats. Treatment at doses of up to 1 000 mg/kg bw/day showed no obvious effect on body weight or food consumption. Clinical signs did not indicate any systemic effect of treatment, and necropsy showed no abnormalities. However, water consumptions were higher for both sexes from all treatment groups compared to controls. This finding was not considered sufficient to preclude a dosage of 1 000 mg/kg bw/day from being utilised as the high dose for this study. Water consumptions was measured gravimetrically during the two weeks prior to pairing to confirm the results of the 14-day preliminary study (Study No. CM06WP). Dose levels of 100 and 300 mg/kg bw/day (followed the OECD guidelines with a regular interval) were considered suitable for this main investigation as the low and intermediate dose levels, respectively.
- Rationale for animal assignment: Random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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 acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were performed on F0 generation animals at the following times in relation to dose administration:
F0 males:
Week 1 - daily
Week 2 onwards - weekly
F0 females:
Week 1 - daily
Week 2 - once
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12
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
Before treatment commenced and during each week of treatment and on Days 0, 6, 13 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 behaviour. Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of animals was recorded as follows:
F0 males:
Weekly during acclimatisation (not reported)
Before dosing on the day that treatment commenced and weekly thereafter.
On the day of necropsy.
F0 females:
Weekly during acclimatisation (not reported)
Before dosing on the day that treatment commenced 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, but recommenced for males once pairing for all animals was completed.
For females after mating food consumption was performed to match the body weight recording (where the end feeder recording matched a body weight day):
Days 0-7, 7-14 and 14-20 after mating.
Days 1-4, 4-7 and 7-13 of lactation.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: During the two weeks prior to pairing, water consumption was recorded by weight (over a 3 day period on each occasion) for each cage of animals, using water bottles fitted with sipper tubes.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at the following occasion: At termination.
- Anaesthetic used for blood collection: Yes. Animals were held under light general anesthesia induce d by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant.
- Animals fasted: Not specified
- How many animals: The five lowest numbered surviving males per group and the first five lactating females with a surviving litter per group.
- Parameters examined: Haematocrit (Hct)*, Haemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell haemoglobin (MCH)*, Mean cell haemoglobin concentration (MCHC)*, Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC); Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC); Platelet count (Plt).
* Derived values calculated in ClinAxys.
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyser. 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 series analyser and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination. 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.
- Animals fasted: Not specified
- How many animals: The five lowest numbered surviving males per group. The first five lactating females with a surviving litter per group.
- Parameters examined: After separation, the plasma was examined using a Roche P Modular Analyser 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.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 5 of treatment: The five lowest numbered surviving males per group.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: The individual samples were examined for the following characteristics:
Using manual methods:
Clarity and Colour (App) - by visual assessment
Volume (Vol) - using a measuring cylinder
pH - using a pH meter
Specific gravity (SG) - by direct refractometry using a SG meter
Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Ketones (Keto), Bile pigments (Bili), Blood pigments (UBld).
Using a Roche P Modular Analyser: Protein (Prot and T-Prot), Sodium (U-Na and T-Na), Potassium (U-K and T-K), Chloride (U-Cl and T-Cl).
A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data asdescribed below.
Epithelial cells (Epi), Leucocytes (WBC), Erythrocytes (RBC), Casts, Other abnormal components (A).
The slide was also examined for abnormalities in spermatozoa and crystals.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed (before dosing) during Week 5 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged.
Motor activity: 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.
- Dose groups that were examined: Sensory reactivity and grip strength: The five lowest numbered surviving males in each group; and on the first five lactating females in each group at Day 7-9 of lactation.
- Battery of functions tested:
Sensory reactivity and grip strength: 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/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 vocalisation without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalisation
4 Exaggerated response e.g. excessive vocalisation, 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
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 twoheight 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.

IMMUNOLOGY: No

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.

THYROID HORMONE ANALYSIS
Blood samples were collected as follows:
At termination: All surviving adult males and females (no samples were taken from females which failed to litter).
Day 4 of age: Offspring: up to two females per litter (where possible):
- one for T4 (serum) (Priority given to T4 sample)
- one for TSH (serum)
No offspring were allocated to these procedures on Day 4 of age if:
- the resultant live litter size would fall below ten offspring
- the resultant number of female pups would fall below three offspring
If only four female offspring were available within a litter but the overall litter size was >10, one female was selected with priority given to the T4 sample.
Day 13 of age: Offspring: two males and two females per litter (where possible):
- two for T4 (serum); where possible one male and one female (Priority given to T4 sample)
- two for TSH (serum); where possible one male and one female
Sequence of blood sampling on each occasion: To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory intergroup comparisons.
Conditions: No overnight deprivation of food.
Anesthetic: Adults: Isoflurane; Offspring: None.
Blood sample site: Adults: Sublingual vein; Offspring: Decapitation.
Parameters: Thyroid stimulating hormone (TSH), Thyroxine (T4).
Anticoagulant: None.
Tubes: Greiner Minicollect tubes with clotting activator.
Blood volume: Adults: 1.0 mL; Offspring: Maximum possible.
Number of blood tubes: One per animal or litter.
Processing: Samples were kept at ambient temperature (15 to 25 °C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions: At 2 000g for ten minutes at 4 °C.
Serum tubes: All available serum was transferred to appropriately labelled polypropylene “cryo” tubes using plastic disposable pipettes, then mixed by gentle 10-fold inversion.
Following mixing, each sample was divided into aliquots.
Aliquot volumes Adults:
Aliquot 1: T4 - 0.2mL of serum
Aliquot 2: TSH - residual serum
Offspring Day 4 and Day 13 of age:
T4 - all available serum
TSH - all available serum
Final storage conditions: Deep frozen (approximately -60 °C to -90 °C).
Thyroid hormone analysis: Samples from offspring on Day 13 of age and adult males were assessed for levels of thyroxine (T4) by the Principal Investigator. The remaining samples were retained pending further analyses.

OETROUS CYCLE
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 fail to exhibit 4 - 5 day cycles were not be allocated to study.
- After pairing until mating (for a maximum of 14 days).
- For four days before scheduled termination.

GROSS PATHOLOGY: Yes. 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. See table below.
- Method of Kill for all adult animals: Carbon dioxide asphyxiation (no exposure to carbon dioxide occured until after the completion of blood sampling for thyroid hormone assays).
- Time of Necropsy
F0 males: After final investigations completed (after at least five weeks of treatment).
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 13 of lactation.
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 intervals.

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.


HISTOPATHOLOGY: Yes
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 the first five lactating females with a surviving litter in Groups 1 and 4 at scheduled termination.
Abnormalities only: All remaining adult animals.
Routine staining: Sections were stained with haematoxylin 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: Groups 1 and 4: First five lowest numbered F0 males and first five lactating F0 females with a surviving litter: All specified tissues.
Scheduled kill: 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. For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted. For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.
Fetal examinations:
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.

TIME OF NECROPSY
Premature deaths:
Where possible, a fresh macroscopic examination with an assessment of stomach for milk content was performed. Grossly externally abnormal tissues were retained.

Day 4 of age:
Offspring with no clinical observations discarded without examination.
Offspring with clinical observations, examined externally and retained pending possible future examination.

Day 13 of age:
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.
Statistics:
Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including oestrous cycles before treatment, gestation index and stage of oestrous cycle at termination, the similarity of the data was such that analyses were not considered to be necessary.
All statistical analyses were carried out separately for males and females. Data relating to food consumption were analysed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/foetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
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 blood sampling) 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: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs seen at detailed physical examination and arena observations which could be considered related to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight and body weight gain for males during Days 1-36 of treatment was similar to Controls across all groups.
Mean body weight and body weight gain for females during Days 1-15 of treatment (before pairing) was similar to Controls across all groups.
The bodyweight gain of females receiving 100, 300 or 1 000 mg/kg/day was similar to that of the Control throughout gestation.
The bodyweight gain of females receiving 1 000 mg/kg/day was slightly lower than that of the Controls during Days 1-4 of lactation; a dose response was not apparent. The body weight gain of these females thereafter was generally similar to that of the Control during Days 4-13 of lactation.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean food consumption for males during Days 1 - 15 and 22 - 36 of treatment was similar to Controls across all groups.
The food consumption of females receiving 100, 300 or 1 000 mg/kg/day was similar to that of the Control during Days 1 - 15 of treatment and throughout gestation and Days 4 - 7 of lactation; food intake was marginally low during Days 1 - 4 and Days 7 - 13 of lactation at 1 000 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Mean water consumption for males and females during Weeks 1 and 2 of treatment was similar to Controls across all groups.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematological investigations in Week 6 for males and on Day 13 of lactation for females did not reveal any findings that were considered related to treatment.
The investigations in Week 6 for males revealed slightly but statistically high mean cell haemoglobinconcentrations in males receiving 100, 300 or 1 000 mg/kg/day when compared with the Control, although a dose response was not apparent. In addition, neutrophil and monocyte counts were slightly but statistically significantly high in males receiving 300 and 1 000 mg/kg/day when compared with Controls. These differences did not show a dose response, so no effect of treatment was inferred.
All other differences were minor, confined to one sex or did not show a relationship to treatment and were therefore attributed to normal biological variation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Biochemical examination of blood plasma in Week 6 for males and on Day 13 of lactation for females did not reveal any findings that were considered related to treatment. The investigations in Week 6 for males revealed slightly but statistically significant low glucose and total protein concentrations when compared with Control in males receiving 1 000 mg/kg/day, and statistically significantly low albumin concentrations in males receiving 100, 300 or 1 000 mg/kg/day.
The biochemical examination of the blood plasma on Day 13 of lactation for females revealed, when compared with the Controls, statistically significantly high phosphorus concentrations in females receiving 1 000 mg/kg/day and statistically significantly high total protein concentrations in females receiving 300 mg/kg/day. These differences occurred in isolation, so no effect of treatment was inferred.
All other differences were minor, confined to one sex or did not show a relationship to treatment and were therefore attributed to normal biological variation.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Examination of urine in Week 5 for males did not reveal any findings that were considered related to treatment.
The investigations in Week 5 for males revealed slightly but statistically significant low total proteinlevels and high chloride concentrations when compared with Control in males receiving 1 000 mg/kg/day.
All other differences were minor, confined to one sex or did not show a relationship to treatment and were therefore attributed to normal biological variation.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory reactivity observations was similar for animals treated at 100, 300 or 1 000 mg/kg/day, when compared with the Controls. For males receiving 1 000 mg/kg/day, the forelimb grip strength was slightly but statistically significantly higher than the Controls. In the absence of a similar effect on hindlimb grip strength or any such difference in females, this finding is considered unlikely to be related to treatment.
Motor Activity, assessed by low and high beam breaks over a 60 minute period, was unaffected by treatment with the test material.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The evaluation of organ weights of males after 5 weeks of treatment and of females on Day 13 of lactation revealed no differences when compared with the Controls.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination performed after 6 weeks of treatment revealed the following changes in the stomach.
Stomach
Depressions of the glandular mucosa was seen in five males receiving 1 000 mg/kg/day.
The incidence and distribution of all other findings were considered incidental and unrelated to test material.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Animals Killed After 6 Weeks of Treatment
Treatment Related Findings
Changes related to treatment with the test material were seen in the stomach.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted. The qualitative examination of the ovary also revealed no abnormality.

Stomach
Mucous cell hypertrophy of the glandular region of the stomach was seen in males given 100, 300 or 1 000 mg/kg/day.

Incidental Findings
Minimal haemorrhage and/ or oedema were seen in a three males given 1 000 mg/kg/day, one male given 300 mg/kg/day and one male given 100 mg/kg/day, and were considered as incidental and unrelated to test material due to the reduced severity and incidence. These are common background findings in rodents and correlated with dark areas seen at necropsy. All other histological changes were considered incidental and unrelated to test material.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age. There was therefore no requirement to measure T4 in the samples obtained from offspring on Day 4 of age or from the adult females and none of the TSH (thyroid stimulating hormone) samples required analysis.

Oestrous Cycles, Pre-Coital Interval, Mating Performance, Fertility and Gestation Length and Index:
All females allocated to study showed normal 4/5 day oestrous cycles during the acclimatization period.
Oestrous cyclicity, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment.

Determination of Thyroxine (T4) in Rat Serum Samples: Compliance of the Bioanalytical Batches
Sample Analysis: All samples taken from Groups 1 to 4 adult at termination males and Day 13 males and females offspring showed T4 concentrations and are consistent among groups.

System Suitability
Qualitatively, retention times and analyte responses were monitored. The system was suitable for analysis in all batches.
Carry-over Test: In each analytical run, the analysis of last unspiked samples injected after the ULOQ showed the absence of peaks in the zone where the corresponding reference and internal standards eluted.

Calibration Curves
All curves used for quantification complied with the acceptance criteria.

Quality Control Samples
The accuracy of the quantification of T4 in spiked surrogate or control matrix samples used as a control of assay quality is reported. The sample analysis batches were accepted since the corresponding quality control sample analyses complied with the acceptance criteria.

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
There was no clear effect of parental treatment with the test item at 100 or 300 mg/kg/day on the mean number of implantations.
For females receiving 1 000 mg/kg/day, the mean number of implantations was lower than that of females receiving 100 or 300 mg/kg/day or the Controls.

Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not examined

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
other: No effects at the highest tolerable dose tested.

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The body weight of male and female offspring on Day 1 of age and subsequent body weight gain until Day 13 of age was not inferior to that of the Control in all treated groups.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no clear effect of parental treatment with the test material at 100 or 300 mg/kg/day on the mean offspring survival indices.

Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no clear effect of parental treatment with the test material at 100 or 300 mg/kg/day on the mean sex ratio or offspring survival indices.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
There was no clear effect of parental treatment with the test material at 100 or 300 mg/kg/day on the mean litter size.

For females receiving 1 000 mg/kg/day, the mean number of implantations was lower than that of females receiving 100 or 300 mg/kg/day or the Controls resulting in a statistically significantly low total litter size in these groups. The mean total litter size is all within the historical control data range with the Control being high, and in the absence of an effect on any other indices, this is considered to have arisen by chance and is unrelated to treatment.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
There was no clear effect of parental treatment with the test material at 100 or 300 mg/kg/day on the mean offspring survival indices.
External malformations:
not examined
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There was no effect of parental treatment with the test material at 100, 300 or 1000 mg/kg/day on the absolute or adjusted ano-genital distances of offspring.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No effects at the highest tolerable dose tested.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Formulation Analysis

The analytical procedure was successfully validated with respect to specificity of the analysis, method accuracy and precision.

The homogeneity was confirmed for the test material in purified water formulations at nominal concentrations of 10 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for up to 4 hours, ambient temperature storage (15 to 25 °C) for 1 day and refrigerated storage (2 to 8 °C) for up to 15 days.

The mean concentrations of the test material in test formulations analysed for the study were within +10/-15 % of nominal concentrations, confirming accurate formulation, with the exception of Week 1 Groups 2 to 4. The difference from mean remained within 4 %, confirming precise analysis, with the exception of Week 1 Groups 2 to 4. The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure.

Mean Serum T4 Concentration (pg/mL)

Group

Treatment

Dose

(mg/kg/day)

Adult terminal males

Male offspring on Day 13 of age

Female offspring on Day 13 of age

1

Control

0

Mean

45800

48200

49200

SD

5290.30

7760.66

8747.08

CV %

11.55%

16.10%

17.78%

N

10

8

8

2

Test Material

100

Mean

50600

51500

54700

SD

9072.33

8489.96

5770.13

CV %

17.93%

16.49%

10.55%

N

10

9

9

3

 

Test Material

300

Mean

49600

52400

53500

SD

5055.48

8645.03

4948.25

CV %

10.19%

16.50%

9.25%

N

10

10

10

4

 

Test Material

1 000

Mean

47900

52900

53700

SD

5055.26

8825.36

10933.17

CV %

10.55%

16.68%

20.6%

N

10

8

8

Summary of Findings in the Stomach for Animals Killed After 6 Weeks of Treatment.

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

100

300

1 000

0

100

300

1 000

Depression(s), Glandular Mucosa

0

0

0

5

0

0

0

0

Number of tissues examined

10

10

10

10

8

9

10

8

The incidence and distribution of all other findings were considered incidental and unrelated to test material.

Summary of Treatment Related Findings in the Stomach of Animals Killed After 6 Weeks of Treatment.

Group/sex

1M

2M

3M

4M

Dose (mg/kg/day)

0

100

300

1000

Hypertrophy, Mucous Cells

 

 

 

 

Minimal

0

6

6

9

Total

0

6

6

9

Number of tissues examined

5

6

6

9

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study the no-observed-adverse-effect-level (NOAEL) for developmental toxicity was considered to be 1 000 mg/kg/day, the highest tolerable dose tested.
Executive summary:

The developmental toxicity of the test material was assessed according to OECD test guideline OECD 422 and in compliance with GLP.

The purpose of this study was an assessment of general systemic toxic potential in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test material by oral gavage administration for at least five weeks. Three groups of ten male and ten female rats received the test material at doses of 100, 300 or 1 000 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 13 of lactation. The F1 generation received no direct administration of the test material; 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, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, water consumption, haematology (peripheral blood), blood chemistry, urinalysis (males only), oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, thyroid hormone analysis, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. Blood samples were collected from selected offspring on Day 4 (not analysed) and Day 13 of age for thyroid hormone analysis.

The homogeneity was confirmed for the test material in purified water formulations at nominal concentrations of 10 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for up to 4 hours, ambient temperature storage (15 to 25 °C) for 1 day and refrigerated storage (2 to 8 °C) for up to 15 days.

The mean concentrations of the test material in test formulations analysed for the study were within +10/-15 % of nominal concentrations, confirming accurate formulation, with the exception of Week 1 Groups 2 to 4. The difference from mean remained within 4 %, confirming precise analysis, with the exception of Week 1 Groups 2 to 4. The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure.

Parental (F0) responses

There was no premature death related to treatment with the test material. Clinical condition, behaviour in the arena, sensory reactivity, grip strength and motor activity were unaffected by treatment. There were no signs seen in association with dosing.

The slight variations of mean body weights and body weight gains in females during the dosing period were considered likely to be related to litter size and not treatment with the test material. The food consumption of all animals was considered unaffected by treatment. Oestrous cycles, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment. There was no effect of treatment on the number of implantations or litter size. Haematological investigations of the plasma and biochemical examinations of the blood did not reveal any findings that could be attributed to treatment. There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 offspring.

The evaluation of organ weights of males after 5 weeks of treatment and of females on Day 13 of lactation revealed no significant differences when compared with the Controls.

At macroscopic examination, depressions of the glandular mucosa were seen in the majority of males receiving 1 000 mg/kg/day. No microscopic correlate was seen for the gross finding of depressions in the stomach of males given 1 000 mg/kg/day. At histopathological examination, mucous cell hypertrophy of the glandular region of the stomach was seen in males given 100, 300 or 1 000 mg/kg/day. This was considered to be an irritant effect of the test material, although considered non-adverse. F1 Litter Responses

The clinical condition of the offspring, offspring survival and sex ratio were unaffected by parental treatment. Litter size and offspring body weight on Day 1 of age were unaffected by treatment; offspring weight gain was slightly high at 100, 300 and 1 000 mg/kg/day. This is considered to be related to the slightly lower litter sizes in these groups and not an effect of treatment with the test material. Ano-genital distance of both male and female offspring on Day 1 of age and male nipple counts on Day 13 of age showed no effects of parental treatment. Macroscopic examination of offspring that died prior to the scheduled termination or were killed on Day 13 of age did not reveal any findings that were considered related to parental treatment at any dose level.

In conclusion, oral administration of the test material to parental Han Wistar rats at dose levels of 100, 300 or 1 000 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 13 of lactation in females was well-tolerated in the adult animals with no treatment related adverse effects observed.

Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no effect of treatment on the number of implantations, litter size or the growth of the offspring. In the context of this study, the test material, the test material, showed no evidence of being an endocrine disruptor.

Under the conditions of the study the no-observed-adverse-effect-level (NOAEL) for systemic toxicity and for developmental toxicity was considered to be 1 000 mg/kg/day, the highest tolerable dose tested.