4,11-diamino-2-(3-methoxypropyl)-1H-naphth[2,3-f]isoindol-1,3,5,10(2H)-tetrone

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Guideline followed

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Animal supply, acclimatisation and allocation
Strain/Species: RccHan™; WIST rat.
Supplier: Envigo RMS Ltd.
Number of animals : 44 males and 44 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatisation: Five days before commencement of treatment.
Age of animals at the start of the study:
Males: 69 to 75 days old
Females: 62 to 68 days old.
Weight range of animals at the start of the study:
Males: 281 to 313 g
Females. 153 to 196 g.
Allocation and identification
Allocation: On arrival, by non-selective allocation to cages.
On Day 1 of study all animals were weighed and body weights were reviewed by Study Management
before dosing commenced. Body weight of animals did not exceed ±20% of the mean for each sex.
Identification of animals: Each adult animal was assigned a number and identified uniquely within the
study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each lit
ter on Day 1 of age, using a toe tattoo.
Identification of cages: Each cage label was colour-coded according to group and was numbered
uniquely with cage and study number, as well as the identity of the occupant(s).
Animal replacement
Individuals rejected during the acclimatisation period were replaced with spare animals of suitable weight
from the same batch; the following replacements were performed:
Two males: To reduce inter/intra group bodyweight variation.
One female: At the extreme of the body weight range
Animal housing, diet and water supply
Environmental control
Rodent facility: Full barrier - to minimise entry of external biological and chemical agents and to minimise t
he 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, with the exception that the humidity was recorded as 36%
on the last day of necropsy.
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 acclimatisation, 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 equalise, as far as possible, environ
mental influences amongst the groups.
Bedding : Solid bottom cages contained softwood based bark-free fibre bedding, which was changed at
appropriate intervals each week.
Number of animals per cage
Pre-pairing: up to five animals per 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 (exce
pt during pairing and lactation) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and
replaced when necessary.
Diet supply
Diet: SDS VRF1 Certified pelleted diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability: Non-restricted (removed overnight before blood sampling for haematology and blood
chemistry investigations).
Water supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were
changed at appropriate intervals.
Availability: Non-restricted.

Route of administration:

oral: gavage

Vehicle:

other: 1% w/v methylcellulose

Details on exposure:

Dose levels were selected based on the results of the 14-day preliminary study performed in these laboratories with this compound (Huntingdon Life Sciences Study Number TMR0051). In the preliminary
study there was no evidence of overt toxicity during routine physical examinations or following dose administration. Bodyweight and food consumption at dose levels up to and including 350 mg/kg/day
showed no adverse effects of treatment. At 500 mg/kg/day overall body weight gain for males was slightly low when compared with the weight gain observed at 350 mg/kg/day, with some females at 500 mg/kg/day showing body weight loss. At macroscopic observation findings were limited to blue staining of the adipose tissue for animals that received 350 followed by 500 mg/kg/day and these animals also showed marginally low liver weights. It was therefore considered that a high dose of 500 mg/kg/day would be appropriate for this combined repeat dose toxicity study and reproductive/developmental toxicity screening study; with low and intermediate doses of 50 and 150 mg/kg/day, respectively.

Formulation
Correction factor: A correction factor of 1.1 was used when calculating quantities of test substance used during dose preparation.
Method of preparation : The test material was ground in a mortar and pestle to a fine powder. Small amounts of vehicle were added and mixed to produce a smooth paste. It was made up to volume with
vehicle and mixed using a high shear homogeniser until homogenous and finally mixed using a magnetic stirrer.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test substance.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated.
Test substance accounting: Detailed records of compound usage were maintained. The amount of test substance 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.

Formulation analysis
Stability and homogeneity: The dose form at 1 and 50 mg/mL (in terms of nominal concentration) was shown to be homogenous and stable for 15 days when stored refrigerated or two days when stored at ambient temperature.
Achieved concentratio: Samples of each formulation prepared for administration in the first and last weeks of treatment were analysed for achieved concentration of the test substance.

Administration
Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg.
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 the treated groups.
Frequency: Once daily, at approximately the same time each day.
Sequence: Groups dosed in ascending order.
Formulation: Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Storage of formulation: Refrigerated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The validation of the analytical procedure (FIA/M041/15) established at Envigo for the determination of FAT 36152/M TE in 1% w/v methylcellulose formulations.
The homogeneity and stability, determined with respect to the level of concentration, of FAT 36152/M TE in 1% w/v methylcellulose at nominal concentrations of 1 mg/mL and 50 mg/mL.
The concentrations of FAT 36152/M TE in test formulations analysed during the study.
The formulations for this study were prepared by Pharmacy personnel. The analytical work was undertaken by Formulation & Inhalation Analysis personnel between 09 April 2015 and 22 August 2015.

Analytical procedure
Apparatus and instrumentation
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Balances fitted with printers: Capability of weighing to 5 or 6 decimal places
General laboratory apparatus and glassware.

Reagents
Control vehicle: 1% w/v methylcellulose
Acetonitrile: HPLC gradient grade
N,N-Dimethylformamide (DMF): HPLC grade
Propyl-4-hydroxy-benzoate (propyl paraben): >99%
Water: Reverse osmosis
Diluent: DMF
Internal standard: 10 mg propyl paraben in DMF (100 mL)
Preparation of standards
A primary standard solution (100 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 5 mg) of FAT 36152/M TE in DMF (50 mL). Solutions for instrument calibration were prepared by
appropriate dilution of the primary standard solution using DMF containing 0.5 mL of stock internal standard solution and contained FAT 36152/M TE at nominal concentrations of 2 μg/mL, 4 μg/mL, 6 μg/
mL, 8 μg/mL and 10 μg/mL.
Calibration solutions were injected onto the HPLC, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the chromatographic section.
Sample process
A representative sample of test formulation (1 mL, accurately weighed) was dissolved using ultrasonic vibration in DMF. The extract was diluted using DMF to provide a solution containing FAT 36152/M TE at an expected concentration within the range 4 μg/mL to 8 μg/mL and internal standard at a concentration of 5 μg/mL.
The concentration of FAT 36152/M TE in the final solution was quantified by HPLC using UV detection as detailed in the chromatographic section.

Typical chromatographic conditions
Column: NUCLEODUR C18 Isis, 3 μm, 4.6 × 250 mm
Column temperature: 50°C
Sample temperature: Ambient
Mobile Phase A: Acetonitrile/water 10/90 v/v
Mobile Phase B: Acetonitrile/water 90/10 v/v
Linear Gradient:
Time (min.) % A % B
0 50 50
8.0 10 90
12 10 90
13 50 50
16 50 50
Flow rate: 1.0 mL/min
Detector wavelength: UV, 254 nm
Injection volume: 5 μL
Run time: 16 minutes
Approximate retention time:
FAT 36152/M TE: 10.0 minutes
Internal standard: 4.9 minutes
Calculation
The peak area response ratio (test item/internal standard) for FAT 36152/M TE in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response ratio versus calibration standard concentration. The concentration of FAT 36152/M TE was determined using the following equation:
Analysed concentration (mg/mL) = ((Y – I) / S) × (V/W) × (D / 1000)
Where
Y = Peak area response ratio for FAT 36152/M TE in test chromatogram
I = Intercept derived from linear regression of calibration data
S = Slope derived from linear regression of calibration data
V = Dilution factor of sample (mL)
W = Sample weight (g)
D = Density (g/mL)
Validation of the analytical procedure
The analytical procedure was validated by determining the following parameters:
The specificity of the chromatographic analysis in control sample chromatograms.
The limit of detection and quantification was estimated by examination of control vehicle chromatograms in order to calculate a test substance concentration based on a peak height response equivalent to three times and ten times baseline noise respectively.
The linearity of detector response over the calibration standard concentration range.
The repeatability of the lowest and highest concentration calibration standards.
The method accuracy and precision, by determining six procedural recoveries at nominal concentrations of 1 mg/mL and 50 mg/mL during the method validation.
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (1% w/v methylcellulose) with known amounts of FAT 36152/M TE. The prepared procedural recoveries were analysed in accordance with the analytical procedure.

Homogeneity and stability in 1% w/v methylcellulose formulations
The homogeneity and stability of FAT 36152/M TE in 1% w/v methylcellulose formulations was assessed at nominal concentrations of 1 mg/mL and 50 mg/mL, during ambient temperature and refrigerated
storage. Freshly prepared specimen formulations (400 mL) were equally sub-divided (4 × 100 mL) into four amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Ambient temperature storage (nominally +21ºC)
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 2 hours, single samples (nominally 1 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 2 days storage the contents were remixed and sampled as detailed above.

Refrigerated storage (nominally +4ºC)
The remaining bottles were refrigerated on receipt and on Day 2 and Day 15, the bottles were 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 1 mL) were removed for analysis from the top, middle and bottom of the continuously stirred formulation.

Concentration in test formulations
For the First and Last week of treatment, freshly prepared test formulations were sampled (4 × 1 mL, 2 × 3 mL for controls accurately weighed) by Pharmacy personnel and submitted for analysis. Duplicate 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.

Results
Method validation
The analytical procedure was successfully validated for FAT 36152/M TE in 1% w/v methylcellulose with respect to the specificity of chromatographic analysis, limit of detection and quantification, the linearity of detector response, repeatability, method accuracy and precision. Results are summarised below:
The specificity of the HPLC assay was demonstrated by the absence of a peak at the characteristic retent ion time for FAT 36152/M TE in the control sample chromatogram.
The limit of detection and quantification was estimated as 0.00521 μg/mL and 0.0173 μg/mL respectively.
Linearity was confirmed over the nominal concentration range 2 μg/mL to 10 μg/mL with a coefficient of determination >0.999;
The repeatability was <1% for six replicate injections of standard solutions containing FAT 36152/M TE at nominal concentrations of 2 μg/mL and 10 μg/mL;
Method accuracy and precision were confirmed: a mean procedural recovery value of 102.1% (CV= 1.29%, n=6) was obtained for 1 mg/mL and 102.1% (CV=0.52%, n=6) was obtained for 50 mg/mL.

Formulation trial
The homogeneity and stability of FAT 36152/M TE in 1% w/v methylcellulose formulations was assessed with respect to the level of concentration at nominal concentrations of 1 mg/mL and 50 mg/mL.
Stability was confirmed during distribution between the bottles and on re-suspension following storage at ambient temperature for 2 days and refrigeration for up to 15 days. At each time-point, the mean analysed concentration for the single samples from top, middle and bottom remained within 5% of the initial time zero value and the coefficient of variation was less than 4%.
Procedural 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 from Day 15 (50 mg/mL). 3 of 4 procedural recoveries were acceptable and results were reported.

Concentration in dose formulations
The mean concentrations of FAT 36152/M TE in test formulations analysed during the study and the deviation of the mean result from the nominal value. The mean concentrations were within applied
limits +10%/-15%, confirming the accuracy of formulation. Difference from mean values were within 1% confirming precise analysis.

Conclusion
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method
accuracy and precision. The stability was confirmed for FAT 36152/M TE in 1% w/v methylcellulose formulations at nominal concentrations of 1 mg/mL and 50 mg/mL at ambient temperature storage for 2 days and refrigerated storage for up to 15 days.
The mean concentrations of FAT 36152/M TE in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation. Difference from mean values
were within 1% confirming precise analysis.

Details on mating procedure:

Paired for mating: 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 before pairing, throughout pairing and up to necropsy after a minimum of five weeks of treatment.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 6 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected based on the results of the 14-day preliminary study performed in these laboratories with this compound (Huntingdon Life Sciences Study Number TMR0051). In the preliminary
study there was no evidence of overt toxicity during routine physical examinations or following dose administration. Bodyweight and food consumption at dose levels up to and including 350 mg/kg/day
showed no adverse effects of treatment. At 500 mg/kg/day overall body weight gain for males was slightly low when compared with the weight gain observed at 350 mg/kg/day, with some females at 500
mg/kg/day showing body weight loss. At macroscopic observation findings were limited to blue staining of the adipose tissue for animals that received 350 followed by 500 mg/kg/day and these animals also
showed marginally low liver weights. It was therefore considered that a high dose of 500 mg/kg/day would be appropriate for this combined repeat dose toxicity study and reproductive/developmental toxicity
screening study; with low and intermediate doses of 50 and 150 mg/kg/day, respectively.

Maternal examinations:

Clinical and behavioural observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of 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.

Signs associated with dosing
Daily during Week 1 of treatment and once each week thereafter and for females on Days 0, 6, 13 and 20 after mating and Day 1 and 6 of lactation, detailed observations were recorded at the following times in relation to dose administration:
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, 6, 13 and 20 after mating and Days 1 and 6 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 behaviour 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.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered males in each group during Week 5 of treatment and on the five lowest numbered lactating females in each group during Days 4 6 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 length of separation of dam from litter was minimised. The cage labels on these individual cages showed only the study and animal number. Animals were not necessarily all tested on the sa me 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 during Days 4-6 of lactation for females, the motor activity of five lowest numbered surviving males and the five lowest numbered lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.5), 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:
Before dosing on the day that treatment commenced (Week 0) and weekly thereafter.
On the day of necropsy.
F0 females
Before dosing on the day that treatment commenced (Week 0) and weekly before pairing.
Days 0, 6, 13 and 20 after mating.
Day 1, 4, and 7 of lactation.
On the day of necropsy.

Food consumption
The weight of food supplied, that remaining and an estimate of any spilled was recorded as follows:
F0 animals
Weekly, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
For females after mating food consumption was performed to match the body weight recording:
Days 0-5, 6-12 and 13-19 after mating
Days 1-3 and 4-6 of lactation
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

Haematology, peripheral blood
Blood samples were collected after overnight withdrawal of food and before dose administration at the following occasion:
Week 2 of treatment (before pairing): The five lowest numbered surviving males and females per group Animals were held under light general anaesthesia 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 analyser:
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)
Morphology:
Anisocytosis
Microcytosis
Macrocytosis
Hypochromasia
Hyperchromasia
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 an ACL 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.

Blood chemistry
At the same time and using the same animals as for peripheral haematology, further blood samples were collected after overnight withdrawal of food and before dose administration at the following occasion:
Week 2 of treatment (before pairing): The five lowest numbered surviving males and females per group Animals were held under light general anaesthesia 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 Analyser in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acid (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 analysed albumin concentration.
Vaginal smears
Wet smears: After pairing until mating, using pipette lavage.

Mating
Paired for mating: 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.
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.

Postmortem examinations
Terminal procedures
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.

Time of necropsy
F0 males: After Week 5 investigations completed.
F0 females failing to litter: Day 25 after mating.
F0 females: Day 7 of lactation.

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

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

Organ weights
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite orga ns 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: Davidson’s fluid
For all animals examined, samples of any abnormal tissues were retained. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region.

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 surviving F0 males and females in Groups 1 and 4 at scheduled termination.
Abnormalities only: All F0 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
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:

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 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 1-7 of age.
Sex ratio of each litter: Recorded on Days 1, 4 and 7 of age.
Individual offspring body weights: Days 1, 4 and 7 of age.

Postmortem examinations (offspring)
Time of necropsy
F1 offspring: Day 7 of age.
Method of kill
Offspring: Intraperitoneal injection of sodium pentobarbitone.
Sequence: To allow satisfactory inter-group comparison.
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
Offspring at scheduled termination: Subject to a complete macroscopic examination.

Statistics:

Refer "Any other information on materials and methods incl. tables"

Indices:

Reproductive indices
Mating performance and fertility
Individual data was tabulated. Group values were calculated for males and females separately for the following:
Percentage mating (%) = (Number animals mating / Animals paired) x 100
Conception rate (%) = (Number animals achieving pregnancy / Animals mated) x 100
Fertility index (%) = (Number animals achieving pregnancy / Animals pairing) x 100

Off spring viability indices
Litter size
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1 and 7 of age. Group mean litter size and SD were calculated from the individual litter values.

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 7 / Number live offspring on Day 1 after littering) 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 and 7 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.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Refer 'details on results'

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Refer 'details on results'

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Refer 'details on results'

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Refer 'details on results'

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Refer 'details on results'

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Refer 'details on results'

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Refer 'details on results'

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

Signs and mortality
Treatment of males and female at all doses of FAT 36152/M TE was associated with blue staining on various parts of the body and blue staining on the cage bedding.
One female receiving 50 mg/kg/day was killed for welfare reasons on Day 22 after mating due to apparent dystocia: clinical signs included cold to touch, irregular breathing, red discharge from the vagina and whole body pallor. Macroscopic examination revealed that one fetus and a placenta were stuck in the cervix, and the internal organs and extremities were pale. As there were no cases of dystocia at higher doses, this single case in the low dose group was concluded to be due to chance and unrelated to treatment. No signs were observed in association with dose administration.

Sensory reactivity and grip strength
During Days 4-6 of lactation, there were three females at 50 mg/kg/day and two females receiving 500 mg/kg/day that showed a weak tail pinch response compared with none in the Control group; in the absence of any other sensory reactivity effects in females or a similar effect on males this is not considered to be related to treatment.
Forelimb grip strength for females receiving 50 mg/kg/day was low when compared with Controls with the difference achieving statistical significance. However, there was no evidence of a dose response and females at 500 mg/kg/day had values that were similar to Controls which suggests that the difference observed at 50 mg/kg/day is due to natural variation.
Group mean sensory reactivity and grip strength values for all groups of treated males during Week 5 of treatment were similar to Controls and considered to be unaffected by treatment.

Motor activity
Overall, group mean activity scores for all treated males and females were similar to the Control values. The high beam score (rearing activity) for males receiving 50 mg/kg/day at the 36-minute time interval was slightly but significantly high when compared with Controls, but this was an isolated incidence and scores at 150 and 500 mg/kg/day were similar to Controls; this difference was therefore attributed to natural variation.

Body weight
Body weight gain for males during the treatment period and for females before pairing was low at 150 or 500 mg/kg/day; all differences attained statistical significance except for females at 150 mg/kg/day. Bodyweight gain at 50 mg/kg/day was unaffected by treatment. There was no effect of treatment on body weight gain of females during gestation or during lactation.

Food consumption
Food consumption for males receiving 150 or 500 mg/kg/day was slightly low during Weeks 1 and 5 of treatment. Food consumption for females at 500 mg/kg/day was slightly low during Week 1 of treatment.
There were no adverse effects of treatment on food consumption of females during gestation or early lactation; females receiving 500 mg/kg/day showed slightly high consumption during Days 1 to 3 of lactation.

Haematology
During Week 2 of treatment animals receiving FAT 36152/M TE showed high reticulocyte counts when compared with Controls, with the difference attaining statistical significance for males at each dose level (p<0.01) and for females at 150 or 500 mg/kg/day (p<0.05 and p<0.01, respectively). In addition females receiving 500 mg/kg/day also showed significantly low haematocrit, haemoglobin and red blood cell count (p<0.01, p<0.01 and p<0.05, respectively) whilst the red cell distribution width and platelet counts were significantly high when compared with Controls (p<0.01). Other differences were considered slight and of no toxicological significance.

Blood chemistry
On separation of the plasma it was noted that the plasma for animals receiving 150 or 500 mg/kg/day was discoloured (blue). Bile acid levels for both males and females receiving FAT 36152/M TE were low when compared with Controls with the differences attaining statistical significance for males at 500 mg/kg/day (p<0.01) and for females at all dose levels (p<0.05); a dose response was not apparent.
In addition potassium levels were high for treated males and A/G ratios were slightly high for females at 150 and 500 mg/kg/day; however for both of these parameters these differences were not seen in the other sex.

Pre-coital interval, mating performance and fertility
All females mated within 4 days of pairing at the first oestrus, with no apparent adverse effect on either mating performance or fertility.

Gestation length
All females had gestation lengths within the normal range of 22 to 23.5 days, however at 500 mg/kg/day there was a significant shift toward a shorter gestation length (p<0.01). The distribution of gestation lengths for females at 50 or 150 mg/kg/day were not dissimilar to the Controls and were therefore considered to be unaffected by treatment. At 50 mg/kg/day one female (no. 53) was killed prematurely on Day 22 of gestation with signs of dystocia (extended parturition, low body temperature, irregular breathing, and pallor); macroscopic examination revealed that a fetus and placenta were lodged in the cervix. There were no other females in the higher dose groups that showed any problems during parturition, this incidence in the low dose group was therefore isolated and considered to be unrelated to treatment; the gestation index was therefore considered to be unaffected by treatment.

Terminal examinations
Parental Organ weights
When compared with Controls the mean adjusted kidney weights were high for males that received FAT 36152/M TE (p<0.05-0.01) and at 500 mg/kg/day the mean adjusted liver weight of males was high (p<0.05).
On Day 7 of lactation the mean adjusted spleen weights of females that received 150 or 500mg/kg/day were high when compared with the Controls; a dose response was apparent (p<0.05 and p<0.01, respectively).

Parental Macropathology
Five males receiving 150 mg/kg/day and 3 males that received 500 mg/kg/day had blue contents in the urinary bladder. Four females that received 500 mg/kg/day had blue adipose tissue.
Blue stained fur was apparent for one male that received 50 mg/kg/day, 6/10 males and 6/10 females that received 150 mg/kg/day and all males and females that received 500 mg/kg/day.

Histopathology
Microscopic changes related to treatment with FAT 36152/M TE were seen in the kidneys: Increased hyaline droplet formation (minimal to slight severity) was present bilaterally in the kidneys of
all males that received 500 mg/kg/day. Increased accumulations of hyaline drops were present in the cytoplasm of the tubular epithelium lining the proximal tubules in the renal cortex.
The following finding was considered to be incidental:
A minimal inflammatory cell infiltrate was present in the urinary bladder of three females that received 500 mg/kg/day FAT 36152/M TE. The infiltrate consisted of small perivascular foci of lymphocytes in the submucosal tissue. It is likely that the appearance of these changes in FAT 36152/M TE treated animals and not in Control animals is a chance event and not related to administration of the test item.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

effects observed, treatment-related

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

There were no adverse effects on pre-coital interval, mating performance, fertility or gestation index.

All females had gestation lengths within the normal range of 22 to 23.5 days, however at 500 mg/kg/day there was a significant shift toward a shorter gestation length.

There was no effect of treatment on the mean number of implantations, litter size, the survival of the offspring or the sex ratio (% males).

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: haematological effects and spleen weight for females at 500 mg/kg/day

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg/day, the offspring body weight on Day 1 of age and subsequent weight gain was marginally lower than expected.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Routine physical examination of the offspring showed an increased incidence of blue/grey discolouration at 500 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Offspring clinical signs

Clinical signs at routine examination that were attributed to treatment were limited to blue/grey discolouration of the offspring at 500 mg/kg/day; this was considered to be related to the colour of the test item.

Litter size, sex ratio and survival indices

One female in each of the 50 or 500 mg/kg/day groups did not litter and were subsequently confirmed to be not pregnant. The mean number of implantations and litter size were slightly high at 150 and 500 mg/kg/day when compared with Controls; with statistical significance attained at 500 mg/kg/day (p<0.05). There was no effect of treatment on survival of the offspring or the sex ratio (% males).

Offspring body weight

At 150 or 500mg/kg/day mean body weights of male and female offspring on Day 1 of age and the subsequent growth of the offspring were low when compared with Controls, with differences attaining statistical significance for the majority of the data points.

Review of the individual litter mean values and live litter size did show some correlation with the larger litter sizes showing lower offspring body weights; however there was some evidence that the low offspring weights were not solely attributable to live litter size. Therefore in the context of this screening study an involvement of treatment with the low offspring bodyweight at 150 and 500 mg/kg/day cannot be ruled out. Offspring body weight at 50 mg/kg/day was unaffected by parental treatment.

Offspring macropathology

Macroscopic examination of offspring that either died before scheduled termination or were killed at scheduled termination on Day 7 of age, revealed an increased incidence of abnormal colour skin/milk at 500 mg/kg/day; this was attributed to the colour of the test item.

Conclusions:

No observed adverse effect level (NOAEL) for developmental toxicity was 500 mg/kg/day.

Executive summary:

The developmental toxicity potential of FAT 36152/M TE (a textile dye) was evaluated in a study conducted according to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test).

Three groups comprising of ten male and ten female rats received FAT 36152/M TE at doses of 50, 150 or 500 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, throughout pairing and 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 6 of lactation. Females were killed on Day 7 of lactation. The F1 generation received no direct administration of the test substance; any exposure wasin uteroor via the milk. A similarly constituted Control group received the vehicle,
1% w/v methylcellulose, at the same volume dose as the treated groups. 

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. 

The clinical condition, litter size and survival, sex ratio, body weight and macropathology for all offspring were also assessed. 

Results

One female receiving 50 mg/kg/day was killed for welfare reasons on Day 22 after mating with dystocia: clinical signs included cold to touch, irregular breathing, red discharge from the vagina and whole body pallor. Macroscopic examination revealed that one fetus and a placenta were stuck in the cervix, and the internal organs and extremities were pale. As there were no cases of dystocia at higher dose levels, this single case in the low dose group was concluded to be due to chance and unrelated to treatment.

Treatment of males and female at all doses of FAT 36152/M TE was associated with blue staining on various parts of the body. No signs were observed in association with dose administration.

Sensory reactivity, grip strength and motor activity showed no adverse effects of treatment.

Body weight gain for males during the treatment period and for females before pairing was low at 150 or 500 mg/kg/day. Body weight gain for animals at 50 mg/kg/day and for treated females at all dose levels during gestation and lactation was unaffected by treatment.

Food consumption for males receiving 150 or 500 mg/kg/day was slightly low during Weeks 1 and 5 of treatment and consumption for females at 500 mg/kg/day was slightly low during Week 1 of treatment. There were no adverse effects of treatment on food consumption for females during gestation or early lactation; females receiving 500 mg/kg/day showed slightly high consumption during Days 1 to 3 of lactation.

During Week 2 of treatment animals receiving FAT 36152/M TE showed high reticulocyte counts when compared with Controls. In addition females receiving 500 mg/kg/day also showed significantly low haematocrit, haemoglobin and red blood cell count and significantly high red cell distribution width and platelet counts.

Bile acid levels for both males and females receiving FAT 36152/M TE were low when compared with Controls. In addition potassium levels were high for treated males and A/G ratios were slightly high for females at 150 and 500 mg/kg/day.

There were no adverse effects on pre-coital interval, mating performance, fertility or gestation index.

All females had gestation lengths within the normal range of 22 to 23.5 days, however at 500 mg/kg/day there was a significant shift toward a shorter gestation length.

Routine physical examination of the offspring showed an increased incidence of blue/grey discolouration at 500 mg/kg/day.

There was no effect of treatment on the mean number of implantations, litter size, the survival of the offspring or the sex ratio (% males).

At 500 mg/kg/day, the offspring body weight on Day 1 of age and subsequent weight gain was marginally lower than expected.

After 5 weeks of treatment males at all dose levels showed high mean adjusted kidney weights and males at 500 mg/kg/day had high mean adjusted liver weight when compared with Controls.

On Day 7 of lactation the mean adjusted spleen weights of females that received 150 or 500mg/kg/day were high when compared with the Controls.

Macroscopic findings at necropsy included blue content in the urinary bladder content for males at 150 and 500 mg/kg/day, blue adipose for females that received 500 mg/kg/day and there was a dose related incidence of blue coat staining. Macroscopic examination of offspring revealed an increased incidence of abnormal colour skin/milk at 500 mg/kg/day.

Microscopic changes related to treatment with FAT 36152/M TE were seen in the kidneys of all males that received 500 mg/kg/day with increased hyaline droplet formation (minimal to slight severity) present bilaterally; increased accumulations of hyaline drops were present in the cytoplasm of the tubular epithelium lining the proximal tubules in the renal cortex.

Conclusion

Administration of FAT 36152/M TE at dose levels of 50, 150 and 500 mg/kg/day was well tolerated with no adverse effects on clinical condition, sensory reactivity, grip strength, motor activity, mating performance, fertility, parturition, gestation index or offspring survival.

At 500 mg/kg/day haematological findings and spleen weights for parental female animals were indicative of anaemia and at 500 mg/kg/day offspring body weight on day 1 of age and subsequent gain up to Day 7 of age was marginally low.

It was therefore concluded that in this screening study the no observed adverse effect level (NOAEL) for:

·        general systemic toxicity was 150 mg/kg/day (haematological effects and spleen weight for females at 500 mg/kg/day)

·        mating performance, fertility and offspring survival was 500 mg/kg/day

.   offspring development was 500 mg/kg/day(marginally low offspring bodyweights and weight gain but no effect on survival, clinical condition or macropathology)

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

None

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal supply, acclimatisation and allocation
Strain/Species: RccHan™; WIST rat.
Supplier: Envigo RMS Ltd.
Number of animals : 44 males and 44 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatisation: Five days before commencement of treatment.
Age of animals at the start of the study:
Males: 69 to 75 days old
Females: 62 to 68 days old.
Weight range of animals at the start of the study:
Males: 281 to 313 g
Females. 153 to 196 g.

Allocation and identification
Allocation: On arrival, by non-selective allocation to cages.
On Day 1 of study all animals were weighed and body weights were reviewed by Study Management before dosing commenced. Body weight of animals did not exceed ±20% of the mean for each sex.
Identification of animals: Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo 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 colour-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal replacement
Individuals rejected during the acclimatisation period were replaced with spare animals of suitable weight from the same batch; the following replacements were performed:
Two males: To reduce inter/intra group bodyweight variation.
One female: At the extreme of the body weight range

Animal housing, diet and water supply
Environmental control
Rodent facility: Full barrier - to minimise entry of external biological and chemical agents and to minimise 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, with the exception that the humidity was recorded as 36% on the last day of necropsy.
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 acclimatisation, 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 equalise, as far as possible, environmental influences amongst the groups.
Bedding : Solid bottom cages contained softwood based bark-free fibre bedding, which was changed at appropriate intervals each week.

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

Environmental enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.

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

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

Route of administration:

oral: feed

Vehicle:

other: 1% w/v methylcellulose

Details on oral exposure:

Treatment groups and doses
Dose levels were selected based on the results of the 14-day preliminary study performed in these laboratories with this compound (Huntingdon Life Sciences Study Number TMR0051). In the preliminary study there was no evidence of overt toxicity during routine physical examinations or following dose administration. Bodyweight and food consumption at dose levels up to and including 350 mg/kg/day showed no adverse effects of treatment. At 500 mg/kg/day overall body weight gain for males was slightly low when compared with the weight gain observed at 350 mg/kg/day, with some females at 500 mg/kg/day showing body weight loss. At macroscopic observation findings were limited to blue staining of the adipose tissue for animals that received 350 followed by 500 mg/kg/day and these animals also showed marginally low liver weights. It was therefore considered that a high dose of 500 mg/kg/day would be appropriate for this combined repeat dose toxicity study and reproductive/developmental toxicity screening study; with low and intermediate doses of 50 and 150 mg/kg/day, respectively.

Formulation
Correction factor: A correction factor of 1.1 was used when calculating quantities of test substance used during dose preparation.
Method of preparation : The test material was ground in a mortar and pestle to a fine powder. Small amounts of vehicle were added and mixed to produce a smooth paste. It was made up to volume with vehicle and mixed using a high shear homogeniser until homogenous and finally mixed using a magnetic stirrer.

A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test substance.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated.
Test substance accounting: Detailed records of compound usage were maintained. The amount of test substance 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.

Formulation analysis
Stability and homogeneity: The dose form at 1 and 50 mg/mL (in terms of nominal concentration) was shown to be homogenous and stable for 15 days when stored refrigerated or two days when stored at ambient temperature.
Achieved concentration: Samples of each formulation prepared for administration in the first and last weeks of treatment were analysed for achieved concentration of the test substance.


Administration
Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg.
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 the treated groups.
Frequency: Once daily, at approximately the same time each day.
Sequence: Groups dosed in ascending order.
Formulation: Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Storage of formulation: Refrigerated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The description of the analytical and sampling procedures used and details of the results obtained are given below:
The validation of the analytical procedure (FIA/M041/15) established at Envigo for the determination of FAT 36152/M TE in 1% w/v methylcellulose formulations.
The homogeneity and stability, determined with respect to the level of concentration, of FAT 36152/M TE in 1% w/v methylcellulose at nominal concentrations of 1 mg/mL and 50 mg/mL.
The concentrations of FAT 36152/M TE in test formulations analysed during the study.
The formulations for this study were prepared by Pharmacy personnel. The analytical work was undertaken by Formulation & Inhalation Analysis personnel between 09 April 2015 and 22 August 2015.

Analytical procedure
Apparatus and instrumentation
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Balances fitted with printers: Capability of weighing to 5 or 6 decimal places
General laboratory apparatus and glassware.

Reagents
Control vehicle: 1% w/v methylcellulose
Acetonitrile: HPLC gradient grade
N,N-Dimethylformamide (DMF): HPLC grade
Propyl-4-hydroxy-benzoate (propyl paraben): >99%
Water: Reverse osmosis
Diluent: DMF
Internal standard: 10 mg propyl paraben in DMF (100 mL)

Preparation of standards
A primary standard solution (100 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 5 mg) of FAT 36152/M TE in DMF (50 mL). Solutions for instrument calibration were prepared by appropriate dilution of the primary standard solution using DMF containing 0.5 mL of stock internal standard solution and contained FAT 36152/M TE at nominal concentrations of 2 μg/mL, 4 μg/mL, 6 μg/mL, 8 μg/mL and 10 μg/mL.
Calibration solutions were injected onto the HPLC, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the chromatographic section.

Sample process
A representative sample of test formulation (1 mL, accurately weighed) was dissolved using ultrasonic vibration in DMF. The extract was diluted using DMF to provide a solution containing FAT 36152/M TE at an expected concentration within the range 4 μg/mL to 8 μg/mL and internal standard at a concentration of 5 μg/mL.
The concentration of FAT 36152/M TE in the final solution was quantified by HPLC using UV detection as detailed in the chromatographic section.

Typical chromatographic conditions
Column: NUCLEODUR C18 Isis, 3 μm, 4.6 × 250 mm
Column temperature: 50°C
Sample temperature: Ambient
Mobile Phase A: Acetonitrile/water 10/90 v/v
Mobile Phase B: Acetonitrile/water 90/10 v/v

Linear Gradient:
Time (min.) % A % B
0 50 50
8.0 10 90
12 10 90
13 50 50
16 50 50
Flow rate: 1.0 mL/min
Detector wavelength: UV, 254 nm
Injection volume: 5 μL
Run time: 16 minutes
Approximate retention time:
FAT 36152/M TE: 10.0 minutes
Internal standard: 4.9 minutes

Calculation
The peak area response ratio (test item/internal standard) for FAT 36152/M TE in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response ratio versus calibration standard concentration. The concentration of FAT 36152/M TE was determined using the following equation:

Analysed concentration (mg/mL) = ((Y – I) / S) × (V/W) × (D / 1000)

Where
Y = Peak area response ratio for FAT 36152/M TE in test chromatogram
I = Intercept derived from linear regression of calibration data
S = Slope derived from linear regression of calibration data
V = Dilution factor of sample (mL)
W = Sample weight (g)
D = Density (g/mL)

Validation of the analytical procedure
The analytical procedure was validated by determining the following parameters:
The specificity of the chromatographic analysis in control sample chromatograms.
The limit of detection and quantification was estimated by examination of control vehicle chromatograms in order to calculate a test substance concentration based on a peak height response equivalent to three times and ten times baseline noise respectively.
The linearity of detector response over the calibration standard concentration range.
The repeatability of the lowest and highest concentration calibration standards.
The method accuracy and precision, by determining six procedural recoveries at nominal concentrations of 1 mg/mL and 50 mg/mL during the method validation.
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (1% w/v methylcellulose) with known amounts of FAT 36152/M TE. The prepared procedural recoveries were analysed in accordance with the analytical procedure.

Homogeneity and stability in 1% w/v methylcellulose formulations
The homogeneity and stability of FAT 36152/M TE in 1% w/v methylcellulose formulations was assessed at nominal concentrations of 1 mg/mL and 50 mg/mL, during ambient temperature and refrigerated storage. Freshly prepared specimen formulations (400 mL) were equally sub-divided (4 × 100 mL) into four amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Ambient temperature storage (nominally +21ºC)
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 2 hours, single samples (nominally 1 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 2 days storage the contents were remixed and sampled as detailed above.

Refrigerated storage (nominally +4ºC)
The remaining bottles were refrigerated on receipt and on Day 2 and Day 15, the bottles were 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 1 mL) were removed for analysis from the top, middle and bottom of the continuously stirred formulation.

Concentration in test formulations
For the First and Last week of treatment, freshly prepared test formulations were sampled (4 × 1 mL, 2 × 3 mL for controls accurately weighed) by Pharmacy personnel and submitted for analysis. Duplicate 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.

Results
Method validation
The analytical procedure was successfully validated for FAT 36152/M TE in 1% w/v methylcellulose with respect to the specificity of chromatographic analysis, limit of detection and quantification, the linearity of detector response, repeatability, method accuracy and precision. Results are summarised below:
The specificity of the HPLC assay was demonstrated by the absence of a peak at the characteristic retention time for FAT 36152/M TE in the control sample chromatogram.
The limit of detection and quantification was estimated as 0.00521 μg/mL and 0.0173 μg/mL respectively.
Linearity was confirmed over the nominal concentration range 2 μg/mL to 10 μg/mL with a coefficient of determination >0.999;
The repeatability was <1% for six replicate injections of standard solutions containing FAT 36152/M TE at nominal concentrations of 2 μg/mL and 10 μg/mL;
Method accuracy and precision were confirmed: a mean procedural recovery value of 102.1% (CV=1.29%, n=6) was obtained for 1 mg/mL and 102.1% (CV=0.52%, n=6) was obtained for 50 mg/mL.

Formulation trial
The homogeneity and stability of FAT 36152/M TE in 1% w/v methylcellulose formulations was assessed with respect to the level of concentration at nominal concentrations of 1 mg/mL and 50 mg/mL.
Stability was confirmed during distribution between the bottles and on re-suspension following storage at ambient temperature for 2 days and refrigeration for up to 15 days. At each time-point, the mean analysed concentration for the single samples from top, middle and bottom remained within 5% of the initial time zero value and the coefficient of variation was less than 4%.
Procedural 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 from Day 15 (50 mg/mL). 3 of 4 procedural recoveries were acceptable and results were reported.

Concentration in dose formulations
The mean concentrations of FAT 36152/M TE in test formulations analysed during the study and the deviation of the mean result from the nominal value. The mean concentrations were within applied limits +10%/-15%, confirming the accuracy of formulation. Difference from mean values were within 1% confirming precise analysis.

Conclusion
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.
The stability was confirmed for FAT 36152/M TE in 1% w/v methylcellulose formulations at nominal concentrations of 1 mg/mL and 50 mg/mL at ambient temperature storage for 2 days and refrigerated storage for up to 15 days.
The mean concentrations of FAT 36152/M TE in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation. Difference from mean values were within 1% confirming precise analysis.

Duration of treatment / exposure:

Males: Two weeks before pairing, throughout pairing and up to necropsy after a minimum of five weeks of treatment.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 6 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 50, 150 and 500 mg/kg/day
Basis:
nominal in diet

No. of animals per sex per dose:

10 / sex / dose

Control animals:

yes, plain diet

Details on study design:

Dose levels were selected based on the results of the 14-day preliminary study performed in these laboratories with this compound (Huntingdon Life Sciences Study Number TMR0051). In the preliminary study there was no evidence of overt toxicity during routine physical examinations or following dose administration. Bodyweight and food consumption at dose levels up to and including 350 mg/kg/day showed no adverse effects of treatment. At 500 mg/kg/day overall body weight gain for males was slightly low when compared with the weight gain observed at 350 mg/kg/day, with some females at 500 mg/kg/day showing body weight loss. At macroscopic observation findings were limited to blue staining of the adipose tissue for animals that received 350 followed by 500 mg/kg/day and these animals also showed marginally low liver weights. It was therefore considered that a high dose of 500 mg/kg/day would be appropriate for this combined repeat dose toxicity study and reproductive/developmental toxicity screening study; with low and intermediate doses of 50 and 150 mg/kg/day, respectively.

Positive control:

None

Observations and examinations performed and frequency:

Clinical and behavioural observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of 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.

Signs associated with dosing
Daily during Week 1 of treatment and once each week thereafter and for females on Days 0, 6, 13 and 20 after mating and Day 1 and 6 of lactation, detailed observations were recorded at the following times in relation to dose administration:
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, 6, 13 and 20 after mating and Days 1 and 6 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 behaviour 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.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered males in each group during Week 5 of treatment and on the five lowest numbered lactating females in each group during Days 4 6 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 length of separation of dam from litter was minimised. 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 during Days 4-6 of lactation for females, the motor activity of five lowest numbered surviving males and the five lowest numbered lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.5), 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:
Before dosing on the day that treatment commenced (Week 0) and weekly thereafter.
On the day of necropsy.

F0 females
Before dosing on the day that treatment commenced (Week 0) and weekly before pairing.
Days 0, 6, 13 and 20 after mating.
Day 1, 4, and 7 of lactation.
On the day of necropsy.

Food consumption
The weight of food supplied, that remaining and an estimate of any spilled was recorded as follows:
F0 animals
Weekly, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
For females after mating food consumption was performed to match the body weight recording:
Days 0-5, 6-12 and 13-19 after mating
Days 1-3 and 4-6 of lactation

From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

Haematology, peripheral blood
Blood samples were collected after overnight withdrawal of food and before dose administration at the following occasion:
Week 2 of treatment (before pairing): The five lowest numbered surviving males and females per group


Animals were held under light general anaesthesia 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 analyser:
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)
Morphology:
Anisocytosis
Microcytosis
Macrocytosis
Hypochromasia
Hyperchromasia

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 an ACL 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.

Blood chemistry
At the same time and using the same animals as for peripheral haematology, further blood samples were collected after overnight withdrawal of food and before dose administration at the following occasion:
Week 2 of treatment (before pairing): The five lowest numbered surviving males and females per group


Animals were held under light general anaesthesia 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 Analyser in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acid (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 analysed albumin concentration.

Vaginal smears
Wet smears: After pairing until mating, using pipette lavage.

Mating
Paired for mating: 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.

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.

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 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 1-7 of age.
Sex ratio of each litter: Recorded on Days 1, 4 and 7 of age.
Individual offspring body weights: Days 1, 4 and 7 of age.

Sacrifice and pathology:

Terminal procedures
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.

Time of necropsy
F0 males: After Week 5 investigations completed.
F0 females failing to litter: Day 25 after mating.
F0 females: Day 7 of lactation.
F1 offspring: Day 7 of age.

Method of kill
All adult animals: Carbon dioxide asphyxiation with subsequent exsanguination.
Offspring: Intraperitoneal injection of sodium pentobarbitone.
Sequence: To allow satisfactory inter-group comparison.

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

Offspring
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
Offspring at scheduled termination: Subject to a complete macroscopic examination.

Organ weights
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: Initially in modified Davidson’s fluid.
Eyes: Davidson’s fluid
For all animals examined, samples of any abnormal tissues were retained. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region.

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 surviving F0 males and females in Groups 1 and 4 at scheduled termination.
Abnormalities only: All F0 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
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

Other examinations:

None

Statistics:

See below

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Adult animal responses
Signs and mortality
Treatment of males and female at all doses of FAT 36152/M TE was associated with blue staining on various parts of the body and blue staining on the cage bedding.
One female receiving 50 mg/kg/day was killed for welfare reasons on Day 22 after mating due to apparent dystocia: clinical signs included cold to touch, irregular breathing, red discharge from the vagina and whole body pallor. Macroscopic examination revealed that one fetus and a placenta were stuck in the cervix, and the internal organs and extremities were pale. As there were no cases of dystocia at higher doses, this single case in the low dose group was concluded to be due to chance and unrelated to treatment.
No signs were observed in association with dose administration.

Sensory reactivity and grip strength
During Days 4-6 of lactation, there were three females at 50 mg/kg/day and two females receiving 500 mg/kg/day that showed a weak tail pinch response compared with none in the Control group; in the absence of any other sensory reactivity effects in females or a similar effect on males this is not considered to be related to treatment.
Forelimb grip strength for females receiving 50 mg/kg/day was low when compared with Controls with the difference achieving statistical significance. However, there was no evidence of a dose response and females at 500 mg/kg/day had values that were similar to Controls which suggests that the difference observed at 50 mg/kg/day is due to natural variation.
Group mean sensory reactivity and grip strength values for all groups of treated males during Week 5 of treatment were similar to Controls and considered to be unaffected by treatment.

Motor activity
Overall, group mean activity scores for all treated males and females were similar to the Control values. The high beam score (rearing activity) for males receiving 50 mg/kg/day at the 36-minute time interval was slightly but significantly high when compared with Controls, but this was an isolated incidence and scores at 150 and 500 mg/kg/day were similar to Controls; this difference was therefore attributed to natural variation.

Body weight
Body weight gain for males during the treatment period and for females before pairing was low at 150 or 500 mg/kg/day; all differences attained statistical significance except for females at 150 mg/kg/day. Bodyweight gain at 50 mg/kg/day was unaffected by treatment
There was no effect of treatment on body weight gain of females during gestation or during lactation.

Food consumption
Food consumption for males receiving 150 or 500 mg/kg/day was slightly low during Weeks 1 and 5 of treatment. Food consumption for females at 500 mg/kg/day was slightly low during Week 1 of treatment.
There were no adverse effects of treatment on food consumption of females during gestation or early lactation; females receiving 500 mg/kg/day showed slightly high consumption during Days 1 to 3 of lactation.

Haematology
During Week 2 of treatment animals receiving FAT 36152/M TE showed high reticulocyte counts when compared with Controls, with the difference attaining statistical significance for males at each dose level (p<0.01) and for females at 150 or 500 mg/kg/day (p<0.05 and p<0.01, respectively). In addition females receiving 500 mg/kg/day also showed significantly low haematocrit, haemoglobin and red blood cell count (p<0.01, p<0.01 and p<0.05, respectively) whilst the red cell distribution width and platelet counts were significantly high when compared with Controls (p<0.01).
Other differences were considered slight and of no toxicological significance.

Blood chemistry
On separation of the plasma it was noted that the plasma for animals receiving 150 or 500 mg/kg/day was discoloured (blue).
Bile acid levels for both males and females receiving FAT 36152/M TE were low when compared with Controls with the differences attaining statistical significance for males at 500 mg/kg/day (p<0.01) and for females at all dose levels (p<0.05); a dose response was not apparent.
In addition potassium levels were high for treated males and A/G ratios were slightly high for females at 150 and 500 mg/kg/day; however for both of these parameters these differences were not seen in the other sex.

Pre-coital interval, mating performance and fertility
All females mated within 4 days of pairing at the first oestrus, with no apparent adverse effect on either mating performance or fertility.

Gestation length
All females had gestation lengths within the normal range of 22 to 23.5 days, however at 500 mg/kg/day there was a significant shift toward a shorter gestation length (p<0.01). The distribution of gestation lengths for females at 50 or 150 mg/kg/day were not dissimilar to the Controls and were therefore considered to be unaffected by treatment.
At 50 mg/kg/day one female (no. 53) was killed prematurely on Day 22 of gestation with signs of dystocia (extended parturition, low body temperature, irregular breathing, and pallor); macroscopic examination revealed that a fetus and placenta were lodged in the cervix. There were no other females in the higher dose groups that showed any problems during parturition, this incidence in the low dose group was therefore isolated and considered to be unrelated to treatment; the gestation index was therefore considered to be unaffected by treatment.


F1 litter responses
Offspring clinical signs
Clinical signs at routine examination that were attributed to treatment were limited to blue/grey discolouration of the offspring at 500 mg/kg/day; this was considered to be related to the colour of the test item.

Litter size, sex ratio and survival indices
One female in each of the 50 or 500 mg/kg/day groups did not litter and were subsequently confirmed to be not pregnant.
The mean number of implantations and litter size were slightly high at 150 and 500 mg/kg/day when compared with Controls; with statistical significance attained at 500 mg/kg/day (p<0.05).
There was no effect of treatment on survival of the offspring or the sex ratio (% males).

Offspring body weight
At 150 or 500mg/kg/day mean body weights of male and female offspring on Day 1 of age and the subsequent growth of the offspring were low when compared with Controls, with differences attaining statistical significance for the majority of the data points.
Review of the individual litter mean values and live litter size did show some correlation with the larger litter sizes showing lower offspring body weights; however there was some evidence that the low offspring weights were not solely attributable to live litter size. Therefore in the context of this screening study an involvement of treatment with the low offspring bodyweight at 150 and 500 mg/kg/day cannot be ruled out.
Offspring body weight at 50 mg/kg/day was unaffected by parental treatment


Terminal examinations
Parental Organ weights
When compared with Controls the mean adjusted kidney weights were high for males that received FAT 36152/M TE (p<0.05-0.01) and at 500 mg/kg/day the mean adjusted liver weight of males was high (p<0.05).
On Day 7 of lactation the mean adjusted spleen weights of females that received 150 or 500mg/kg/day were high when compared with the Controls; a dose response was apparent (p<0.05 and p<0.01, respectively).

Parental Macropathology
Five males receiving 150 mg/kg/day and 3 males that received 500 mg/kg/day had blue contents in the urinary bladder.
Four females that received 500 mg/kg/day had blue adipose tissue.
Blue stained fur was apparent for one male that received 50 mg/kg/day, 6/10 males and 6/10 females that received 150 mg/kg/day and all males and females that received 500 mg/kg/day.

Offspring macropathology
Macroscopic examination of offspring that either died before scheduled termination or were killed at scheduled termination on Day 7 of age, revealed an increased incidence of abnormal colour skin/milk at 500 mg/kg/day; this was attributed to the colour of the test item.

Histopathology
Microscopic changes related to treatment with FAT 36152/M TE were seen in the kidneys:
Increased hyaline droplet formation (minimal to slight severity) was present bilaterally in the kidneys of all males that received 500 mg/kg/day. Increased accumulations of hyaline drops were present in the cytoplasm of the tubular epithelium lining the proximal tubules in the renal cortex.

The following finding was considered to be incidental:
A minimal inflammatory cell infiltrate was present in the urinary bladder of three females that received 500 mg/kg/day FAT 36152/M TE. The infiltrate consisted of small perivascular foci of lymphocytes in the submucosal tissue. It is likely that the appearance of these changes in FAT 36152/M TE treated animals and not in Control animals is a chance event and not related to administration of the test item.

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Haematological effects and spleen weight for females were observed at 500 mg/kg/day

Critical effects observed:

not specified

The mean concentrations of FAT 36152/M TE in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation and the difference from mean values were within 1% confirming precise analysis.

Conclusions:

The no observed adverse effect level (NOAEL) for general systemic toxicity was 150 mg/kg/day (haematological effects and spleen weight for females at 500 mg/kg/day).

Executive summary:

The general systemic toxicity potential of FAT 36152/M TE (a textile dye) was evaluated in a study conducted according to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test).

Three groups comprising of ten male and ten female rats received FAT 36152/M TE at doses of 50, 150 or 500 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, throughout pairing and 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 6 of lactation. Females were killed on Day 7 of lactation. The F1 generation received no direct administration of the test substance; any exposure wasin uteroor via the milk. A similarly constituted Control group received the vehicle, 1% w/v methylcellulose, at the same volume dose as the treated groups. 

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. 

The clinical condition, litter size and survival, sex ratio, body weight and macropathology for all offspring were also assessed. 

 

Results:

One female receiving 50 mg/kg/day was killed for welfare reasons on Day 22 after mating with dystocia: clinical signs included cold to touch, irregular breathing, red discharge from the vagina and whole body pallor. Macroscopic examination revealed that one fetus and a placenta were stuck in the cervix, and the internal organs and extremities were pale. As there were no cases of dystocia at higher dose levels, this single case in the low dose group was concluded to be due to chance and unrelated to treatment.

Treatment of males and female at all doses of FAT 36152/M TE was associated with blue staining on various parts of the body. No signs were observed in association with dose administration.

Sensory reactivity, grip strength and motor activity showed no adverse effects of treatment.

Body weight gain for males during the treatment period and for females before pairing was low at 150 or 500 mg/kg/day. Body weight gain for animals at 50 mg/kg/day and for treated females at all dose levels during gestation and lactation was unaffected by treatment.

Food consumption for males receiving 150 or 500 mg/kg/day was slightly low during Weeks 1 and 5 of treatment and consumption for females at 500 mg/kg/day was slightly low during Week 1 of treatment. There were no adverse effects of treatment on food consumption for females during gestation or early lactation; females receiving 500 mg/kg/day showed slightly high consumption during Days 1 to 3 of lactation.

During Week 2 of treatment animals receiving FAT 36152/M TE showed high reticulocyte counts when compared with Controls. In addition females receiving 500 mg/kg/day also showed significantly low hematocrit, hemoglobin and red blood cell count and significantly high red cell distribution width and platelet counts.

Bile acid levels for both males and females receiving FAT 36152/M TE were low when compared with Controls. In addition potassium levels were high for treated males and A/G ratios were slightly high for females at 150 and 500 mg/kg/day.

There were no adverse effects on pre-coital interval, mating performance, fertility or gestation index.

All females had gestation lengths within the normal range of 22 to 23.5 days, however at 500 mg/kg/day there was a significant shift toward a shorter gestation length.

Routine physical examination of the offspring showed an increased incidence of blue/grey discolouration at 500 mg/kg/day.

There was no effect of treatment on the mean number of implantations, litter size, the survival of the offspring or the sex ratio (% males).

At 500 mg/kg/day, the offspring body weight on Day 1 of age and subsequent weight gain was marginally lower than expected.

After 5 weeks of treatment males at all dose levels showed high mean adjusted kidney weights and males at 500 mg/kg/day had high mean adjusted liver weight when compared with Controls.

On Day 7 of lactation the mean adjusted spleen weights of females that received 150 or 500mg/kg/day were high when compared with the Controls.

Macroscopic findings at necropsy included blue content in the urinary bladder content for males at 150 and 500 mg/kg/day, blue adipose for females that received 500 mg/kg/day and there was a dose related incidence of blue coat staining. Macroscopic examination of offspring revealed an increased incidence of abnormal colour skin/milk at 500 mg/kg/day.

Microscopic changes related to treatment with FAT 36152/M TE were seen in the kidneys of all males that received 500 mg/kg/day with increased hyaline droplet formation (minimal to slight severity) present bilaterally; increased accumulations of hyaline drops were present in the cytoplasm of the tubular epithelium lining the proximal tubules in the renal cortex.

Conclusion

Administration of FAT 36152/M TE at dose levels of 50, 150 and 500 mg/kg/day was well tolerated with no adverse effects on clinical condition, sensory reactivity, grip strength, motor activity, mating performance, fertility, parturition, gestation index or offspring survival.

At 500 mg/kg/day haematological findings and spleen weights for parental female animals were indicative of anaemia and at 500 mg/kg/day offspring body weight on day 1 of age and subsequent gain up to Day 7 of age was marginally low.

It was therefore concluded that in this screening study the no observed adverse effect level (NOAEL) for

· general systemic toxicity was 150 mg/kg/day (haematological effects and spleen weight for females at 500 mg/kg/day)

· mating performance, fertility and offspring survival was 500 mg/kg/day offspring development was 500 mg/kg/day(marginally low offspring bodyweights and weight gain but no effect on survival, clinical condition or macropathology)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,11-diamino-2-(3-methoxypropyl)-1H-naphth[2,3-f]isoindol-1,3,5,10(2H)-tetrone

EC Number:

235-402-7

EC Name:

4,11-diamino-2-(3-methoxypropyl)-1H-naphth[2,3-f]isoindol-1,3,5,10(2H)-tetrone

Cas Number:

12217-80-0

Molecular formula:

C20H17N3O5

IUPAC Name:

4,11-diamino-2-(3-methoxypropyl)-1H-naphtho[2,3-f]isoindole-1,3,5,10(2H)-tetrone

Test material form:

solid: particulate/powder

Details on test material:

None

Specific details on test material used for the study:

None

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal supply, acclimatisation and allocation
Strain/Species: RccHan™; WIST rat.
Supplier: Envigo RMS Ltd.
Number of animals : 44 males and 44 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatisation: Five days before commencement of treatment.
Age of animals at the start of the study:
Males: 69 to 75 days old
Females: 62 to 68 days old.
Weight range of animals at the start of the study:
Males: 281 to 313 g
Females. 153 to 196 g.

Allocation and identification
Allocation: On arrival, by non-selective allocation to cages.
On Day 1 of study all animals were weighed and body weights were reviewed by Study Management before dosing commenced. Body weight of animals did not exceed ±20% of the mean for each sex.
Identification of animals: Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo 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 colour-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal replacement
Individuals rejected during the acclimatisation period were replaced with spare animals of suitable weight from the same batch; the following replacements were performed:
Two males: To reduce inter/intra group bodyweight variation.
One female: At the extreme of the body weight range

Animal housing, diet and water supply
Environmental control
Rodent facility: Full barrier - to minimise entry of external biological and chemical agents and to minimise 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, with the exception that the humidity was recorded as 36% on the last day of necropsy.
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 acclimatisation, 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 equalise, as far as possible, environmental influences amongst the groups.
Bedding : Solid bottom cages contained softwood based bark-free fibre bedding, which was changed at appropriate intervals each week.

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

Environmental enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.

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

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% w/v methylcellulose

Details on exposure:

Treatment groups and doses
Dose levels were selected based on the results of the 14-day preliminary study performed in these laboratories with this compound (Huntingdon Life Sciences Study Number TMR0051). In the preliminary study there was no evidence of overt toxicity during routine physical examinations or following dose administration. Bodyweight and food consumption at dose levels up to and including 350 mg/kg/day showed no adverse effects of treatment. At 500 mg/kg/day overall body weight gain for males was slightly low when compared with the weight gain observed at 350 mg/kg/day, with some females at 500 mg/kg/day showing body weight loss. At macroscopic observation findings were limited to blue staining of the adipose tissue for animals that received 350 followed by 500 mg/kg/day and these animals also showed marginally low liver weights. It was therefore considered that a high dose of 500 mg/kg/day would be appropriate for this combined repeat dose toxicity study and reproductive/developmental toxicity screening study; with low and intermediate doses of 50 and 150 mg/kg/day, respectively.

Formulation
Correction factor: A correction factor of 1.1 was used when calculating quantities of test substance used during dose preparation.
Method of preparation : The test material was ground in a mortar and pestle to a fine powder. Small amounts of vehicle were added and mixed to produce a smooth paste. It was made up to volume with vehicle and mixed using a high shear homogeniser until homogenous and finally mixed using a magnetic stirrer.

A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test substance.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated.
Test substance accounting: Detailed records of compound usage were maintained. The amount of test substance 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.

Formulation analysis
Stability and homogeneity: The dose form at 1 and 50 mg/mL (in terms of nominal concentration) was shown to be homogenous and stable for 15 days when stored refrigerated or two days when stored at ambient temperature.
Achieved concentratio: Samples of each formulation prepared for administration in the first and last weeks of treatment were analysed for achieved concentration of the test substance.


Administration
Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg.
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 the treated groups.
Frequency: Once daily, at approximately the same time each day.
Sequence: Groups dosed in ascending order.
Formulation: Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Storage of formulation: Refrigerated.

Details on mating procedure:

Paired for mating: 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:

This report describes the analytical and sampling procedures used and details the results obtained for:
The validation of the analytical procedure (FIA/M041/15) established at Envigo for the determination of FAT 36152/M TE in 1% w/v methylcellulose formulations.
The homogeneity and stability, determined with respect to the level of concentration, of FAT 36152/M TE in 1% w/v methylcellulose at nominal concentrations of 1 mg/mL and 50 mg/mL.
The concentrations of FAT 36152/M TE in test formulations analysed during the study.
The formulations for this study were prepared by Pharmacy personnel. The analytical work was undertaken by Formulation & Inhalation Analysis personnel between 09 April 2015 and 22 August 2015.

Analytical procedure
Apparatus and instrumentation
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Balances fitted with printers: Capability of weighing to 5 or 6 decimal places
General laboratory apparatus and glassware.

Reagents
Control vehicle: 1% w/v methylcellulose
Acetonitrile: HPLC gradient grade
N,N-Dimethylformamide (DMF): HPLC grade
Propyl-4-hydroxy-benzoate (propyl paraben): >99%
Water: Reverse osmosis
Diluent: DMF
Internal standard: 10 mg propyl paraben in DMF (100 mL)

Preparation of standards
A primary standard solution (100 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 5 mg) of FAT 36152/M TE in DMF (50 mL). Solutions for instrument calibration were prepared by appropriate dilution of the primary standard solution using DMF containing 0.5 mL of stock internal standard solution and contained FAT 36152/M TE at nominal concentrations of 2 μg/mL, 4 μg/mL, 6 μg/mL, 8 μg/mL and 10 μg/mL.
Calibration solutions were injected onto the HPLC, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the chromatographic section.

Sample process
A representative sample of test formulation (1 mL, accurately weighed) was dissolved using ultrasonic vibration in DMF. The extract was diluted using DMF to provide a solution containing FAT 36152/M TE at an expected concentration within the range 4 μg/mL to 8 μg/mL and internal standard at a concentration of 5 μg/mL.
The concentration of FAT 36152/M TE in the final solution was quantified by HPLC using UV detection as detailed in the chromatographic section.

Typical chromatographic conditions
Column: NUCLEODUR C18 Isis, 3 μm, 4.6 × 250 mm
Column temperature: 50°C
Sample temperature: Ambient
Mobile Phase A: Acetonitrile/water 10/90 v/v
Mobile Phase B: Acetonitrile/water 90/10 v/v

Linear Gradient:
Time (min.) % A % B
0 50 50
8.0 10 90
12 10 90
13 50 50
16 50 50
Flow rate: 1.0 mL/min
Detector wavelength: UV, 254 nm
Injection volume: 5 μL
Run time: 16 minutes
Approximate retention time:
FAT 36152/M TE: 10.0 minutes
Internal standard: 4.9 minutes

Calculation
The peak area response ratio (test item/internal standard) for FAT 36152/M TE in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response ratio versus calibration standard concentration. The concentration of FAT 36152/M TE was determined using the following equation:

Analysed concentration (mg/mL) = ((Y – I) / S) × (V/W) × (D / 1000)

Where
Y = Peak area response ratio for FAT 36152/M TE in test chromatogram
I = Intercept derived from linear regression of calibration data
S = Slope derived from linear regression of calibration data
V = Dilution factor of sample (mL)
W = Sample weight (g)
D = Density (g/mL)

Validation of the analytical procedure
The analytical procedure was validated by determining the following parameters:
The specificity of the chromatographic analysis in control sample chromatograms.
The limit of detection and quantification was estimated by examination of control vehicle chromatograms in order to calculate a test substance concentration based on a peak height response equivalent to three times and ten times baseline noise respectively.
The linearity of detector response over the calibration standard concentration range.
The repeatability of the lowest and highest concentration calibration standards.
The method accuracy and precision, by determining six procedural recoveries at nominal concentrations of 1 mg/mL and 50 mg/mL during the method validation.
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (1% w/v methylcellulose) with known amounts of FAT 36152/M TE. The prepared procedural recoveries were analysed in accordance with the analytical procedure.

Homogeneity and stability in 1% w/v methylcellulose formulations
The homogeneity and stability of FAT 36152/M TE in 1% w/v methylcellulose formulations was assessed at nominal concentrations of 1 mg/mL and 50 mg/mL, during ambient temperature and refrigerated storage. Freshly prepared specimen formulations (400 mL) were equally sub-divided (4 × 100 mL) into four amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Ambient temperature storage (nominally +21ºC)
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 2 hours, single samples (nominally 1 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 2 days storage the contents were remixed and sampled as detailed above.

Refrigerated storage (nominally +4ºC)
The remaining bottles were refrigerated on receipt and on Day 2 and Day 15, the bottles were 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 1 mL) were removed for analysis from the top, middle and bottom of the continuously stirred formulation.

Concentration in test formulations
For the First and Last week of treatment, freshly prepared test formulations were sampled (4 × 1 mL, 2 × 3 mL for controls accurately weighed) by Pharmacy personnel and submitted for analysis. Duplicate 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.

Results
Method validation
The analytical procedure was successfully validated for FAT 36152/M TE in 1% w/v methylcellulose with respect to the specificity of chromatographic analysis, limit of detection and quantification, the linearity of detector response, repeatability, method accuracy and precision. Results are summarised below:
The specificity of the HPLC assay was demonstrated by the absence of a peak at the characteristic retention time for FAT 36152/M TE in the control sample chromatogram.
The limit of detection and quantification was estimated as 0.00521 μg/mL and 0.0173 μg/mL respectively.
Linearity was confirmed over the nominal concentration range 2 μg/mL to 10 μg/mL with a coefficient of determination >0.999;
The repeatability was <1% for six replicate injections of standard solutions containing FAT 36152/M TE at nominal concentrations of 2 μg/mL and 10 μg/mL;
Method accuracy and precision were confirmed: a mean procedural recovery value of 102.1% (CV=1.29%, n=6) was obtained for 1 mg/mL and 102.1% (CV=0.52%, n=6) was obtained for 50 mg/mL.

Formulation trial
The homogeneity and stability of FAT 36152/M TE in 1% w/v methylcellulose formulations was assessed with respect to the level of concentration at nominal concentrations of 1 mg/mL and 50 mg/mL.
Stability was confirmed during distribution between the bottles and on re-suspension following storage at ambient temperature for 2 days and refrigeration for up to 15 days. At each time-point, the mean analysed concentration for the single samples from top, middle and bottom remained within 5% of the initial time zero value and the coefficient of variation was less than 4%.
Procedural 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 from Day 15 (50 mg/mL). 3 of 4 procedural recoveries were acceptable and results were reported.

Concentration in dose formulations
The mean concentrations of FAT 36152/M TE in test formulations analysed during the study and the deviation of the mean result from the nominal value. The mean concentrations were within applied limits +10%/-15%, confirming the accuracy of formulation. Difference from mean values were within 1% confirming precise analysis.

Conclusion
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.
The stability was confirmed for FAT 36152/M TE in 1% w/v methylcellulose formulations at nominal concentrations of 1 mg/mL and 50 mg/mL at ambient temperature storage for 2 days and refrigerated storage for up to 15 days.
The mean concentrations of FAT 36152/M TE in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation. Difference from mean values were within 1% confirming precise analysis.

Duration of treatment / exposure:

Males: Two weeks before pairing, throughout pairing and up to necropsy after a minimum of five weeks of treatment.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 6 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Daily

Details on study schedule:

- Age at mating of the mated animals in the study:
Males: Minimum of 83 - 89 days old
Females: Minimum of 76 - 82 days old

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/ sex / dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected based on the results of the 14-day preliminary study performed in these laboratories with this compound (Huntingdon Life Sciences Study Number TMR0051). In the preliminary study there was no evidence of overt toxicity during routine physical examinations or following dose administration. Bodyweight and food consumption at dose levels up to and including 350 mg/kg/day showed no adverse effects of treatment. At 500 mg/kg/day overall body weight gain for males was slightly low when compared with the weight gain observed at 350 mg/kg/day, with some females at 500 mg/kg/day showing body weight loss. At macroscopic observation findings were limited to blue staining of the adipose tissue for animals that received 350 followed by 500 mg/kg/day and these animals also showed marginally low liver weights. It was therefore considered that a high dose of 500 mg/kg/day would be appropriate for this combined repeat dose toxicity study and reproductive/developmental toxicity screening study; with low and intermediate doses of 50 and 150 mg/kg/day, respectively.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

Clinical and behavioural observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of 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.

Signs associated with dosing
Daily during Week 1 of treatment and once each week thereafter and for females on Days 0, 6, 13 and 20 after mating and Day 1 and 6 of lactation, detailed observations were recorded at the following times in relation to dose administration:
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, 6, 13 and 20 after mating and Days 1 and 6 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 behaviour 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.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered males in each group during Week 5 of treatment and on the five lowest numbered lactating females in each group during Days 4 6 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 length of separation of dam from litter was minimised. 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 during Days 4-6 of lactation for females, the motor activity of five lowest numbered surviving males and the five lowest numbered lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.5), 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:
Before dosing on the day that treatment commenced (Week 0) and weekly thereafter.
On the day of necropsy.

F0 females
Before dosing on the day that treatment commenced (Week 0) and weekly before pairing.
Days 0, 6, 13 and 20 after mating.
Day 1, 4, and 7 of lactation.
On the day of necropsy.

Food consumption
The weight of food supplied, that remaining and an estimate of any spilled was recorded as follows:
F0 animals
Weekly, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
For females after mating food consumption was performed to match the body weight recording:
Days 0-5, 6-12 and 13-19 after mating
Days 1-3 and 4-6 of lactation

From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

Haematology, peripheral blood
Blood samples were collected after overnight withdrawal of food and before dose administration at the following occasion:
Week 2 of treatment (before pairing): The five lowest numbered surviving males and females per group

Animals were held under light general anaesthesia 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 analyser:
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)
Morphology:
Anisocytosis
Microcytosis
Macrocytosis
Hypochromasia
Hyperchromasia

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 an ACL 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.

Blood chemistry
At the same time and using the same animals as for peripheral haematology, further blood samples were collected after overnight withdrawal of food and before dose administration at the following occasion:
Week 2 of treatment (before pairing): The five lowest numbered surviving males and females per group


Animals were held under light general anaesthesia 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 Analyser in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acid (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 analysed albumin concentration.

Vaginal smears
Wet smears: After pairing until mating, using pipette lavage.

Mating
Paired for mating: 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.

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):

No data

Sperm parameters (parental animals):

No data

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 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 1-7 of age.
Sex ratio of each litter: Recorded on Days 1, 4 and 7 of age.
Individual offspring body weights: Days 1, 4 and 7 of age.

Postmortem examinations (parental animals):

Terminal procedures
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.

Time of necropsy
F0 males: After Week 5 investigations completed.
F0 females failing to litter: Day 25 after mating.
F0 females: Day 7 of lactation.

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

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

Organ weights
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: Initially in modified Davidson’s fluid.
Eyes: Davidson’s fluid
For all animals examined, samples of any abnormal tissues were retained. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region.

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 surviving F0 males and females in Groups 1 and 4 at scheduled termination.
Abnormalities only: All F0 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
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.

Postmortem examinations (offspring):

Time of necropsy
F1 offspring: Day 7 of age.

Method of kill
Offspring: Intraperitoneal injection of sodium pentobarbitone.
Sequence: To allow satisfactory inter-group comparison.

Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
Offspring at scheduled termination: Subject to a complete macroscopic examination.

Statistics:

None

Reproductive indices:

Reproductive indices
Mating performance and fertility

Individual data was tabulated. Group values were calculated for males and females separately for the following:

Percentage mating (%) = (Number animals mating / Animals paired) x 100

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

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

Offspring viability indices:

Off spring viability indices
Litter size
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1 and 7 of age. Group mean litter size and SD were calculated from the individual litter values.

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 7 / Number live offspring on Day 1 after littering) 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 and 7 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

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Refer 'details on results'

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Refer 'details on results'

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Refer 'details on results'

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Refer 'details on results'

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

Adult animal responses
Signs and mortality
Treatment of males and female at all doses of FAT 36152/M TE was associated with blue staining on various parts of the body and blue staining on the cage bedding.
One female receiving 50 mg/kg/day was killed for welfare reasons on Day 22 after mating due to apparent dystocia: clinical signs included cold to touch, irregular breathing, red discharge from the vagina and whole body pallor. Macroscopic examination revealed that one fetus and a placenta were stuck in the cervix, and the internal organs and extremities were pale. As there were no cases of dystocia at higher doses, this single case in the low dose group was concluded to be due to chance and unrelated to treatment.
No signs were observed in association with dose administration.

Sensory reactivity and grip strength
During Days 4-6 of lactation, there were three females at 50 mg/kg/day and two females receiving 500 mg/kg/day that showed a weak tail pinch response compared with none in the Control group; in the absence of any other sensory reactivity effects in females or a similar effect on males this is not considered to be related to treatment.
Forelimb grip strength for females receiving 50 mg/kg/day was low when compared with Controls with the difference achieving statistical significance. However, there was no evidence of a dose response and females at 500 mg/kg/day had values that were similar to Controls which suggests that the difference observed at 50 mg/kg/day is due to natural variation.
Group mean sensory reactivity and grip strength values for all groups of treated males during Week 5 of treatment were similar to Controls and considered to be unaffected by treatment.

Motor activity
Overall, group mean activity scores for all treated males and females were similar to the Control values. The high beam score (rearing activity) for males receiving 50 mg/kg/day at the 36-minute time interval was slightly but significantly high when compared with Controls, but this was an isolated incidence and scores at 150 and 500 mg/kg/day were similar to Controls; this difference was therefore attributed to natural variation.

Body weight
Body weight gain for males during the treatment period and for females before pairing was low at 150 or 500 mg/kg/day; all differences attained statistical significance except for females at 150 mg/kg/day. Bodyweight gain at 50 mg/kg/day was unaffected by treatment
There was no effect of treatment on body weight gain of females during gestation or during lactation.

Food consumption
Food consumption for males receiving 150 or 500 mg/kg/day was slightly low during Weeks 1 and 5 of treatment. Food consumption for females at 500 mg/kg/day was slightly low during Week 1 of treatment.
There were no adverse effects of treatment on food consumption of females during gestation or early lactation; females receiving 500 mg/kg/day showed slightly high consumption during Days 1 to 3 of lactation.

Haematology
During Week 2 of treatment animals receiving FAT 36152/M TE showed high reticulocyte counts when compared with Controls, with the difference attaining statistical significance for males at each dose level (p<0.01) and for females at 150 or 500 mg/kg/day (p<0.05 and p<0.01, respectively). In addition females receiving 500 mg/kg/day also showed significantly low haematocrit, haemoglobin and red blood cell count (p<0.01, p<0.01 and p<0.05, respectively) whilst the red cell distribution width and platelet counts were significantly high when compared with Controls (p<0.01).
Other differences were considered slight and of no toxicological significance.

Blood chemistry
On separation of the plasma it was noted that the plasma for animals receiving 150 or 500 mg/kg/day was discoloured (blue).
Bile acid levels for both males and females receiving FAT 36152/M TE were low when compared with Controls with the differences attaining statistical significance for males at 500 mg/kg/day (p<0.01) and for females at all dose levels (p<0.05); a dose response was not apparent.
In addition potassium levels were high for treated males and A/G ratios were slightly high for females at 150 and 500 mg/kg/day; however for both of these parameters these differences were not seen in the other sex.

Pre-coital interval, mating performance and fertility
All females mated within 4 days of pairing at the first oestrus, with no apparent adverse effect on either mating performance or fertility.

Gestation length
All females had gestation lengths within the normal range of 22 to 23.5 days, however at 500 mg/kg/day there was a significant shift toward a shorter gestation length (p<0.01). The distribution of gestation lengths for females at 50 or 150 mg/kg/day were not dissimilar to the Controls and were therefore considered to be unaffected by treatment.
At 50 mg/kg/day one female (no. 53) was killed prematurely on Day 22 of gestation with signs of dystocia (extended parturition, low body temperature, irregular breathing, and pallor); macroscopic examination revealed that a fetus and placenta were lodged in the cervix. There were no other females in the higher dose groups that showed any problems during parturition, this incidence in the low dose group was therefore isolated and considered to be unrelated to treatment; the gestation index was therefore considered to be unaffected by treatment.

Terminal examinations
Parental Organ weights
When compared with Controls the mean adjusted kidney weights were high for males that received FAT 36152/M TE (p<0.05-0.01) and at 500 mg/kg/day the mean adjusted liver weight of males was high (p<0.05).
On Day 7 of lactation the mean adjusted spleen weights of females that received 150 or 500mg/kg/day were high when compared with the Controls; a dose response was apparent (p<0.05 and p<0.01, respectively).

Parental Macropathology
Five males receiving 150 mg/kg/day and 3 males that received 500 mg/kg/day had blue contents in the urinary bladder.
Four females that received 500 mg/kg/day had blue adipose tissue.
Blue stained fur was apparent for one male that received 50 mg/kg/day, 6/10 males and 6/10 females that received 150 mg/kg/day and all males and females that received 500 mg/kg/day.

Histopathology
Microscopic changes related to treatment with FAT 36152/M TE were seen in the kidneys:
Increased hyaline droplet formation (minimal to slight severity) was present bilaterally in the kidneys of all males that received 500 mg/kg/day. Increased accumulations of hyaline drops were present in the cytoplasm of the tubular epithelium lining the proximal tubules in the renal cortex.

The following finding was considered to be incidental:
A minimal inflammatory cell infiltrate was present in the urinary bladder of three females that received 500 mg/kg/day FAT 36152/M TE. The infiltrate consisted of small perivascular foci of lymphocytes in the submucosal tissue. It is likely that the appearance of these changes in FAT 36152/M TE treated animals and not in Control animals is a chance event and not related to administration of the test item.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on mating performance, fertility and offspring survival.

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

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:

no effects observed

Haematological findings:

no effects observed

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

Histopathological findings:

no effects observed

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

F1 litter responses
Offspring clinical signs
Clinical signs at routine examination that were attributed to treatment were limited to blue/grey discolouration of the offspring at 500 mg/kg/day; this was considered to be related to the colour of the test item.

Litter size, sex ratio and survival indices
One female in each of the 50 or 500 mg/kg/day groups did not litter and were subsequently confirmed to be not pregnant.
The mean number of implantations and litter size were slightly high at 150 and 500 mg/kg/day when compared with Controls; with statistical significance attained at 500 mg/kg/day (p<0.05).
There was no effect of treatment on survival of the offspring or the sex ratio (% males).

Offspring body weight
At 150 or 500mg/kg/day mean body weights of male and female offspring on Day 1 of age and the subsequent growth of the offspring were low when compared with Controls, with differences attaining statistical significance for the majority of the data points.
Review of the individual litter mean values and live litter size did show some correlation with the larger litter sizes showing lower offspring body weights; however there was some evidence that the low offspring weights were not solely attributable to live litter size. Therefore in the context of this screening study an involvement of treatment with the low offspring bodyweight at 150 and 500 mg/kg/day cannot be ruled out.
Offspring body weight at 50 mg/kg/day was unaffected by parental treatment

Offspring macropathology
Macroscopic examination of offspring that either died before scheduled termination or were killed at scheduled termination on Day 7 of age, revealed an increased incidence of abnormal colour skin/milk at 500 mg/kg/day; this was attributed to the colour of the test item.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Formulation analysis

The mean concentrations of FAT 36152/M TE in test formulations analysed for the study were within +10 %/ -15 % of nominal concentrations, confirming accurate formulation and the difference from mean values were within 1 % confirming precise analysis.

Histopathology

Summary of treatment related findings in the kidneys for males killed after 5 weeks of treatment and females on Day 7 of lactation
Group/sex	1M	4M	1F	4F
Dose (mg/kg/day)	0	500	0	500
Accumulation, Hyaline Droplets
Minimal	0	2	0	0
Slight	0	3	0	0
Total	0	5	0	0
Number of tissues examined	5	5	5	5

Applicant's summary and conclusion

Conclusions:

No observed adverse effect level (NOAEL) for mating performance, fertility and offspring survival was 500 mg/kg/day.

Executive summary:

The reproductive toxicity potential of FAT 36152/M TE (a textile dye) was evaluated in a study conducted according to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test).

Three groups comprising of ten male and ten female rats received FAT 36152/M TE at doses of 50, 150 or 500 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, throughout pairing and 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 6 of lactation. Females were killed on Day 7 of lactation. The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle,
1% w/v methylcellulose, at the same volume dose as the treated groups. 

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. 

The clinical condition, litter size and survival, sex ratio, body weight and macropathology for all offspring were also assessed. 

Results

One female receiving 50 mg/kg/day was killed for welfare reasons on Day 22 after mating with dystocia: clinical signs included cold to touch, irregular breathing, red discharge from the vagina and whole body pallor. Macroscopic examination revealed that one fetus and a placenta were stuck in the cervix, and the internal organs and extremities were pale. As there were no cases of dystocia at higher dose levels, this single case in the low dose group was concluded to be due to chance and unrelated to treatment.

Treatment of males and female at all doses of FAT 36152/M TE was associated with blue staining on various parts of the body. No signs were observed in association with dose administration.

Sensory reactivity, grip strength and motor activity showed no adverse effects of treatment.

Body weight gain for males during the treatment period and for females before pairing was low at 150 or 500 mg/kg/day. Body weight gain for animals at 50 mg/kg/day and for treated females at all dose levels during gestation and lactation was unaffected by treatment.

Food consumption for males receiving 150 or 500 mg/kg/day was slightly low during Weeks 1 and 5 of treatment and consumption for females at 500 mg/kg/day was slightly low during Week 1 of treatment. There were no adverse effects of treatment on food consumption for females during gestation or early lactation; females receiving 500 mg/kg/day showed slightly high consumption during Days 1 to 3 of lactation.

During Week 2 of treatment animals receiving FAT 36152/M TE showed high reticulocyte counts when compared with Controls. In addition females receiving 500 mg/kg/day also showed significantly low haematocrit, haemoglobin and red blood cell count and significantly high red cell distribution width and platelet counts.

Bile acid levels for both males and females receiving FAT 36152/M TE were low when compared with Controls. In addition potassium levels were high for treated males and A/G ratios were slightly high for females at 150 and 500 mg/kg/day.

There were no adverse effects on pre-coital interval, mating performance, fertility or gestation index.

All females had gestation lengths within the normal range of 22 to 23.5 days, however at 500 mg/kg/day there was a significant shift toward a shorter gestation length.

Routine physical examination of the offspring showed an increased incidence of blue/grey discolouration at 500 mg/kg/day.

There was no effect of treatment on the mean number of implantations, litter size, the survival of the offspring or the sex ratio (% males).

At 500 mg/kg/day, the offspring body weight on Day 1 of age and subsequent weight gain was marginally lower than expected.

After 5 weeks of treatment males at all dose levels showed high mean adjusted kidney weights and males at 500 mg/kg/day had high mean adjusted liver weight when compared with Controls.

On Day 7 of lactation the mean adjusted spleen weights of females that received 150 or 500mg/kg/day were high when compared with the Controls.

Macroscopic findings at necropsy included blue content in the urinary bladder content for males at 150 and 500 mg/kg/day, blue adipose for females that received 500 mg/kg/day and there was a dose related incidence of blue coat staining. Macroscopic examination of offspring revealed an increased incidence of abnormal colour skin/milk at 500 mg/kg/day.

Microscopic changes related to treatment with FAT 36152/M TE were seen in the kidneys of all males that received 500 mg/kg/day with increased hyaline droplet formation (minimal to slight severity) present bilaterally; increased accumulations of hyaline drops were present in the cytoplasm of the tubular epithelium lining the proximal tubules in the renal cortex.

Conclusion

Administration of FAT 36152/M TE at dose levels of 50, 150 and 500 mg/kg/day was well tolerated with no adverse effects on clinical condition, sensory reactivity, grip strength, motor activity, mating performance, fertility, parturition, gestation index or offspring survival.

At 500 mg/kg/day haematological findings and spleen weights for parental female animals were indicative of anaemia and at 500 mg/kg/day offspring body weight on day 1 of age and subsequent gain up to Day 7 of age was marginally low.

It was therefore concluded that in this screening study the no observed adverse effect level (NOAEL) for:

·        general systemic toxicity was 150 mg/kg/day (haematological effects and spleen weight for females at 500 mg/kg/day)

·        mating performance, fertility and offspring survival was 500 mg/kg/day

.    offspring development was 500 mg/kg/day(marginally low offspring bodyweights and weight gain but no effect on survival, clinical condition or macropathology)