Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 23 June 2015 and 14 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422
Deviations:
yes
Remarks:
See below
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Test substance: FAT 41001-H TE
Test substance identity (including alternative names):
Trisodium 1-amino-4-[[3-[[4-chloro-6-[(sulphonatophenyl) amino]-1,3,5-triazin-2-yl]amino]-2,4,6-trimethyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate
NOVACRON BLUE P-3R (Reactive Blue 049)

CAS number: 72214-18-7
EC number: 276-481-8
Intended use: Textile dye
Appearance: A solid powder
Storage conditions: Frozen (nominally -20°C)
Supplier: Sponsor
Batch number: BOP 01-12 (Lot: BS-1106872)
Expiry date: 31 January 2017
Purity: 77.3%
Purity/weighing factor: 1.29
pH: 7.2
Supplier’s responsibilities: Characterisation of the test substance and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 g representative sample was taken from the batch of test substance. This sample was placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Animal supply, acclimatisation and allocation
Strain/Species: RccHan™; WIST rat.
Supplier: Harlan (UK) 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: 257 to 289 g
Females: 159 to 189 g

Allocation and identification
Allocation : On arrival and non-selective allocation to cages.
On Day 1 of study all animals were weighed and body weights were reviewed before dosing commenced by Study Management. 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 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 19-23ºC and 40-70%. There were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.

Animal accommodation
Cages:
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the 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 of one sex
Pairing: one male and one female
Males after mating: up to five animals
Gestation: one female
Lactation: one female + litter

Environmental enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced 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:
water
Details on exposure:
Vehicle
The vehicle used was purified water.

Correction factor: A correction factor of 1.29 was used when calculating quantities of test substance used during dose preparation (including purity and ratio salt/base).
Vehicle : Purified water.
Method of preparation : Sufficient vehicle was added to the test material to obtain a solution and it was mixed by hand using a spatula. It was mixed using a magnetic stirrer to dissolve the test material. It was made up to volume with the remaining vehicle and mixed with a magnetic stirrer.
A series of solutions at the required concentrations were prepared by dilution of individual weighings of the test substance.
Frequency of preparation: Weekly.
Storage of formulation: Ambient temperature (nominally +21ºC) for two days or refrigerated (nominally +4ºC) for 15 days.
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 stability of a solution of the test substance in the vehicle at concentrations of 10 mg/mL and 100 mg/mL was demonstrated over a period of 15 days at nominally +4ºC and two days at nominally +21ºC.
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: The stability was confirmed following storage at ambient temperature (nominally 21ºC) for up to two days, and refrigeration (nominally 2-8ºC) for up to 15 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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: Purified water
Acetonitrile (ACN): Gradient HPLC grade
N,N-Dimethylformamide (DMF): HPLC grade
Tetra-n-Butylammonium bromide: Reagent plus, >99.0%
Water: Reverse osmosis
Diluent: DMF/water 20/80 v/v

Preparation of standards
A primary standard solution (1000 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of FAT 41001-H TE in diluent (50 mL). A secondary standard solution (250 μg/mL) was prepared by diluting primary standard solution with diluent (20 mL). Solutions for instrument calibration were prepared by appropriate dilution of the secondary standard solution using diluent and contained FAT 41001-H TE at nominal concentrations of 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL and 25 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 (accurate volume for stability trial)) was diluted using diluent to provide a solution containing FAT 41001-H TE at an expected concentration within the range 10 μg/mL to 20 μg/mL.
The concentration of FAT 41001-H TE in the final solution was quantified by HPLC using UV detection as detailed in the chromatographic section.

Typical chromatographic conditions
Column: Hypersil BDS, C18, 5 μm 4.6 × 250 mm
Column temperature: 40°C
Sample temperature: 10°C
Mobile Phase A: Tetra-n-butylammonium bromide (2 g/L) in ACN/water 35/65 v/v
Mobile Phase B: Tetra-n-butylammonium bromide (2 g/L) in ACN/water 90/10 v/v

Linear Gradient:
Time (min.) % A % B
0 100 0
1.0 100 0
20.0 40 60
20.5 100 0
23.0 100 0

Flow rate: 1.2 mL/min
Detector wavelength: UV, 254 nm
Injection volume: 10 μL
Run time: 23 minutes
Approximate retention time:
FAT40061-F TE peak 1: 15.1 minutes
FAT40061-F TE peak 2: 15.3 minutes

Calculation
The peak area response for FAT 41001-H TE in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The concentration of FAT 41001-H TE was determined using the following equation:

Analysed concentration (mg/mL) = ((Y – I) / S) x (V / 1000)

Where
Y = Peak area response for FAT 41001-H 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

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 10 mg/mL and 100 mg/mL during the method validation.
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (purified water) with known amounts of FAT 41001-H TE. The prepared procedural recoveries were analysed in accordance with the analytical procedure.

Stability in purified water formulations
The homogeneity and stability of FAT 41001-H TE in purified water formulations was assessed at nominal concentrations of 10 mg/mL and 100 mg/mL, during ambient temperature and refrigerated storage. Freshly prepared specimen formulations (100 mL) were equally sub-divided (2 × 50 mL) into two 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 was mixed by 20-fold inversion (representing 0 hour) and 2 hours, duplicate samples (1 mL accurate volume) were removed for analysis from the middle of the 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 bottle was refrigerated on receipt and on Day 2 and Day 15, the bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottle were mixed by 20-fold inversion and single samples (1 mL accurate volume) were removed for analysis from the middle of the 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 41001-H TE in purified water 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 41001-H TE in the control sample
chromatogram.
The limit of detection and quantification was estimated as 0.0225 μg/mL and 0.0751 μg/mL respectively.
Linearity was confirmed over the nominal concentration range 5 μg/mL to 25 μg/mL with a coefficient of determination >0.999;
The repeatability was <1% for six replicate injections of standard solutions containing FAT 41001-H TE at nominal concentrations of 5 μg/mL and 25 μg/mL
;
Method accuracy and precision were confirmed: a mean procedural recovery value of 101.2% (CV=0.82%, n=6) was obtained for 10 mg/mL and 99.9% (CV=1.31%, n=6) was obtained for 100 mg/mL.

Formulation trial
The stability of FAT 41001-H TE in purified water formulations was assessed with respect to the level of concentration at nominal concentrations of 10 mg/mL and 100 mg/mL. Stability was confirmed 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 duplicate samples remained within 3% of the initial time zero value and difference from mean values were less than 2%.

Concentration in dose formulations
The mean concentrations of FAT 41001-H TE in test formulations analysed during the study and the deviation of the mean result from the nominal value are presented. The mean concentrations were within applied limits ±10%, confirming the accuracy of formulation. Difference from mean values were within 5% 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 41001-H TE in purified water formulations at nominal concentrations of 10 mg/mL and 100 mg/mL at ambient temperature storage for 2 days and refrigerated storage for up to 15 days.
The mean concentrations of FAT 41001-H TE in test formulations analysed for the study were within ±10% of nominal concentrations, confirming accurate formulation. Difference from mean values were within 5% confirming precise analysis.
Details on mating procedure:
Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Duration of treatment / exposure:
At least four weeks
Frequency of treatment:
Daily
Duration of test:
At least four weeks
No. of animals per sex per dose:
10 / sex / dose
Control animals:
yes
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 TMR0055).
In this preliminary study, FAT 41001 H TE was well tolerated for 14 days at doses up to and including 1000 mg/kg/day. Overall body weight gain and food consumption were satisfactory and there was no evidence of toxicity during routine physical examinations or following dose administration. Kidney weights were marginally high in males given 500 or 1000 mg/kg/day and spleen weights were low amongst males given 1000 mg/kg/day. Macroscopic examination revealed blue or dark colouration in the kidneys, caecum, colon, ileum, jejunum, mesenteric lymph nodes, rectum, stomach and on the fur, tail or paws from the majority of animals which was attributed to the properties of FAT 41001-H TE, which is a blue dye.
Based on these results 1000 mg/kg/day was considered to be an appropriate high dose for this main OECD 422 study. A low and intermediate dose of 100 and 330 mg/kg/day, respectively, have been selected to investigate a dose response and with reference to the Globally Harmonized System of Classification and Labelling of Chemicals (2013).

Examinations

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 at 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 at 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 prior to dosing at the following occasions:
Week 2 prior to pairing: The five lowest numbered surviving males and females per group
Week 4 (to coincide with first gestation Day 17 occasion)#: The five lowest numbered surviving males per group
Day 17 of gestation#: The five lowest numbered surviving females per group
# Citrate samples only

Animals were held under light general anaesthesia induced by isoflurane after overnight withdrawal of food for Week 2 prior to pairing only. 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 prior to dosing at the following occasion:
Week 2 prior to 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
Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

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.

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 produce a viable 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.
The organs weighed, tissue samples fixed and sections examined microscopically are detailed as follows for F0 animals:

Females
The following were recorded:
Each uterine horn: Number of implantation sites.
Offspring: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.

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: In 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 numbered surviving F0 males and females in Groups 1 and 4 at scheduled termination.
Abnormalities only: All F0 animals.
Kidneys, mesenteric and left axillary lymph nodes, stomach and epididymis: The five lowest numbered surviving F0 males and females in Groups 2 and 3 at scheduled termination.
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.
The epididymis of two Control males and two Group 4 males were also be stained with Periodic Acid Schiffs (PAS) in order to confirm results observed at the initial histological examination.

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.
Ovaries and uterine content:
The following were recorded:
Each uterine horn: Number of implantation sites.
Fetal examinations:
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.

Time of necropsy
F1 offspring: Day 7 of age.

Method of kill
Offspring : Intraperitoneal injection of sodium pentobarbitone.
Statistics:
See below
Indices:
See below

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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, non-treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
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
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Maternal developmental toxicity

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:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: See results

Details on maternal toxic effects:
Adult animal responses
Signs
Signs seen in association with the administration of FAT 41001-H TE were restricted to rales in one male receiving 330 mg/kg/day on Day 3 of treatment. This was observed from 1 2 hours after dosing up to the end of the working day but was no longer evident the following day.
At the detailed physical examination and arena observations, signs seen which were considered to be related to the administration of FAT 41001-H TE included blue staining of various body parts of all males receiving FAT 41001-H TE at 330 or 1000 mg/kg/day, all females receiving 1000 mg/kg/day, nine females receiving 330 mg/kg/day and four males receiving 100 mg/kg/day.
Irritable behaviour was observed in two females receiving 100 mg/kg/day. Vocalisation was evident in females across all groups (inclusive of one control female) and for one control males, however, vocalisation had been observed in a small number of females prior to the commencement of treatment.
Salivation was seen in one male and one female receiving 1000 mg/kg/day, however, this is a common sign observed with oral gavage dosing and is not considered to be a direct response to treatment with FAT 41001-H TE.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments did not reveal any response to treatment with FAT 41001-H TE.

Motor activity
Motor activity assessments during Week 5 of treatment, did not reveal any response to treatment in male animals receiving FAT 41001-H TE.
For females treated with FAT 41001-H TE, motor activity assessments between Day 4 and Day 6 of lactation revealed small inter- and intra-group differences in the activity scores. This is not unusual for lactating females that have been separated from their pups and therefore these differences are considered to be a consequence of natural biological variation.

Body weight
Group mean body weight gain for males and females receiving FAT 41001-H TE at 100 mg/kg/day were similar to controls and were considered to be unaffected by treatment.
The group mean weight gain of males receiving FAT 41001-H TE at 1000 mg/kg/day was low during Week 1 of treatment when compared with controls; thereafter until Week 4, the mean weight gains of these animals improved and were similar to the gains of the controls. During Weeks 4-5, group mean weight gain was significantly lower in males receiving FAT 41001-H TE at 330 or 1000 mg/kg/day when compared with controls and subsequently overall weight gain (Weeks 0-5) appeared low in these animals. For females receiving FAT 41001-H TE at 300 or 1000 mg/kg/day, body weight gain during the two weeks of treatment prior to pairing was slightly low when compared with the control animals however these differences were minor and did not demonstrate a clear dose response.
Body weight gain during the gestation period (Days 0-20), for females receiving FAT 41001 H TE at 100, 330 or 1000 mg/kg/day, were similar to that of the Control and showed no effect of treatment.
For the period Days 1-4 of lactation, group mean body weight gain was low in females receiving 100 mg/kg/day, however, this was attributed to a large bodyweight loss (32g) in female No. 56 for this period, resulting in overall lower gains for the period Days 1-7 of lactation. Body weight gain during Days 1-4 of lactation was high at 330 or 1000 mg/kg/day but differences did not attain statistical significance, and were not dose-related.

Food consumption
There was no effect of treatment with FAT 41001-H TE on food consumption during the first two weeks of treatment (prior to pairing) in either sex. During treatment Week 4 males treated with FAT 41001 H TE at 100, 330, or 1000 mg/kg/day appeared to consume more diet than controls however these differences were minor and lacked dose relationship; food consumption was not recorded whilst animals were paired for mating (Week 3).
No effect of treatment on food consumption was observed in treated females, during the period of gestation (Days 0-19). Between Day 6 and Day 19 of gestation, the mean food consumption of all groups of treated females was slightly higher than in controls and all differences attained statistical significance except for food intake at 100 mg/kg/day during Days 13-19 of gestation.
No effect of treatment on food consumption was observed in females receiving 100 mg/kg/day, during the period of lactation (Days 1-7). In females receiving 330 or 1000 mg/kg/day food consumption was slightly higher throughout lactation, with the slight increase being dose dependent.

Water consumption
No formal water consumption measurements/assessments were performed on this study however, on two occasions (at the water bottle change) a marked increase in water consumption was observed amongst animals receiving 1000 mg/kg/day when compared with Controls. These observations were recorded within the study day book, and are not reported in a Table or Appendix.

Haematology
Haematological examination during Week 2 of treatment, prior to pairing, and the examination of the clotting parameters in Week 4 of treatment for the males and on gestation Day 17 for the females, revealed no significant response to treatment with FAT 41001-H TE.
A small, but statistically significant increase in mean cell haemoglobin concentration was apparent in males at all dose levels when compared with Controls. A small but statistically significant shortening of the prothrombin time was apparent in males receiving 1000 mg/kg/day.
All other differences from control were minor, lacked dose relationship and were found to be within background control data ranges; therefore these changes were attributed to normal biological variation.

Blood chemistry
Biochemical examination of the blood plasma in Week 2 of treatment, prior to pairing revealed low bile acid concentrations amongst all treated groups of males and females when compared with the control animals. There was no dose response evident in the females with the most pronounced effects being apparent in females receiving 100 or 330 mg/kg/day. Cholesterol concentration was high in animals receiving 330 or 1000 mg/kg/day and creatinine concentration was high in females at all dose levels. Glucose and urea concentrations were low in males receiving 1000 mg/kg/day when compared with control animals, however, in the case of the urea concentration this was partially attributed to both a high value in one control male (Animal No. 23; plasma urea = 11.62 mmol/L) and a low value in one male receiving 1000 mg/kg/day (Animal No. 1; plasma urea = 5.66 mmol/L); as such these differences in urea concentration were not considered to be of toxicological significance.
High calcium concentration was evident in all treated male groups when compared with the control group; there was no similar effect in the females.
Other changes that attained statistical significance included the low plasma alkaline phosphatase activity in males receiving 1000 mg/kg/day and the low alanine aminotransferase activity in females at all dose levels. As low levels of enzyme activity are not considered to be biologically relevant, no toxicological significance is inferred.

Pre-coital interval, mating performance, fertility and gestation length
There was no effect of treatment with FAT 41001-H TE, on pre-coital interval, mating performance or fertility.
There was no effect of treatment on gestation length or index.

Organ weights
After five weeks of treatment, absolute and body weight adjusted kidney weights of males receiving 1000 mg/kg/day were marginally higher than Controls. Absolute testes weights were also high in these animals and in males receiving 330 mg/kg/day.
On Day 7 of lactation, absolute and body weight adjusted adrenal weights for all treated groups of females were low when compared with Controls, attaining statistical significance for adjusted adrenal weights for all dose levels. Absolute and body weight adjusted kidney weights of females receiving 1000 mg/kg/day, were higher than Controls, attaining statistical significance for adjusted kidney weights only.

Macropathology
Macroscopic examination performed in males after five weeks of treatment and in females on Day 7 of lactation, did not reveal any treatment-related findings for animals receiving FAT 41001-H TE at 100 mg/kg/day.

In males receiving 330 or 1000 mg/kg/day, blue colouration (including blue colouration of the contents) of the rectum, ileum, caecum, colon, stomach, kidneys and mesenteric lymph nodes was evident with the incidence higher at 1000 mg/kg/day. Further blue coloration was evident at 1000 mg/kg/day in the skin, jejunum, lymph nodes (mandibular, pancreatic, inguinal, axillary and lumbar), testes and epididymides. In females receiving 330 or 1000 mg/kg/day, blue colouration (including blue coloration of the contents) of the rectum, caecum, kidneys and colon was evident with the incidence higher at 1000 mg/kg/day. Further blue colouration was evident at 1000 mg/kg/day in the skin (including pinnae), jejunum, ileum, mesenteric lymph nodes, uterus and uterine cervix, vagina, thymus, stomach and duodenum. These findings were considered to be due to the administration of the test article, FAT 41001 H TE, which is a blue dye.
In addition, two females receiving 1000 mg/kg/day were observed to have a dark colon with abnormally dark contents; this is also considered to be in relation to the administration of the test article, FAT 41001-H TE.

Histopathology
For males killed after four weeks of treatment and females killed on Day 7 of lactation, changes related to administration with FAT 41001-H TE were seen in the kidneys, mesenteric and left axillary lymph nodes, stomach and epididymides:

Kidneys
Vacuolation of the cortical tubules with granular deposits was seen in males and females given 330 or 1000 mg/kg/day. Increased severity of the vacuolation was recorded in the females when compared with the males given 1000 mg/kg/day. Hyaline droplets in the cortical tubules were recorded in the males given 330 or 1000 mg/kg/day.

Lymph nodes
Vacuolated macrophages were recorded in the majority of males and females given 1000 mg/kg/day.

Stomach
Foveolar hyperplasia was recorded in the stomach of three males and the majority of females given 1000 mg/kg/day.

Epididymides
Epithelial vacuolation was recorded in the epididymides of males given 1000 mg/kg/day. A PAS stain was carried out in two control males and two males treated with 1000 mg/kg/day. In the controls all clear cells stained strongly PAS positive, whereas in the treated animals only some of the clear cells stained PAS positive.
In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.
All other histological changes were considered to be unrelated to treatment.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: Absence of any evidence for general systemic toxicity or effects on reproductive performance/offspring development

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
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:
not examined
Visceral malformations:
not examined
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Offspring clinical signs
Clinical signs at routine examination that were observed in relation to treatment with FAT 41001 H-TE were limited to dark areas on the lower ventral abdomen of offspring at 100, 330 and 1000 mg/kg/day; in addition, blue staining was observed in offspring at 330 and 1000 mg/kg/day, this was considered to be related to the colour of the test item.

Litter size, sex ratio and survival indices
Litter size, offspring survival and sex ratio were considered to be unaffected by parental exposure to FAT 41001-H TE.
For sex ratio (%M), the group mean Control data were atypically low; consequently the apparent changes from Control for these parameters were considered to be of natural biological variation and not a response to treatment.

Offspring body weight
There was no effect of treatment in offspring body weight on Day 1 of age; growth thereafter was also considered to be unaffected by treatment with FAT 41001-H TE.

Offspring macropathology
At macroscopic examination, findings observed in relation to treatment were confined to dark contents of the gastrointestinal tract of offspring at 1000 mg/kg/day. In addition, one litter at 1000 mg/kg/day were observed to have blue skin, this was considered to be related to the colour of the test item.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of treatment-related effects in the offspring.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Formulation analysis

The stability was confirmed for FAT 41001-H TE in purified water formulations at nominal concentrations of 10 mg/mL and 100 mg/mL at ambient temperature storage for 2 days and refrigerated storage for up to 15 days.  The mean concentrations of FAT 41001-H TE in test formulations analysed for the study were within ±10% of nominal concentrations, confirming accurate formulation. Differences from mean values were within 5% confirming precise analysis.

Histopathology

Kidneys

Summary of treatment related findings in the kidneys for animals killed after four weeks of treatment and females killed on Day 7 of lactation

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

100

330

1000

0

100

330

1000

 

 

 

 

 

 

 

 

 

Cortical Tubular Vacuolation

 

 

 

 

 

 

 

 

Minimal

0

0

5

0

0

0

6

0

Slight

0

0

0

4

0

0

1

1

Moderate

0

0

0

1

0

0

0

4

 

 

 

 

 

 

 

 

 

Total

0

0

5

5

0

0

7

5

 

 

 

 

 

 

 

 

 

Cortical Tubular Hyaline Droplets

 

 

 

 

 

 

 

 

Minimal

0

0

3

3

0

0

0

0

Slight

0

0

0

2

0

0

0

0

 

 

 

 

 

 

 

 

 

Total

0

0

3

5

0

0

0

0

 

 

 

 

 

 

 

 

 

Number of tissues examined

5

5

5

5

5

5

7

5

 

 

 

 

 

 

 

 

 

Lymph nodes

Summary of treatment related findings in the mesenteric and axillary lymph nodes for animals killed after four weeks of treatment and females killed on Day 7 of lactation

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

100

330

1000

0

100

330

1000

 

 

 

 

 

 

 

 

 

Mesenteric lymph nodes

Vacuolated macrophages

 

 

 

 

 

 

 

 

Minimal

0

0

0

2

0

0

0

4

Slight

0

0

0

2

0

0

0

0

Moderate

0

0

0

1

0

0

0

0

Total

0

0

0

5

0

0

0

4

 

Left axillary lymph nodes

 

 

 

 

 

 

 

 

Vacuolated macrophages

 

 

 

 

 

 

 

 

Minimal

0

0

0

4

0

0

0

3

Total

0

0

0

4

0

0

0

3

 

 

 

 

 

 

 

 

 

Number of tissues examined

5

5

5

4

5

5

5

5

 

 

 

 

 

 

 

 

 

Stomach

Summary of treatment related findings in the stomach for animals killed after four weeks of treatment and females killed on Day 7 of lactation

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

100

330

1000

0

100

330

1000

 

 

 

 

 

 

 

 

 

Foveolar hyperplasia

 

 

 

 

 

 

 

 

Minimal

0

0

0

3

0

0

0

4

Total

0

0

0

3

0

0

0

4

 

 

 

 

 

 

 

 

 

Number of tissues examined

5

5

5

5

5

5

5

5

 

 

 

 

 

 

 

 

 

 

Epididymides

Summary of findings in the epididymides for animals killed after four weeks of treatment

Group/sex

1M

2M

3M

4M

Dose (mg/kg/day)

0

100

330

1000

 

 

 

 

 

Epithelial vacuolation

 

 

 

 

Minimal

0

0

0

1

Slight

0

0

0

4

Total

0

0

0

5

 

 

 

 

 

Number of tissues examined

5

6

5

5

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
It was concluded that in the absence of any evidence for general systemic toxicity or effects on reproductive performance/offspring development that the no observed adverse effect level was 1000 mg/kg/day.
Executive summary:

This study was designed to assess the general systemic toxic potential of FAT 41001-H TE (a blue textile dye) in rats, including a screen for reproductive/developmental effects.

Three groups, each comprising ten male and ten female rats received FAT 41001-H TE at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing up to necropsy, after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 6 of lactation. Females were killed on Day 7 of lactation. A similarly constituted Control group received the vehicle (purified water) at the same volume dose as the treated groups. 

The F1 generation received no direct administration of the test substance; any exposure wasin uteroor via the milk.

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, macroscopic pathology and histopathology investigations were undertaken. 

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

Results

Oral administration of FAT 41001-H TE at doses of 100, 330 or 1000 mg/kg/day was generally well tolerated with no mortalities related to treatment. There were no adverse effects attributed to treatment on sensory reaction, grip strength or motor activity; in addition all of the reproductive/developmental endpoints assessed were unaffected by treatment and included, pre-coital interval, mating performance, fertility, gestation length, offspring weights, litter size, sex ratio and offspring survival.

Signs in association with the administration of FAT 41001-H TE were restricted to rales in one male receiving 330 mg/kg/day on Day 3 of treatment. At the detailed physical examination and arena observations, signs seen in relation to treatment were confined to blue staining of various body parts at all dose levels (100, 330 or 1000 mg/kg/day), with the magnitude of incidence increasing as the dose level increased. 

Group mean body weight gain for males and females receiving FAT 41001-H TE at 100 mg/kg/day were similar to controls throughout the study and were considered unaffected by treatment.  In males treated at 330 or 1000 mg/kg/day, overall group mean body weight gain was low when compared with Controls however this was predominately a result of low weight gains in Week 0-1 (males treated at 1000 mg/kg/day) and Week 4-5 (males treated at 330 or 1000 mg/kg/day). For females receiving FAT 41001‑H TE there was no conclusive effect of treatment on body weight gain.  

There was no effect of treatment on food consumption in males, or in females prior to pairing.

During Days 6-19 gestation, food consumption of all groups of treated females was slightly high. Throughout lactation, food consumption was slightly higher in females receiving 330 or 1000 mg/kg/day, with the increase being dose dependent.  

On two occasions during treatment, a marked increase in water consumption was observed amongst animalsreceiving 1000 mg/kg/day when compared with Controls.  

Haematological examination during Week 2 of treatment, prior to pairing, and the examination of the clotting parameters in Week 4 of treatment for the males and on gestation Day 17 for the females, revealed no significant response to treatment with FAT 41001-H TE.

Biochemical examination of the blood plasma in Week 2 of treatment revealed lowbile acid concentrations amongst all treated groups of males and females when compared with the control animals. Cholesterol concentration was high in animals receiving 330 or 1000 mg/kg/day and creatinine concentration was high in females at all dose levels. Glucose concentrations were low in males receiving 1000 mg/kg/day. High calcium concentration was evident in all treated male groups when compared with the control group; there was no similar effect in the females. 

At routine examination of the offspring, signs that were observed in relation to treatment with FAT 41001 H-TE were limited to dark areas on the lower ventral abdomen at 100, 330 and 1000 mg/kg/day and blue staining was observed in offspring at 330 and 1000 mg/kg/day; this was considered to be related to the colour of the test item.

After five weeks of treatment, absolute and body weight adjusted kidney weights of F0 males and females receiving 1000 mg/kg/day were marginally higher than Controls.  Absolute testes weights were also high in these males and in males receiving 330 mg/kg/day. On Day 7 of lactation, absolute and body weight adjusted adrenal weights for all treated groups of F0 females were low when compared with Controls. 

Macroscopic examination performed in F0 males after five weeks of treatment and in F0 females on Day 7 of lactation, did not reveal any treatment-related findings for animals receiving FAT 41001-H TE at 100 mg/kg/day. Findings observed at macroscopic examination of the males and females treated at 330 or 1000 mg/kg/day were confined to blue or dark coloration (including some blue or dark colouration of the contents)in a variety of tissues; this was considered to be due to the coloured nature of the compound and was not representative of any pathological change. At macroscopic examination of the offspring, findings observed in relation to treatment were confined to dark contents of the gastrointestinal tract at 1000 mg/kg/day. In addition, one litter at 1000 mg/kg/day were observed to have blue skin.

Microscopic examination revealed treatment related changes within the kidneys, mesenteric and left axillary lymph nodes, stomach of both sexes and epididymides of the males. Renal cortical tubular vacuolation was recorded in males and females treated with 330 or 1000 mg/kg/day, accompanied by hyaline droplets in the males at both dose levels. Vacuolated macrophages were recorded in the mesenteric and left axillary lymph nodes of most males and females given 1000 mg/kg/day. Foveolar hyperplasia was recorded in the stomach of both sexes given 1000 mg/kg/day. Epithelial vacuolation of the epididymides was recorded in all males treated with 1000 mg/kg/day.

Conclusion

It was concluded that in the absence of any evidence for general systemic toxicity or effects on reproductive performance/offspring development that the no observed adverse effect level was 1000 mg/kg/day.