Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 11 January 2017. Experimental completion date 12 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 422 guideline for testing of chemicals adopted 22 March 1996: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

EC Number:

289-056-7

EC Name:

Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

Cas Number:

85959-61-1

Molecular formula:

C162H180N12O18Cl6+O3.P2O5.xWO3.yMoO3

IUPAC Name:

heptakis(N-[(3E)-6-(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-3H-xanthen-3-ylidene]ethan-1-aminium) dioxomolybdenumbis(olate) dioxotungstenbis(olate) phosphate

Test material form:

other: granula solid

Specific details on test material used for the study:

Test item: Pigment Red 81:4
Test item identity (including alternative names): CT RED 9302W
Reference number: PP0310A220.
C.I. Pigment Red 81:4
CAS number: 85959-61-1.
Intended use: Pigment.
Appearance: Magenta (bluish-red) powder.
Storage conditions: At ambient temperature (15 to 25°C).
Supplier: Sponsor.
Batch number: 64078.
Expiry date: 1 November 2017.

Test animals

Species:

rat

Strain:

other: RccHan™;WIST (Han Wistar)

Details on test animals or test system and environmental conditions:

Animals
Strain/Species RccHan™;WIST rat.
Supplier Envigo.
Number of animals ordered: 44 males and 44 females. Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization: Six days before commencement of treatment.
Age of the animals at the start of the study: Males 70 to 76 days old. Females 63 to 69 days old.
Weight range of the animals at the start of the study: Males 270 to 316 g. Females 164 to 194 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 color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Environmental Control
Rodent facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%. There were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.

Animal Accommodation
Cages: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Cage distribution: The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage: Pre-pairing up to five animals of one sex. Pairing one male and one female Males after mating up to five animals Gestation one female Lactation one female + litter

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

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

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:

DMSO

Details on exposure:

Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly. On Day 1 of Lactation animal number 41 Group 2 (30 mg/kg/day) was noted to be difficult to dose due to
an apparent blockage in throat, for welfare reasons this animal was therefore not dosed on this day of treatment.
Treated at: Constant doses in mg/kg/day.
Volume dose: 1.25 mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Formulation: A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 100 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix
Achieved concentration Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analyzed for achieved concentration of the test item.

Preparation of Calibration Standards
A primary standard solution (100 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 5 mg) of Pigment Red 81:4 in extract solvent (50 mL).
Solutions for instrument calibration were prepared by appropriate dilution of the primary standard using diluent and contained Pigment Red 81:4 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.

Preparation of Test Samples
A representative sample of test formulation (1 mL, accurately weighed) and dissolved using ultrasonic vibration in a suitable volume of extract solvent. The extract was diluted using diluent, to provide a solution containing Pigment Red 81:4 at an expected concentration within the range 4 μg/mL to 8 μg/mL.
The concentration of Pigment Red 81:4 in the final solution was quantified by HPLC using UV detection as detailed in the chromatographic section.

Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (DMSO) with known amounts of Pigment Red 81:4. The prepared procedural recoveries were analyzed in accordance with the analytical procedure.

Instrumentation Parameters
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Poroshell 120 SB-C18, 2.7μm, 4.6 ×100 mm
Column temperature: 45°C
Sample temperature: Ambient
Mobile Phase: 0.05% Formic Acid in Acetonitrile/Water 45/55 v/v
Flow rate: 1 mL/min
Rinse solvent/Needle wash: Acetonitrile/Water 50/50 v/v
Detector wavelength: 270 nm
Injection volume: 10 μL
Run time: 6.0 minutes
Approximate retention time: 4.5 minutes

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 DMSO chromatograms in order to calculate a Pigment Red 81:4 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 five procedural recoveries at nominal concentrations of Low nominal concentration mg/mL and High nominal concentration mg/mL during the method validation.
Calibration standard stability was assessed by analyzing stored standards with freshly prepared calibration standards after refrigerated storage for 9 days.
Extract stability was assessed by re-analyzing the method accuracy and precision samples with freshly prepared calibration standards after refrigerated storage for 9 days.

Homogeneity and Stability in DMSO Formulations
The homogeneity and stability of Pigment Red 81:4 in DMSO formulations was assessed at nominal concentrations of 1 mg/mL and 100 mg/mL, during ambient (+15 to +25°C) and refrigerated storage (+2 to +8°C). Freshly prepared specimen formulations (400 mL) were equally sub divided into 4 amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Ambient Temperature Storage (+15 to +25°C)
On receipt, the contents of one bottle of each formulation were mixed by 20-fold inversion followed by magnetic stirring. After stirring for 20 minutes (representing 0 hour) and 4 hours, single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the continuously stirred formulation. Upon sampling the 4 hour time point for formulations at 100 mg/mL, it was noted that the formulation was no longer stirring. The formulation was remixed by 20-fold inversion followed by magnetic stirring for 5 minutes before sampling. The remainder of the bottle was stored at ambient temperature (+15 to +25°C) and after 24 hours and 8 days storage the contents were remixed and sampled as detailed above. Following zero hour sampling, an additional 4 × 1 mL aliquots (weight recorded) were taken directly into volumetric flasks and stored for analysis on Day 8 and Day 15 (2 aliquots each day).

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

Concentration of Dose Formulations
For Week 1 and Week 5, freshly prepared test formulations were sampled by Pharmacy personnel and submitted for analysis.
Group 1 was sampled (2 × 3 mL, accurately weighed) from the middle of the formulation. Duplicate samples were analyzed in accordance with the analytical procedure, and the remaining samples were retained for contingency.
Groups 2, 3 and 4 were samples (4 × 1 mL, accurately weighed) from the middle of the formulation. Two samples were analyzed in accordance with the analytical procedure, and the remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.

Method Validation
The analytical procedure was successfully validated for Pigment Red 81:4 in DMSO with respect to the specificity of chromatographic analysis, limit of detection and quantification,linearity of detector response, repeatability, method accuracy and precision. Results are summarized below:
The specificity of the HPLC assay was demonstrated by the absence of a peak at the characteristic retention time for Pigment Red 81:4 in the control samplechromatogram.
The limit of detection and quantification was estimated as 0.0532 μg/mL and 0.177 μg/mL respectively.
Linearity was confirmed over the nominal concentration range 2 μg/mL to 10 μg/mL with a coefficient of determination >0.995;
The repeatability was <0.5% for six replicate injections of standard solutions containing Pigment Red 81:4 at nominal concentrations of 2 μg/mL and 10 μg/mL;
Method accuracy and precision were confirmed a mean procedural recovery value of 99.9% (CV=0.65%, n=5) was obtained for 2 mg/mL and 99.6% (CV=0.36%, n=5) was obtained for 100 mg/mL.
Calibration standard stability was confirmed after refrigerated storage (+2 to +8°C) for 9 days.
Extract stability was confirmed after refrigerated storage (+2 to +8°C) for 9 days.

Homogeneity and Stability of Dose Formulations
The homogeneity and stability of Pigment Red 81:4 in DMSO formulations were assessed with respect to the level of concentration at nominal concentrations of 1 mg/mL and 100 mg/mL.
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 4 hours, and on re-suspension following storage at ambient temperature (+15 to +25°C) for up to 8 days and refrigeration (+2 to +8°C) for up to 15 days. At each time-point, the mean analyzed concentration for the four samples remained within 3% of the initial time zero value and the coefficient of variation was less than 5%.
Discrete sample stability was proven for up to 15 days refrigerated.
Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method.

Concentration of Dose Formulations
The mean concentrations were within ±1%, confirming the accuracy of formulation. The % difference from mean values were within 3% of the mean 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, calibration standard and extract stability.
The homogeneity and stability was confirmed for Pigment Red 81:4 in DMSO formulations at nominal concentrations of 1 mg/mL and 100 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage for up to 8 days and refrigerated storage for up to 15 days.
The mean concentrations of Pigment Red 81:4 in test formulations analyzed for the study were within ±1% of nominal concentrations, confirming accurate formulation.

Details on mating procedure:

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.

Vaginal Smears
Wet smears were taken as follows:
Wet smears After pairing until mating, using pipette lavage.

Duration of treatment / exposure:

Males Two weeks pre-pairing up to necropsy after minimum of five weeks.
Females Two weeks before pairing, then throughout pairing and gestation until Day 6 of lactation.

Frequency of treatment:

Once daily at approximately the same time each day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

125 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females at each dose

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection
The doses used in this study (0, 30, 62.5 and 125 mg/kg/day) were selected in conjunction with the Sponsor.
Dose levels were selected following the completion of the preliminary study RM98QW. In that study there were no effects on body weight, food consumption, water consumption (visual assessment), necropsy changes or organ weights, with the exception of one female receiving 100 mg/kg/day which lost a moderate amount of body weight which was of uncertain association with treatment. An earlier
OECD 420 acute oral toxicity study was performed on this compound using a single dose of 300 mg/kg administered to a single fasted female; the animal was found dead two days after dosing following
clinical signs of hunched posture, piloerection, increased lachrymation and hypothermia, body weight loss and orange/red faeces were also recorded and at necropsy red staining of internal organs was
observed. In order to make every effort to induce minor toxicity in the high dose animals the dose level was increased to 125 mg/kg/day with dose levels at approximately 0.5 fold intervals below of 62.5 and 30 mg/kg/day to establish any dose relationship to any effects potentially observed.

Examinations

Maternal examinations:

Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

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

Signs Associated with Dosing
Daily during the first week of treatment, weekly thereafter and for females on Days 0, 6, 13 and 20 of gestation and Days 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
A cage observation was made at the time of cage clean out.

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 behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity.
Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior. Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the five lowest numbered surviving lactating females in each group at Day 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 cage labels on these individual cages showed only the study and animal number. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing. The following measurements, reflexes and responses were recorded:
Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores probe/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction
Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response
Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor
Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression
Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.

Motor activity
During Week 5 of treatment for males and at Day 4-6 of lactation for females, the motor activity of the five lowest numbered surviving males and the five lowest numbered surviving lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo. Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

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

Hematology, Peripheral Blood
Blood samples were collected at the following occasions:
Week 2 before pairing The five lowest numbered surviving males and females per group.
Termination (citrate sample only) The five lowest numbered surviving males and females per group.
Samples collected before pairing were collected following overnight deprivation of food and prior to dosing. Deprivation of food was not required at the termination blood sampling.
Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct) *
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH) *
Mean cell hemoglobin concentration (MCHC) *
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)
* Derived values calculated in ClinAxys
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5 mL before pairing or 2 x 0.6 mL at termination) were taken into tubes containing citrate anticoagulant and examined using a Stago compact max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood Chemistry
Blood samples were collected after overnight withdrawal of food and prior to dosing at the following occasion:
Week 2 before pairing The five lowest numbered surviving males and females per group.
Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile Acids (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

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

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
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.
The organs weighed, tissue samples fixed and sections examined microscopically are detailed in section any other information on materials and methods for F0 animals.
Females
The following were recorded:
Each uterine horn Number of implantation sites was counted and confirmed if none were visible at visual inspection.

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

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

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

Light Microscopy
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Premature deaths All F0 animals from all groups. All specified in tabel below
Scheduled kill Five lowest numbered F0 surviving All specified in tabel below
males and females in Groups 1 and 4
All F0 animals. Abnormalities.
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings. For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted. For the assessment of the ovaries a qualitative evaluation of one section from each ovary was
made.

Ovaries and uterine content:

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.

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 maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-7 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.

Method of Kill
Offspring Intraperitoneal injection of sodium pentobarbitone.

Necropsy
F1 offspring Day 7 of age.
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: Examined externally, if found to be normal offspring were discarded without further examination. Any externally abnormal offspring were also examined internally. Abnormal tissues were retained in an appropriate fixative.

Statistics:

Please see any other information on materials and methods.

Indices:

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

Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of matingfor calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:
Gestation index (%) = Number of live litters born/Number pregnant x 100

Litter Size
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 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, 4 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: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs observed included mainly staining with the test item; this sign is considered not to be adverse.
Pink bedding and pink feces were recorded on all animals receiving Pigment Red 81:4.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Group 3 female 69 was killed for welfare reasons on Day 18 of treatment. This female had lost approximately 10 g in the second week of study but had experienced more notable weight loss following pairing with an additional 32g lost in the last 3 days. The animal had shown signs of irregular respiration and rales from Day 16 of study. At necropsy pink material was noted throughout the GI tract which is attributed to the colour of the test material. Other signs included partially blocked nasal turbinates and the GI tract was distended with gas. Rats are obligate nasal breathers and if they have to breathe through their mouths then gas distension does occur.
Group 3 female 62 was killed for welfare reasons on Day 15 of gestation. It was confirmed to be pregnant with 10 fetuses. This animal had shown signs of rales from Day 13 as well as irregular respiration and gasping on Day 15 of gestation. At necropsy pink material was noted in the trachea, stomach, colon, caecum and ileum which is attributed to the colour of the test material. Pale areas were noted in the lungs and bronchi and the adrenals were enlarged. There were no fetal abnormalities.
In the absence of a dose relationship, the clinical signs and macropathology findings seen in these animals are thought to be due to the dosing procedure not test item related.
Group 3 female 64 was sent to necropsy on Day 25 gestation having failed to litter. At necropsy pink material was noted in the stomach, colon, caecum and rectum. This is attributed to the colour of the test material.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no adverse effect of Pigment Red 81:4 on body weight performance in males throughout or females before pairing, during gestation and lactation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no indication of a food consumption effect on either males throughout or females before pairing, during gestation or lactation.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At haematology evaluation during Week 2 of study males receiving Pigment Red 81:4 at 125 mg/kg/day had a statistically significantly higher large unstained cell count compared to Control. A dose relationship is not apparent and in the absence of a similar effect in females this is of uncertain relationship to treatment. No other parameters attained statistical differences to the Control.
There was an issue with the citrate samples provided for clotting factor analysis during Week 2 which resulted in a high proportion of the samples being unsuitable for analysis. As a result of this another citrate sample was taken and analysed at study termination. The samples at termination had a decrease in prothrombin time in all groups of treated males when compared to controls, however in the absence of a dose relationship this is of uncertain significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At blood chemistry evaluation during Week 2 of study all groups of treated females had statistically significantly lower albumin concentration compared to Control females. A dose relationship is not apparent and in the absence of a similar effect in males this is of uncertain relationship to treatment.
Males receiving 62.5 or 125 mg/kg/day had slightly higher triglyceride levels, no statistical different was attained, however, a dose relationship was apparent.
No other parameters attained statistical differences to the Control.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory reactivity and grip strength were considered to be unaffected by Pigment 81:4 at all dose levels investigated.
The motor activity assessment conducted on males during Week 5 of treatment and females during Days 4-6 of lactation revealed no treatment related effects at all dose levels. The mean total high beam score for females receiving 125 mg/kg/day was statistically significantly higher than in Controls; however, since the value was within the Historical control data range no effect of treatment is inferred.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Absolute and body weight adjusted organ weights were generally similar across all treated groups for both males and females and considered unaffected by treatment with Pigment Red 81:4 at all dose
levels investigated.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Abnormal colour of the caecum was observed in F0 males receiving Pigment Red 81:4 at 62.5 mg/kg/day 125 mg/kg/day. Abnormal colour of the rectum was observed in all treated male groups as well as in one female receiving 125 mg/kg/day. Abnormal pink contents were seen in the caecum of males receiving 30 mg/kg/day and in the caecum and rectum of both males and females receiving 62.5 or 125 mg/kg/day. Abnormal pink contents were also seen in the colon of male and females receiving 125 mg/kg/day and males receiving 62.5 mg/kg/day. Abnormal colour of the trachea was seen in one male treated at 125 mg/kg/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Oral administration of Pigment Red 81:4 to rats for 5 weeks resulted in no test item related findings. No microscopic correlates were seen for the pink coloured and pink contents/gas of the gastroint
estinal tract tissues and trachea reported at necropsy. This discolouration was attributed to the colour of the test item.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Two females receiving 125 mg/kg/day and one receiving 62.5 mg/kg/day showed piloerection following dose administration on Day 8 of treatment. In isolation this sign is considered not to be adverse and of uncertain relationship to treatment.

Maternal developmental toxicity

Number of abortions:

not examined

Description (incidence and severity):

not applicable in rats

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no effect of Pigment Red 81:4 on offspring survival

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

gestation lenght was unaffected by treatment

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

fertility was unaffected by treatment

Details on maternal toxic effects:

Mating performance, pre coital interval, fertility, gestation length and index were unaffected by treatment.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical biochemistry

clinical signs

dead fetuses

effects on pregnancy duration

gross pathology

haematology

histopathology: non-neoplastic

maternal abnormalities

mortality

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

not examined

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no effect of Pigment Red 81:4 on litter size

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of Pigment Red 81:4 on sex ratio.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no effect of Pigment Red 81:4 on litter size.

On Day 1 of age the body weights of all groups of offspring derived from females receiving Pigment Red 81:4 were similar to Control. Subsequent offspring body weight gain during Days 1-7 of age was slightly lower than Control at the 125 mg/kg/day dose level. For both male and female offspring derived from females receiving 125 mg/kg/day body weight gain was 11% lower than Control during Days 1-7 of age. Although this may be attributable to treatment, it is considered not to be adverse at the degree observed.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There was no effect of Pigment Red 81:4 on offspring survival to Day 7 of age.

External malformations:

no effects observed

Description (incidence and severity):

Macroscopic examination of offspring that either died prematurely or at scheduled termination on Day 7 of age did not reveal any findings that could be attributed to treatment.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

It was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity and reproductive/developmental toxicity was 125 mg/kg/day. Administration of Pigment Red 81:4 at dose levels of 30, 62.5 or 125 mg/kg/day had no effect on clinical condition, bodyweight, food consumption, motor activity, sensory reactivity and grip strength, pre-coital interval, mating performance, fertility, gestation length, organ weights, histopathology, offspring survival, sex ratio, offspring clinical signs or offspring macropathology. The macropathology findings seen in the F0 adults are thought to be attributed to the coloration of
the test material.

Executive summary:

The purpose of the study was an assessment of general systemic toxic potential in rats, including a screen for reproductive/developmental effects, with administration of the test item by oral administration for at least four weeks.

Three groups of ten male and ten female Han Wistar rats received Pigment Red 81:4 at doses of 30, 62.5 or 125 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 allowed to litter, rear their offspring and were killed on Day 7 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, DMSO, 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, hematology (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

Two females (3F 62 and 3F 69) receiving 62.5 mg/kg/day were killed for welfare for reasons during the treatment period. Animal 69 was terminated on Day 18 of treatment, due to bodyweight loss and signs of irregular respiration and rales. At necropsy pink material was noted throughout the GI tract which is attributed to the colour of the test material. Other signs included partially blocked nasal turbinates and the GI tract was distended with gas. Rats are obligate nasal breathers and if they have to breathe through their mouths then gas distension does occur. Animal 62 was terminated on Day 15 of gestation. It was confirmed to be pregnant with 10 fetuses. This animal had shown signs of rales from Day 13 as well as irregular respiration and gasping on Day 15 of gestation. At necropsy pink material was noted in the trachea, stomach, colon, caecum and ileum which is attributed to the colour of the test material. Pale areas were noted in the lungs and bronchi and the adrenals were enlarged. There were no fetal abnormalities. In the absence of a dose relationship, the clinical signs and macropathology findings seen in these animals are thought to be due to the dosing procedure not test item related.

Administration of Pigment Red 81:4 at dose levels of 30, 62.5 or 125 mg/kg/day had no effect on clinical condition, bodyweight, food consumption, motor activity, sensory reactivity and grip strength, pre-coital interval, mating performance, fertility, gestation length, organ weights or histopathology.

At haematology evaluation study males receiving Pigment Red 81:4 at 125 mg/kg/day had a statistically significantly higher large unstained cell count compared to Control. A dose relationship was not apparent and in the absence of a similar effect in females this is of uncertain relationship to treatment.

At blood chemistry evaluation all groups of treated females had statistically significantly lower albumin concentration compared to Control females. A dose relationship is not apparent and in the absence of a similar effect in males this is of uncertain relationship to treatment. Males receiving 62.5 or 125 mg/kg/day had slightly higher triglyceride levels, no statistical different was attained, however a dose relationship was apparent.

Abnormal colour of the caecum was observed in F0 males receiving Pigment Red 81:4 at 62.5 mg/kg/day 125 mg/kg/day. Abnormal colour of the rectum was observed in all treated male groups as well as in one female receiving 125 mg/kg/day. Abnormal pink contents were seen in the caecum of males receiving 30 mg/kg/day and in the caecum and rectum of both males and females receiving 62.5 or 125 mg/kg/day. Abnormal pink contents were also seen in the colon of male and females receiving 125 mg/kg/day and males receiving 62.5 mg/kg/day. Abnormal colour of the trachea was seen in one male treated at 125 mg/kg/day.

There was no effect of Pigment Red 81:4 on litter size, offspring survival to Day 7 of age, sex ratio, offspring clinical signs or macropathology.

Offspring body weight gain during Days 1-7 of age was slightly lower than Control at the 125 mg/kg/day dose level. For both male and female offspring derived from females receiving 125 mg/kg/day body weight gain was 11% lower than Control during Days 1-7 of age. Although this may be attributable to treatment, it is considered not to be adverse at the degree observed.

Conclusion

It was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity and reproductive/developmental toxicity was 125 mg/kg/day. Administration of Pigment Red 81:4 at dose levels of 30, 62.5 or 125 mg/kg/day had no effect on clinical condition, bodyweight, food consumption, motor activity, sensory reactivity and grip strength, pre-coital interval, mating performance, fertility, gestation length, organ weights, histopathology, offspring survival, sex ratio, offspring clinical signs or offspring macropathology. The macropathology findings seen in the F0 adults are thought to be attributed to the coloration of the test material.