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

Endpoint summary

Description of key information

In a key study, the subject material was tested by oral gavage in male and female rats following OECD guideline 422 and following accepted GLP standards.

It was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity was 125 mg/kg/day.

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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.

Species:

rat

Strain:

other: RccHan™;WIST (Han Wistar)

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The RccHan™;WIST (Han Wistar) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

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.

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was chosen to simulate a condition of possible human exposure.

Vehicle:

DMSO

Details on oral 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.

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

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

62.5 mg/kg bw/day (nominal)

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.

Time Schedule
Experimental start date 11 January 2017
Animal arrival 11 January 2017
Treatment commenced 17 January 2017
F0 pairing commenced 31 January 2017
F0 necropsy
Males 27 February 2017
Females 1 to 5 March 2017
Experimental completion date 12 April 2017

Observations and examinations performed and frequency:

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.

Sacrifice and pathology:

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

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.
F1 offspring Day 7 of age.
The organs weighed, tissue samples fixed and sections examined microscopically are detailed in section any other information on materials and methods for F0 animals:

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.

Statistics:

Please see any other information on materials and methods.

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 gastrointestinal 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.

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Critical effects observed:

no

In this study Pigment Red 81:4 was orally administered to Han Wistar rats at doses of 30, 62.5 or 125 mg/kg/day. Males were dosed for 5 weeks and females for 2 weeks before pairing, throughout gestation and up to Day 6 of lactation. Treatment was well tolerated. Two females treated at 62.5 mg/kg/day were euthanized during the study for welfare reasons, 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 and not test item related. There were no clinical signs or effects on behavioral parameters. There were no effects on body weight, food consumption, organ weights or macropathology.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) conducted in 2015 with no deficiencies and assigned Klimisch 1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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.

Justification for classification or non-classification

Pigment Red 81:4 is not classified for specific organ toxicity, repeated exposure, as the NOAEL for rats is 125 mg/kg/day, according to CLP classification.