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EC number: 436-060-3 | CAS number: - FC 84508 PK
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
Administration of Disperse Orange FC84508 at doses up to and including 1000 mg/kg bw/day was well tolerated and no adverse effects were apparent. It was therefore concluded that the No-Observed-Adverse-Effect (NOAEL) for reproductive performance and for parental toxicity lies above 1000 mg/kg bw/day.
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Experimental start date (Animal arrival) 21 April 2021
Experimental completion date (T4 Bioanalysis) 09 October 2021 - Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- 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 Sprague-Dawley [Crl:CD(SD)] 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:
- Supplier Charles River (UK) Ltd.
Duration of acclimatization
Males: Six days before commencement of treatment.
Females: 20 days before commencement of treatment.
Age of the animals at the start of treatment
Males 69 to 75 days old.
Females 84 to 90 days old.
Weight range of the animals at the start of treatment
Males 337 to 410 g.
Females 239 to 303 g.
Animal 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. Minimum of 15 supply air changes per hour.
Temperature and relative humidity
Monitored and maintained within the range of 20-24°C 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 throughout the study except during pairing.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.
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 four animals of one sex
Pairing one male and one female
Males after mating up to four animals
Gestation one female
Lactation one female + litter
Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study (except during late gestation and littering) and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing and late gestation and littering) and replaced at the same time as the cages.
Paper shavings Approximately two handfuls of paper shavings were provided to each cage as nesting material from Day 20 after mating and throughout lactation. Shavings were replaced at the same frequency as the bedding.
Diet Supply
Diet SDS VRF1 Certified pelleted.
A sample (100 g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30°C). Samples will be discarded after finalization of the report.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.
Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.
Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen chew blocks.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed. - Route of administration:
- oral: gavage
- Vehicle:
- other: Aqueous 0.5% (w/v) methylcellulose.
- Details on exposure:
- Method of preparation
The required amount of test item was ground in a mortar using a pestle to a fine powder and mixed with a small amount of the vehicle to form a paste. Any agglomerates were broken down. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was transferred to a measuring cylinder, which had been wetted with vehicle, the mortar was rinsed with vehicle, and this was added to the measuring cylinder. Vehicle was added to achieve the final volume and the suspension was transferred to a beaker and mixed using a high shear homogenizer. The suspension was transferred to the final containers, via syringe whilst magnetically stirring.
Formulated in ascending order of concentration.
Frequency of preparation
Weekly, subdivided into daily aliquots and was prepared up to eight days in advance of the first day of dosing.
Storage of formulation Refrigerated (2 to 8°C) for up to 15 days.
Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.
Stability and homogeneity Before commencement of treatment, the suitability of the proposed mixing procedures was determined, and specimen formulations at 1 to 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix (Labcorp Study number 8451440).
The stability of a homogenous suspension of the test item in the vehicle were demonstrated at ambient temperature (15 to 25°C) for four hours and refrigerated (2 to 8°C) for 15 days. - 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. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The analytical method involved dilution with tetrahydrofuran followed by final dilution with water/methanol/formic acid solution prior to quantitation performed using liquid chromatography with tandem mass spectrometric detection (LC MS/MS).
Sample concentrations were determined with reference to external calibration standards freshly prepared in the concentration range 2.5 ng/mL to 25 ng/mL.
LC-MS/MS conditions
Instrument: Sciex API4000 (Analyst version 1.4.2 software) coupled to Waters Acquity UPLC system
Ionisation mode: Ionspray positive
Ion monitoring details: Primary transition m/z 447>359
Parameters: Curtain gas flow: 30 Collision gas pressure: 6
Nebulizer gas 1: 40 Ionspray voltage: 5000
Nebulizer gas 2: 40 Declustering potential: 66
Desolvation temperature: 400 Collision energy m/z 447>359: 33
Column: Acquity UPLC® BEH C18 (2.1 mm x 50 mm, 1.7μm), or equivalent
Column temperature: 45°C
Mobile phase A: Water:methanol:formic acid (90:10:0.1 v:v:v) + 0.01M ammonium formate
Mobile phase B: Methanol:formic acid (100:0.1 v:v)
Gradient: Time (mins) %A %B
0 10 90
0.2 10 90
2.0 2 98
4.5 2 98
5.0 10 90
6.0 10 90
Cycle time: 6 minutes
Weak needle wash: Methanol:water:formic acid (90:10:0.1 v:v:v)
Strong needle wash: Propan-2-ol:dimethyl sulfoxide:water:methanol:formic acid (25:25:25:25:1 v:v)
Injection volume: 5 μL
Flow rate: 0.5 mL/min
Retention time: Approximately 1.0 minute
Concentration of Dose Formulations
The first and last week formulations were sampled. For groups 1 to 4, 4 x 1 mL were sampled from the middle of the formulation by Pharmacy personnel.
Duplicate aliquots from all groups were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency and stored refrigerated at approximately +4°C.
Re-dilution analysis was performed for Group 3 last week formulations due to an outlier result for one replicate which suggested a dilution error during the analytical procedure. Re dilution of the sample from initial extract confirmed the analytical error.
Samples were disposed of once satisfactory results were achieved.
RESULTS AND CONCLUSION
The mean concentrations of Disperse Orange FC84508in test formulations analyzed during the study and the deviation of the mean result from the nominal value are detailed in Table 1.
Re-dilution analysis was performed for Group 3 last week formulations due to a dilution error in the original analysis resulting in an outlier result (less than the limit of detection) for one replicate.
The final mean concentrations were within the ideal nominal accuracy limits of +10/ 15% for all formulation occasions tested. - Duration of treatment / exposure:
- Males Two weeks pre-pairing up to necropsy after minimum of four weeks.
Females Two weeks before pairing, then throughout pairing and gestation until Day 12 of lactation.
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly. - Frequency of treatment:
- Once daily at approximately the same time each day.
- Details on study schedule:
- Study initiation (Study Plan signed by Study Director) 13 April 2021
Experimental start date (Animal arrival) 21 April 2021
Animal arrival
Females 21 April 2021
Males 05 May 021
Estrous cycle evaluation commenced 27 April 2021
Treatment commenced 11 May 2021
F0 pairing commenced 25 May 2021
F0 necropsy
Males 10 to 11 June 2021
Females 29 June 2021 to 04 July 2021
Experimental completion date (T4 Bioanalysis) 09 October 2021 - Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Control
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 10 males, 10 females per dose group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- Rationale for Dose Level Selection
In an acute oral (gavage) toxicity study, a limit dose of 2000 mg/kg body weight test substance as a 20% suspension in Tylose H 4000 G4 PHA (0.5 % in deionized water) was tested in five male and five female Sprague-Dawley rats according to OECD guideline 401. With exception of discolored orange feces, between four hours and one day after administration, no clinical signs were observed until the end of the study. Development of body weight was not impaired, except one female, which showed body weight loss between Day 8 and Day 15. No abnormalities were noted at necropsy.
The sub-acute toxicity of the test substance was examined in an oral (gavage) 28-day repeat dose toxicity study in Sprague-Dawley rats according to OECD guideline 407 followed by a 14 day recovery period. Groups of male and female rats received test substance by oral gavage at dose levels of 0, 62.5, 250 or 1000 mg/kg bw/day for a period of 28 days. On Day 29, five males and five females from each group were killed and necropsied. Five males and five females from the control and high dose group were killed and necropsied after a recovery period of 14 days. No deaths occurred throughout the study. Behavior, state of health, neurotoxicological, hematological and blood serum parameter analysis remained unaffected by the administration of the test substance in all dose groups. Body weight gain, organ weights and food and water consumption were comparable in control and dose groups. No test substance related changes were detected by urine analysis or at necropsy or histopathology. In conclusion, repeated administration of test substance did not cause any substance related changes at the doses administered. Hence, under the test conditions, NOAEL and NOEL of the test substance were determined to be 1000 mg/kg bw/day in rats.
Based on these data, dose levels of 100, 300, and 1000 mg/kg bw/day were selected for this study in order to fulfill the 2-fold to 4-fold dosing interval as specified in the test guideline.
Allocation and Identification
Allocation On arrival and non-selective allocation to cages.
Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the study.
On Day 1 of study all animals were weighed and body weights were reviewed by Study Management. The groups were adjusted to reduce inter /intra-group variation.
Identification of animals Each adult animal was assigned a number and identified uniquely within the study by a microchip 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).
Animal Replacement
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups.
Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before allocation Atypical estrous cycles Four females
Body weight range extremes Two males
- - Parental animals: Observations and 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 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
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males
Week 1 - daily
Weeks 2 to 4 - twice each week (middle and end of week)
Week 5 onwards - once each week
F0 females
Week 1 - daily
Week 2 - twice (middle and end of week)
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12
Detailed observations were recorded at the following times in relation to dose administration:
• Pre-dose observation
• One to two hours after completion of dosing (See Section 4)
• As late as possible in the working day.
Clinical Signs
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
F0 males Once each week
F0 females Once each week until pairing
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 7 and 13
Body Weight
The weight of animals was recorded as follows:
F0 males
Before dosing on the day that treatment commenced (Day 1) and weekly thereafter.
On the day of necropsy.
F0 females
Before dosing on the day that treatment commenced (Day 1) and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7, and 13 of lactation.
On the day of necropsy.
Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals
Weekly, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Week 3) but recommenced for males in Week 4.
For females after mating food consumption was measured to match the body weight recording:
Days 0-7, 7-14 and 14-20 after mating
Days 1-4, 4-7 and 7-13 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.
Parturition Observations and Gestation Length
Duration of gestation Time elapsing between the detection of mating and commencement of parturition.
Parturition observations From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded. - Oestrous cyclicity (parental animals):
- Dry smears For 15 days before pairing using cotton swabs
Wet smears
Using pipette lavage during the following phases:
• For 14 days before treatment; animals that failed to exhibit 4-5 day cycles were not allocated to the study.
• After pairing until mating (for a maximum of 14 days).
• Females showing no evidence of mating: following completion of the pairing period females were separated from the males and vaginal smearing continued for up to five days or until the first estrus smear was seen. If a female showed an estrus smear during this period, she was killed as soon as practically possible and subject to macroscopic examination.
• For four days before scheduled termination (nominally Days 10 to 13 of lactation).
Estrous Cycle
The incidence and percentage females showing the following classifications of estrous cycles before treatment commenced are presented:
Regular: All observed cycles of 4 or 5 days
Irregular: At least one cycle of 2, 3 or 6 to 10 days
Acyclic: At least 10 days without estrus
Vaginal smearing prior to termination is presented in terms of numbers of females that showed estrus during this period and the cycle stage at termination
Pre-Coital Interval
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing. - Sperm parameters (parental animals):
- 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.
- Litter observations:
- Records Made During Littering Phase
Clinical observations of offspring Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights Days 1, 4, 7, 11 and 13 of age.
Ano-genital distance Day 1 - all F1 offspring.
Nipple/areolae count Day 13 of age - male offspring. - Postmortem examinations (parental animals):
- 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 at least 4 weeks of treatment.
F0 females failing to produce a viable litter Day 25 after mating.
F0 females Day 13 of lactation.
Pathology procedures for all F0 parental animals
Tissue and regions examined Necropsy Histology Pathology
Weigh Fix Light microscopy
Abnormalities * * *
Epididymides (caput, corpus and cauda)** * *
Ovaries * * *
Pituitary *
Prostate * *
Seminal vesicles (with coagulation gland)* *
Testes * * * *
Thyroid *
Uterus with cervix and oviducts *c
*
Seminal vesicles (with coagulation gland)* *
Testes * * * *
Thyroid *
Uterus with cervix and oviducts *c
* Organs weighed, samples fixed or sections examined microscopically.
c Excluding females which were not pregnant, or fail to litter.
Animal ID retained
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.
All adult animals killed prematurely.
All adult animals in Groups 1 and 4 at scheduled termination.
Abnormalities only All adult 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.
Females
The following were recorded (including premature decedents):
Each uterine horn Number of implantation sites was counted and confirmed if none were visible at visual inspection after modified Salewski staining. - Postmortem examinations (offspring):
- Offspring
Premature deaths Where possible, a fresh macroscopic examination (external) with an assessment of stomach for milk content was performed. Grossly externally abnormal pups were retained pending possible future examination.
F1 offspring on Day 4 of age Externally normal offspring discarded without examination.
Externally abnormal offspring identified on despatch to necropsy; examined externally, and retained pending possible future examination.
F1 offspring on Day 13 of age All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Abnormalities were retained in appropriate fixative.
Thyroid glands were preserved from one male and one female in each litter, where possible. - Statistics:
- Please refer to "Any other information on materials and methods"
- Reproductive indices:
- Mating Performance and Fertility
Individual data was tabulated. Group values were calculated for males and females separately for the following:
Percentage mating (%) = (Number 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 paired) 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 mating for 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 (before and after blood sampling), 7, 11 and 13 of age. Group mean litter size and SD were calculated from the individual litter values. - Offspring viability indices:
- 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 4/ Number live offspring on Day 1 after littering ) x 100
Lactation index (%) = (Number of live offspring on Day 13 after littering/ Number of live offspring on Day 4) x 100
Group mean values were calculated from individual litter values
Offspring Sex Ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after blood sampling) and 13 of age.
Percentage males = (Number of males in litter / Total number of offspring in litter) x 100
Group mean values were calculated from individual litter values. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- Administration of Disperse Orange FC84508 at dose levels up to and including 1000 mg/kg bw/day was generally well tolerated in adult animals. There were no clear treatment-related changes in clinical condition or signs observed in relation to dose administration at any dose level investigated.
- Mortality:
- no mortality observed
- Description (incidence):
- One female (2F 111) was killed for welfare reasons on Gestation Day 18 showing signs of irregular breathing, piloerection, whole body pallor and dull eyes. Microscopic examination revealed marked abscessation of the thoracic cavity and marked epicardial inflammation, which correlated with the necropsy findings of adhesion(s) of the thoracic cavity and thickened pericardium respectively. The thoracic abscessation was considered to have been the major factor contributing to poor clinical condition of this animals and its early death.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Body weight gain for males during treatment and for females before pairing was unaffected by treatment at all dose levels investigated.
During the gestation phase, females receiving 1000 mg/kg bw/day showed a consistent but slightly higher bodyweight gain when compared to Controls, and resulted in a slightly higher overall mean bodyweight gain (GD0-20). There was no effect of treatment on bodyweight gain at 100 or 300 mg/kg bw/day during gestation.
Overall mean body weight gain at 100, 300 or 1000 mg/kg bw/day during lactation was slightly low when compared to Controls, although the differences were considered to be minor and were unrelated to treatment.
PLEASE REFER TO THE TABLES IN ANY OTHER INFORMATION ON RESULTS- "Body weight and body weight changes - Parental generation" - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Food consumption for the two-week period before pairing for males and females and post-pairing for males, and for females during gestation and lactation was considered to be unaffected by treatment at all dose levels investigated.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Endocrine findings:
- no effects observed
- Description (incidence and severity):
- Analysis of serum T4 concentration revealed low group mean T4 concentration in F0 males given 300 or 1000 mg/kg bw/day, when compared to Controls. Mean serum T4 concentration in samples obtained from F0 males given 100 mg/kg bw/day were unaffected by treatment. Assessment of individual T4 values showed that the majority of values in F0 males at 300 or 1000 mg/kg bw/day were within the 95-percentile historical control range. However, four Control values were above this range, resulting in an apparent difference between Control and treated groups. Therefore, the observed differences at 300 or 1000 mg/kg bw/day, when compared to the Control group mean concentration in F0 males, were not attributed to treatment.
When compared to Controls, the mean serum T4 concentration for male and female F1 offspring on Day 13 of age derived from parental animals treated with Disperse Orange FC84508 at doses up to and including 1000 mg/kg bw/day were unaffected by treatment
PLEASE REFER TO THE ATTACHED TABLES "Thyroid Hormone Analysis" - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- All microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), an/or their severity was as expected for Sprague-Dawley rats of this age. Therefore, all findings were considered not test item related.
The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present. - Histopathological findings: neoplastic:
- not examined
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- Estrous cycles during treatment, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by parental treatment. At termination all females were confirmed to be in diestrus except one female (3F 123) was confirmed not pregnant.
Please refer to the results tables in "Any other information on results" - Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- effects observed, non-treatment-related
- Description (incidence and severity):
- One female (No. 111) given 100 mg/kg bw /day was killed for welfare reasons, and one female (No. 123) that received 300 mg/kg bw/day was recorded as failed to litter and was dispatched to necropsy on Day 25 after mating confirming non-pregnancy for this female. Therefore, the following litter assessment is based on 10, 9, 9 and 10 litters at 0, 100, 300 and 1000 mg/kg bw/day.
The mean number of implantations and subsequently litter size for females receiving 1000 mg/kg bw/day was slightly high when compared to Controls; with differences attaining statistical significance. Mean number of implantations and litter size for females receiving 100 or 300 mg/kg bw/day was similar to Controls - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- There were no clinical signs that were considered to be related to treatment.
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At 1000 mg/kg bw/day, overall mean bodyweight gain for offspring (Day 1-13 of age) was slightly low for male and female offspring when compared to Controls although these differences did not attain statistical significance and was considered to reflect the slightly higher litter size at 1000 mg/kg bw/day.
Offspring bodyweights at 100 or 300 mg/kg bw/day was unaffected by parental treatment. - Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Anogenital distance (AGD):
- no effects observed
- Description (incidence and severity):
- Ano-genital distance for both male and female offspring was unaffected by parental treatment.
- Nipple retention in male pups:
- no effects observed
- Description (incidence and severity):
- No nipples were apparent for male offspring on Day 13 of age.
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Macroscopic findings observed in offspring killed or that died prior to scheduled termination were predominantly the absence of milk in the stomach.
There were no macroscopic findings for offspring at scheduled termination on Day 13 of age. - Histopathological findings:
- not examined
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Treatment related:
- no
- Conclusions:
- Administration of Disperse Orange FC84508 at doses up to and including 1000 mg/kg bw/day was well tolerated and no adverse effects were apparent. It was therefore concluded that the No-Observed-Adverse-Effect (NOAEL) for reproductive performance and for parental toxicity is above 1000 mg/kg bw/day.
- Executive summary:
The purpose of this study was a screening test for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item, Disperse Orange FC84508, by oral gavage administration for at least four weeks.
Three groups of ten male and ten female rats (F0 generation) received Disperse Orange FC84508 at doses of 100, 300 or 1000 mg/kg bw/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The offspring (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, aqueous 0.5% (w/v) methylcellulose, at the same volume dose (10 mL/kg bw/day) as treated groups.
During the study, clinical condition, body weight, food consumption, estrous cycles, pre-coital interval, mating performance, gestation length, thyroid hormone analysis, organ weight and macroscopic pathology and histopathology investigations were undertaken.
The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. Blood samples were collected from selected offspring on Day 4 and Day 13 of age for thyroid hormone analysis.
Results
Parental responsesAnalysis of serum T4 concentration revealed low group mean T4 concentration in F0 males given 300 or 1000 mg/kg bw/day, when compared to Controls. Mean serum T4 concentration in samples obtained from F0 males given 100 mg/kg bw/day were unaffected by treatment. Assessment of individual T4 values showed that the majority of values in F0 males at 300 or 1000 mg/kg bw/day were within the 95-percentile historical control range. However, four Control values were above this range, resulting in an apparent difference between Control and treated groups. Therefore, the observed differences at 300 or 1000 mg/kg bw/day, when compared to the Control group mean concentration in F0 males, were not attributed to treatment with the test substance.
Administration of Disperse Orange FC84508 at dose levels up to and including 1000 mg/kg bw/day was generally well tolerated in adult animals. There were no clear treatment-related changes in clinical condition or signs observed in relation to dose administration at any dose level investigated. One female given 100 mg/kg bw/day was killed for welfare reasons on Gestation Day 18, and the major contributing factor to its poor clinical condition was thoracic abscessation. This finding was considered to be incidental and was not related to treatment with Disperse Orange FC84508.
Body weight gain and food consumption for males during treatment and for females before pairing, during gestation and lactation were unaffected by treatment at all dose levels investigated.
Estrous cycles during treatment, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by parental treatment.
Litter responses
The clinical condition and survival of the F1 offspring, with litter size, sex ratio, offspring birth weights, offspring body weight gain and ano-genital distance were not adversely affected by parental treatment of Disperse Orange FC84508. There were no macroscopic findings for offspring and there were no nipples apparent for male offspring on Day 13 of age. Mean serum T4 concentration for male and female F1 offspring on Day 13 of age were unaffected by parental treatment.
Conclusion
Administration of Disperse Orange FC84508 at doses up to and including 1000 mg/kg bw/day was well tolerated and no adverse effects were apparent. It was therefore concluded that the No-Observed-Adverse-Effect (NOAEL) for reproductive performance and for parental toxicity is above 1000 mg/kg bw/day.
Reference
Formulation Analysis
The mean concentrations of formulation samples taken during this study were within +10/-15% of the nominal concentration, confirming accurate formulation and the percentage difference from mean values remained within 7%, confirming precise analysis.
Procedural recovery values were within the validated range confirming the continued accuracy of the analytical method.
Thyroid Hormone Analysis
The purpose of this phase of the study was to measure total Thyroxine (T4) concentrations in rat serum using liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) using an analytical method validated in Labcorp Study Number FF58YR and documented in Bioanalytical Method Number BM/2016/0632. T4 is an endogenous serum biomarker therefore, concentrations of T4 were measured using a surrogate matrix (4% BSA and 0.2% Tween20 in Phosphate Buffered Saline) with a calibration range of 700 to 70000 pg/mL.
The mean rat serum concentrations of T4 in the samples obtained from F0 male (adult) animals in Groups 1 to 4 in week 5 and the F1 male and female offspring on Day 13 of age following daily oral gavage administration of Disperse Orange FC84508 to adults are summarized the attached "Thyroid Hormone Analysis"
| Body weight and body weight change - group mean values (g) for males and for females before pairing (F0) | Request ID: 5447388 |
|
|
|
| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Group |
| Day |
|
|
|
|
| Change | Change | Change | Change | Change | Change |
/Sex |
| 1 | 8 | 15 | 22 | 29 |
| 1-8 | 8-15 | 15-22 | 22-29 | 1-15 | 1-29 |
Statistics | test | Av | Wi | Wi | Wi | Wi |
| Wi | Wi | Wi | Wi | Wi | Wi |
1M | Mean | 361 | 385 | 410 | 432 | 450 |
| 24 | 24 | 22 | 18 | 49 | 88 |
| SD | 15.3 | 15.9 | 17.3 | 20.3 | 21.1 |
| 9.8 | 6.7 | 7.7 | 6.2 | 14.4 | 16.9 |
| N | 10 | 10 | 10 | 10 | 10 |
| 10 | 10 | 10 | 10 | 10 | 10 |
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2M | Mean | 359 | 384 | 405 | 422 | 441 |
| 25 | 21 | 17 | 19 | 46 | 82 |
| SD | 12.0 | 14.3 | 18.5 | 21.0 | 23.4 |
| 5.5 | 4.8 | 5.8 | 5.5 | 9.5 | 14.0 |
| N | 10 | 10 | 10 | 10 | 10 |
| 10 | 10 | 10 | 10 | 10 | 10 |
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3M | Mean | 368 | 392 | 414 | 431 | 450 |
| 24 | 22 | 17 | 19 | 46 | 82 |
| SD | 12.2 | 13.8 | 19.0 | 15.6 | 19.1 |
| 5.7 | 8.6 | 8.1 | 9.1 | 13.0 | 17.6 |
| N | 10 | 10 | 10 | 10 | 10 |
| 10 | 10 | 10 | 10 | 10 | 10 |
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4M | Mean | 367 | 388 | 415 | 433 | 452 |
| 21 | 27 | 18 | 19 | 48 | 84 |
| SD | 20.5 | 24.2 | 31.0 | 35.8 | 37.2 |
| 7.5 | 8.3 | 8.7 | 9.0 | 12.6 | 22.5 |
| N | 10 | 10 | 10 | 10 | 10 |
| 10 | 10 | 10 | 10 | 10 | 10 |
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(cont) | Body weight and body weight change - group mean values (g) for males and for females before pairing (F0) | Request ID: 5447388 |
| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Group |
| Day |
|
|
| Change | Change | Change |
/Sex |
| 1 | 8 | 15 |
| 1-8 | 8-15 | 1-15 |
Statistics | test | Av | Wi | Wi |
| Wi | Wi | Wi |
1F | Mean | 269 | 274 | 278 |
| 4 | 4 | 8 |
| SD | 16.7 | 19.0 | 19.7 |
| 5.0 | 4.9 | 6.5 |
| N | 10 | 10 | 10 |
| 10 | 10 | 10 |
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|
2F | Mean | 267 | 267 | 272 |
| 1 | 5 | 5 |
| SD | 18.4 | 16.0 | 19.2 |
| 7.6 | 6.9 | 4.4 |
| N | 10 | 10 | 10 |
| 10 | 10 | 10 |
|
|
|
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|
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|
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|
3F | Mean | 273 | 277 | 282 |
| 4 | 5 | 9 |
| SD | 11.1 | 7.1 | 8.3 |
| 6.8 | 6.1 | 8.9 |
| N | 10 | 10 | 10 |
| 10 | 10 | 10 |
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|
4F | Mean | 273 | 279 | 288 |
| 6 | 9 | 15 |
| SD | 19.2 | 17.3 | 22.0 |
| 11.1 | 8.7 | 13.1 |
| N | 10 | 10 | 10 |
| 10 | 10 | 10 |
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| Body weight and body weight change - group mean values (g) for females during gestation (F0) | Request ID: 5447389 |
| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Group |
| Day |
|
|
|
| Change | Change | Change | Change |
/Sex |
| 0 | 7 | 14 | 20 |
| 0-7 | 7-14 | 14-20 | 0-20 |
Statistics | test | Wi | Wi | Wi | Wi |
| Wi | Wi | Wi | Wi |
1F | Mean | 279 | 307 | 338 | 412 |
| 28 | 31 | 75 | 133 |
| SD | 17.5 | 18.5 | 23.8 | 26.3 |
| 5.9 | 9.0 | 11.2 | 16.2 |
| N | 10 | 10 | 10 | 10 |
| 10 | 10 | 10 | 10 |
|
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|
2F | Mean | 273 | 302 | 333 | 411 |
| 29 | 31 | 74 | 139 |
| SD | 21.0 | 26.0 | 25.8 | 29.0 |
| 15.4 | 4.8 | 10.4 | 14.3 |
| N | 10 | 10 | 10 | 9 |
| 10 | 10 | 9 | 9 |
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|
3F | Mean | 286 | 314 | 348 | 422 |
| 29 | 34 | 73 | 136 |
| SD | 7.9 | 12.5 | 17.4 | 25.8 |
| 7.9 | 8.4 | 14.8 | 23.6 |
| N | 9 | 9 | 9 | 9 |
| 9 | 9 | 9 | 9 |
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4F | Mean | 289 | 322 | 358 | 441 |
| 33 | 36 | 83 | 152 |
| SD | 22.0 | 24.4 | 28.4 | 39.9 |
| 7.4 | 9.7 | 13.7 | 25.2 |
| N | 10 | 10 | 10 | 10 |
| 10 | 10 | 10 | 10 |
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| Body weight and body weight change - group mean values (g) for females during lactation (F0) | Request ID: 5447390 |
| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Group |
| Day |
|
|
|
| Change | Change | Change |
/Sex |
| 1 | 4 | 7 | 13 |
| 1-7 | 7-13 | 1-13 |
Statistics | test | Wi | Wi | Wi | Wi |
| Wi | Wi | Wi |
1F | Mean | 310 | 318 | 332 | 347 |
| 22 | 15 | 36 |
| SD | 20.8 | 21.7 | 22.0 | 17.6 |
| 4.8 | 10.0 | 9.6 |
| N | 10 | 10 | 10 | 10 |
| 10 | 10 | 10 |
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2F | Mean | 312 | 315 | 331 | 345 |
| 19 | 14 | 33 |
| SD | 23.5 | 26.5 | 26.7 | 29.4 |
| 6.0 | 13.7 | 9.9 |
| N | 9 | 9 | 9 | 9 |
| 9 | 9 | 9 |
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3F | Mean | 330 | 336 | 347 | 357 |
| 17 | 10 | 27 |
| SD | 21.8 | 22.9 | 26.2 | 19.5 |
| 10.9 | 18.1 | 15.6 |
| N | 9 | 9 | 9 | 9 |
| 9 | 9 | 9 |
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4F | Mean | 333 | 341 | 351 | 363 |
| 18 | 13 | 31 |
| SD | 33.7 | 28.0 | 30.2 | 25.2 |
| 5.2 | 11.3 | 14.5 |
| N | 10 | 10 | 10 | 10 |
| 10 | 10 | 10 |
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| Estrous cycles - group values before treatment (F0) |
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| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
| Number |
| Regular |
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|
| of |
| 4 or 5 day | Irregular |
|
Group | animals |
| cycles | cycle l | Acyclic y |
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1 | 10 | N | 10 | 0 | 0 |
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| (%) | (100) |
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2 | 10 | N | 10 | 0 | 0 |
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| (%) | (100) |
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3 | 10 | N | 10 | 0 | 0 |
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| (%) | (100) |
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4 | 10 | N | 10 | 0 | 0 |
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| (%) | (100) |
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l At least one cycle of two, three or six to ten days
y At least ten days without estrus
| Estrous cycles - group values during treatment (F0) |
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| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
| Number |
| Regular |
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|
| of |
| 4 or 5 day | Irregular |
|
Group | animals |
| cycles | cycle l | Acyclic y |
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1 | 10 | N | 8 | 2 | 0 |
|
| (%) | (80) | (20) |
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2 | 10 | N | 9 | 1 | 0 |
|
| (%) | (90) | (10) |
|
3 | 10 | N | 10 | 0 | 0 |
|
| (%) | (100) |
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4 | 10 | N | 8 | 2 | 0 |
|
| (%) | (80) | (20) |
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l At least one cycle of two, three or six to ten days
y At least ten days without estrus
| Pre-coital interval - group values (F0) |
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| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
| Number |
|
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|
| of |
| Pre-coital interval (days) | |||
Group | animals |
| 1-4 | 5-8 | 9-12 | 13-14 |
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1 | 10 | N | 10 | 0 | 0 | 0 |
|
| (%) | (100) |
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2 | 10 | N | 9 | 1 | 0 | 0 |
|
| (%) | (90) | (10) |
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3 | 10 | N | 10 | 0 | 0 | 0 |
|
| (%) | (100) |
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4 | 10 | N | 10 | 0 | 0 | 0 |
|
| (%) | (100) |
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| Mating performance and fertility - group values (F0) |
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| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Group |
|
| Number |
|
|
|
and | Number | Number | achieving | Percentage | Conception | Fertility |
sex | paired | mating | pregnancy | mating | rate (%) | index (%) |
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1M | 10 | 10 | 10 | 100 | 100 | 100 |
2M | 10 | 10 | 10 | 100 | 100 | 100 |
3M | 10 | 10 | 9 | 100 | 90 | 90 |
4M | 10 | 10 | 10 | 100 | 100 | 100 |
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|
1F | 10 | 10 | 10 | 100 | 100 | 100 |
2F | 10 | 10 | 10 | 100 | 100 | 100 |
3F | 10 | 10 | 9 | 100 | 90 | 90 |
4F | 10 | 10 | 10 | 100 | 100 | 100 |
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| Gestation length and gestation index - group values (F0) |
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| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
| Number of |
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|
|
| Number of complete |
Gestation |
| pregnant |
| Gestation length (days) | live litters | index | |||
Group | animals |
| 22 | 22.5 | 23 | 23.5 | born | (%) |
Statistics test |
| Lt |
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| ||||
1 | 10 | N | 3 | 5 | 2 | 0 | 10 | 100 |
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| (%) | (30) | (50) | (20) |
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2 | 10A | N | 2 | 2 | 5 | 0 | 9 | 90 |
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| (%) | (22) | (22) | (56) |
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3 | 9 | N | 1 | 2 | 5 | 1 | 9 | 100 |
|
| (%) | (11) | (22) | (56) | (11) |
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4 | 10 | N | 2 | 1 | 7 | 0 | 10 | 100 |
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| (%) | (20) | (10) | (70) |
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A Percentage distribution of gestation lengths calculated from nine animals - one pregnant female euthanized for welfare reasons
| Stage of estrous cycle at termination - group values (F0) |
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| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
| Number |
|
| Estrus before |
| Cycle stage at termination | |||
Group | smeared |
|
| termination |
| M | D | P | E |
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1 | 10 | N |
| 0 |
| 0 | 10 | 0 | 0 |
|
| (%) |
|
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| (100) |
|
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2 | 9 | N |
| 0 |
| 0 | 9 | 0 | 0 |
|
| (%) |
|
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|
| (100) |
|
|
3 | 9 | N |
| 0 |
| 0 | 9 | 0 | 0 |
|
| (%) |
|
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|
| (100) |
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4 | 10 | N |
| 0 |
| 0 | 10 | 0 | 0 |
|
| (%) |
|
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| (100) |
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D Diestrus
E Estrus
M Metestrus
P Pro-estrus
| Litter size - group mean values (F1) | Request ID: 5447416 |
| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Group |
| Implantations | Total @ | Live on Day |
|
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| |
/Sex |
|
|
| Before bleed | After bleed |
|
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| |
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|
|
| 1 | 4 | 4 | 7 | 11 | 13 |
Statistics | test | Wi | Wi | Wi | Wi |
|
|
|
|
1F | Mean | 15.9 | 14.7 | 14.2 | 14.1 | 12.2 | 12.2 | 12.2 | 12.2 |
| SD | 2.18 | 2.41 | 2.20 | 2.08 | 1.93 | 1.93 | 1.93 | 1.93 |
| N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
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|
2F | Mean | 15.4 | 14.9 | 14.6 | 14.4 | 12.6 | 12.6 | 12.6 | 12.6 |
| SD | 1.81 | 2.03 | 1.88 | 1.94 | 2.01 | 2.01 | 2.01 | 2.01 |
| N | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 |
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3F | Mean | 15.8 | 14.3 | 14.3 | 14.2 | 12.4 | 12.4 | 12.4 | 12.4 |
| SD | 2.77 | 2.45 | 2.45 | 2.33 | 1.81 | 1.81 | 1.81 | 1.81 |
| N | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 |
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4F | Mean | 16.8 | 16.5 | 16.4* | 16.2* | 14.2 | 14.2 | 14.2 | 14.2 |
| SD | 2.20 | 2.37 | 2.50 | 2.15 | 2.15 | 2.15 | 2.15 | 2.15 |
| N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
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@ - Includes offspring that died prior to the designated Day 1 of age |
| Offspring survival indices - group mean values (F1) | Request ID: 5447417 |
| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Group |
| Post implantation | Live birth | Viability index (%) | Lactation index (%) |
/Sex |
| survival index (%) | index (%) | Day 4 | Day 13 |
Statistics | test | Wi | Ch | Ch |
|
1F | Mean | 92.2 | 97.4 | 98.9 | 100.0 |
| SD | 4.18 | 4.45 | 2.41 | 0.00 |
| N | 10 | 10 | 10 | 10 |
| N<100% | 9 | 3 | 2 | 0 |
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|
2F | Mean | 95.6 | 98.0 | 99.2 | 100.0 |
| SD | 5.65 | 4.26 | 2.38 | 0.00 |
| N | 9 | 9 | 9 | 9 |
| N<100% | 4 | 2 | 1 | 0 |
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|
3F | Mean | 91.3 | 100.0 | 99.3 | 100.0 |
| SD | 6.09 | 0.00 | 1.96 | 0.00 |
| N | 9 | 9 | 9 | 9 |
| N<100% | 7 | 0 | 1 | 0 |
|
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4F | Mean | 98.1* | 99.3 | 99.0 | 100.0 |
| SD | 3.06 | 2.26 | 3.01 | 0.00 |
| N | 10 | 10 | 10 | 10 |
| N<100% | 3 | 1 | 1 | 0 |
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The following data were used for the statistics tests, animal indices for post implantation survival index and animal indices dichotomized to 1 when 100% and 0 otherwise for live birth and viability indices. |
| Offspring sex ratio - group mean values (F1) | Request ID: 5447418 |
| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Group |
| Total @ |
|
| Live (before bleed) on Day |
|
| Live (after bleed) on Day |
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| |||||
/Sex |
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| 1 |
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| 4 |
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| 4 |
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| 13 |
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| M | F | %M | M | F | %M | M | F | %M | M | F | %M | M | F | %M |
Statistics | test |
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| Wi |
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| Wi |
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| Wi |
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1F | Mean | 5.6 | 9.0 | 38.5 | 5.5 | 8.8 | 38.5 | 5.4 | 8.7 | 38.4 | 5.4 | 6.8 | 44.5 | 5.4 | 6.8 | 44.5 |
| SD | 1.26 | 1.89 | 7.17 | 1.27 | 1.75 | 6.89 | 1.17 | 1.64 | 6.90 | 1.17 | 1.55 | 8.44 | 1.17 | 1.55 | 8.44 |
| N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
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2F | Mean | 7.1 | 7.8 | 46.9 | 7.0 | 7.6 | 47.5 | 6.9 | 7.6 | 47.0 | 6.9 | 5.7 | 53.9 | 6.9 | 5.7 | 53.9 |
| SD | 2.98 | 2.39 | 16.82 | 2.78 | 2.51 | 17.20 | 2.85 | 2.51 | 17.41 | 2.85 | 2.35 | 18.72 | 2.85 | 2.35 | 18.72 |
| N | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 |
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3F | Mean | 7.1 | 7.2 | 48.9 | 7.1 | 7.2 | 48.9 | 7.0 | 7.2 | 48.7 | 7.0 | 5.4 | 55.5 | 7.0 | 5.4 | 55.5 |
| SD | 3.02 | 2.39 | 15.94 | 3.02 | 2.39 | 15.94 | 2.83 | 2.39 | 15.64 | 2.83 | 2.24 | 18.27 | 2.83 | 2.24 | 18.27 |
| N | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 |
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4F | Mean | 8.8 | 7.7 | 52.5* | 8.7 | 7.7 | 52.1* | 8.6 | 7.6 | 52.2* | 8.6 | 5.6 | 59.6 | 8.6 | 5.6 | 59.6 |
| SD | 2.57 | 1.06 | 9.03 | 2.71 | 1.06 | 9.64 | 2.55 | 1.07 | 9.79 | 2.55 | 1.07 | 10.43 | 2.55 | 1.07 | 10.43 |
| N | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
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@ - Includes offspring that died prior to the designated Day 1 of age and excludes unsexed offspring |
Ano-genital distance - group mean absolute and adjusted values for offspring (F1) |
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| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Group | Body weight (g) | Ano-genital | |
/Sex |
| Day 1 | distance (mm) |
Statistics test | Wi |
| |
1M | Mean | 7.1 | 4.2 |
| SD | 0.66 | 0.28 |
| N | 10 | 10 |
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2M | Mean | 7.0 | 4.1 |
| SD | 0.58 | 0.24 |
| N | 9 | 9 |
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3M | Mean | 7.4 | 4.2 |
| SD | 0.75 | 0.35 |
| N | 9 | 9 |
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4M | Mean | 7.0 | 4.2 |
| SD | 0.57 | 0.25 |
| N | 10 | 10 |
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Statistics test |
| Wi | |
1M | Adjusted Mean | 4.2 | |
2M | Adjusted Mean | 4.1 | |
3M | Adjusted Mean | 4.2 | |
4M | Adjusted Mean | 4.2 | |
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(cont), | Ano-genital distance - group mean absolute and adjusted values for offspring (F1) |
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| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Group | Body weight (g) | Ano-genital | |
/Sex |
| Day 1 | distance (mm) |
Statistics test | Wi |
| |
1F | Mean | 6.6 | 2.2 |
| SD | 0.75 | 0.10 |
| N | 10 | 10 |
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2F | Mean | 6.7 | 2.1 |
| SD | 0.56 | 0.07 |
| N | 9 | 9 |
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3F | Mean | 7.1 | 2.2 |
| SD | 0.73 | 0.16 |
| N | 9 | 9 |
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4F | Mean | 6.6 | 2.2 |
| SD | 0.63 | 0.09 |
| N | 10 | 10 |
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Statistics test |
| Wi | |
1F | Adjusted Mean | 2.2 | |
2F | Adjusted Mean | 2.1 | |
3F | Adjusted Mean | 2.2 | |
4F | Adjusted Mean | 2.2 |
| Body weight and body weight change - group mean values (g) for offspring (F1) | Request ID: 5447419 |
| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Group |
| Day of age (before bleed) | Day of age (after bleed) |
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| Change | Change | Change | Change | |||
/Sex |
| 1 | 1 @ | 4 | 4 | 7 | 11 | 13 |
| 1-4 | 4-7 | 11-13 | 1-13 |
Statistics | test | Wi | Wi | Wi | Wi | Wi | Wi | Wi |
| Wi | Wi | Wi | Wi |
1M | Mean | 7.1 | 7.1 | 9.9 | 9.9 | 15.0 | 22.9 | 27.2 |
| 2.8 | 5.0 | 4.3 | 20.1 |
| SD | 0.66 | 0.66 | 1.42 | 1.42 | 2.51 | 3.15 | 3.67 |
| 0.88 | 1.19 | 0.63 | 3.19 |
| N | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| 10 | 10 | 10 | 10 |
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2M | Mean | 7.0 | 7.0 | 9.7 | 9.7 | 14.7 | 22.4 | 26.4 |
| 2.6 | 5.0 | 4.0 | 19.3 |
| SD | 0.58 | 0.58 | 0.53 | 0.53 | 1.22 | 2.01 | 3.02 |
| 0.36 | 0.92 | 1.18 | 2.81 |
| N | 9 | 9 | 9 | 9 | 9 | 9 | 9 |
| 9 | 9 | 9 | 9 |
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3M | Mean | 7.4 | 7.4 | 10.5 | 10.5 | 15.8 | 24.2 | 28.2 |
| 3.1 | 5.3 | 3.9 | 20.8 |
| SD | 0.75 | 0.75 | 1.39 | 1.39 | 2.02 | 3.39 | 3.68 |
| 0.79 | 0.78 | 0.65 | 3.20 |
| N | 9 | 9 | 9 | 9 | 9 | 9 | 9 |
| 9 | 9 | 9 | 9 |
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4M | Mean | 7.0 | 7.0 | 9.6 | 9.6 | 14.1 | 21.2 | 25.0 |
| 2.6 | 4.5 | 3.9 | 18.0 |
| SD | 0.57 | 0.58 | 0.93 | 0.93 | 1.77 | 2.76 | 2.99 |
| 0.45 | 0.97 | 0.47 | 2.63 |
| N | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| 10 | 10 | 10 | 10 |
@ - Includes only those pups surviving after the bleed |
(cont) | Body weight and body weight change - group mean values (g) for offspring (F1) | Request ID: 5447419 |
| Control | Disperse Orange FC84508 | ||
Dose Group | 1 | 2 | 3 | 4 |
Dose (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Group |
| Day of age (before bleed) | Day of age (after bleed) |
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| Change | Change | Change | Change | |||
/Sex |
| 1 | 1 @ | 4 | 4 | 7 | 11 | 13 |
| 1-4 | 4-7 | 11-13 | 1-13 |
Statistics | test | Wi | Wi | Wi | Wi | Wi | Wi | Wi |
| Wi | Wi | Wi | Wi |
1F | Mean | 6.6 | 6.6 | 9.3 | 9.3 | 14.2 | 22.2 | 26.0 |
| 2.7 | 4.9 | 3.8 | 19.5 |
| SD | 0.77 | 0.73 | 1.36 | 1.37 | 2.33 | 2.96 | 3.47 |
| 0.74 | 1.04 | 0.77 | 2.96 |
| N | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| 10 | 10 | 10 | 10 |
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2F | Mean | 6.7 | 6.8 | 9.3 | 9.3 | 14.2 | 21.9 | 25.8 |
| 2.5 | 4.9 | 3.9 | 19.0 |
| SD | 0.56 | 0.58 | 0.58 | 0.58 | 1.23 | 2.03 | 2.64 |
| 0.33 | 0.79 | 0.79 | 2.48 |
| N | 9 | 9 | 9 | 9 | 9 | 9 | 9 |
| 9 | 9 | 9 | 9 |
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3F | Mean | 7.1 | 7.1 | 10.1 | 10.2 | 15.3 | 23.5 | 27.5 |
| 3.1 | 5.2 | 4.0 | 20.4 |
| SD | 0.73 | 0.72 | 1.12 | 1.14 | 1.81 | 3.08 | 3.44 |
| 0.61 | 0.83 | 0.60 | 3.15 |
| N | 9 | 9 | 9 | 9 | 9 | 9 | 9 |
| 9 | 9 | 9 | 9 |
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4F | Mean | 6.6 | 6.5 | 9.0 | 8.9 | 13.3 | 20.4 | 24.0 |
| 2.4 | 4.3 | 3.6 | 17.5 |
| SD | 0.63 | 0.64 | 1.11 | 1.06 | 2.04 | 2.92 | 3.32 |
| 0.57 | 1.14 | 0.53 | 2.93 |
| N | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| 10 | 10 | 10 | 10 |
@ - Includes only those pups surviving after the bleed |
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Study duration:
- subacute
- Species:
- rat
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
The purpose of this study was a screening test for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item, Disperse Orange FC84508, by oral gavage administration for at least four weeks.
Three groups of ten male and ten female rats (F0 generation) received Disperse Orange FC84508 at doses of 100, 300 or 1000 mg/kg bw/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The offspring (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, aqueous 0.5% (w/v) methylcellulose, at the same volume dose (10 mL/kg bw/day) as treated groups.
During the study, clinical condition, body weight, food consumption, estrous cycles, pre-coital interval, mating performance, gestation length, thyroid hormone analysis, organ weight and macroscopic pathology and histopathology investigations were undertaken.
The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. Blood samples were collected from selected offspring on Day 4 and Day 13 of age for thyroid hormone analysis.
Results
Parental responses
Analysis of serum T4 concentration revealed low group mean T4 concentration in F0 males given 300 or 1000 mg/kg bw/day, when compared to Controls. Mean serum T4 concentration in samples obtained from F0 males given 100 mg/kg bw/day were unaffected by treatment. Assessment of individual T4 values showed that the majority of values in F0 males at 300 or 1000 mg/kg bw/day were within the 95-percentile historical control range. However, four Control values were above this range, resulting in an apparent difference between Control and treated groups. Therefore, the observed differences at 300 or 1000 mg/kg bw/day, when compared to the Control group mean concentration in F0 males, were not attributed to treatment with Disperse Orange FC84508.
Administration of Disperse Orange FC84508 at dose levels up to and including 1000 mg/kg bw/day was generally well tolerated in adult animals. There were no clear treatment-related changes in clinical condition or signs observed in relation to dose administration at any dose level investigated. One female given 100 mg/kg bw/day was killed for welfare reasons on Gestation Day 18, and the major contributing factor to its poor clinical condition was thoracic abscessation. This finding was considered to be incidental and was not related to treatment with Disperse Orange FC84508.
Body weight gain and food consumption for males during treatment and for females before pairing, during gestation and lactation were unaffected by treatment at all dose levels investigated.
Estrous cycles during treatment, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by parental treatment.
Litter responses
The clinical condition and survival of the F1 offspring, with litter size, sex ratio, offspring birth weights, offspring body weight gain and ano-genital distance were not adversely affected by parental treatment of Disperse Orange FC84508. There were no macroscopic findings for offspring and there were no nipples apparent for male offspring on Day 13 of age. Mean serum T4 concentration for male and female F1 offspring on Day 13 of age were unaffected by parental treatment.
Conclusion
Administration of Disperse Orange FC84508 at doses up to and including 1000 mg/kg bw/day was well tolerated and no adverse effects were apparent. It was therefore concluded that the No-Observed-Adverse-Effect (NOAEL) for reproductive performance and for parental toxicity was above 1000 mg/kg bw/day.
Effects on developmental toxicity
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
No effects on reproduction observed, no classification necessary
Additional information
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.