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EC number: 221-897-7 | CAS number: 3271-76-9
- 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
Repeated dose toxicity: oral
Administrative data
- Endpoint:
- short-term repeated dose toxicity: oral
- Remarks:
- combined repeated dose and reproduction / developmental screening
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27 May 2015 to 10 March 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Cross-referenceopen allclose all
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27 May 2015 to 10 March 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- not specified
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on species / strain selection:
- The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and because there is ample experience and background data on this species and strain.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- A total of 125 Sprague Dawley [Crl:CD(SD)BR] rats (65 males and 60 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, were ordered from and supplied by Charles River Italia S.p.A., Calco (Lecco), Italy.After arrival, on 21 May 2015, the weight range for each sex was determined (189-216 g for females, 226-250 g for males, slightly outside the range at order) and the animals were temporarily identified within the cage by means of a coloured mark on the tail.A health check was then performed by a veterinarian. An acclimatisation period of at least 14 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C +/- 2°C and 55% +/- 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
- Route of administration:
- oral: gavage
- Vehicle:
- other: Sesame oil
- Details on exposure:
- The required amount of Vat Black 25 was dissolved/suspended in the vehicle. The formulations were prepared daily (concentrations of 12,5, 50 and 200 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
- Details on mating procedure:
- Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred or 14 days had elapsed. The pairing combination of one animal which did not have positive identification of mating after 14 days of pairing was changed within the treatment group (female no. 5). The subsequent pairing was monitored for mating as described above.
- Analytical verification of doses or concentrations:
- no
- Details on analytical verification of doses or concentrations:
- Analysis was not performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory as the test item is neither extractable in hydrophilic nor in lipophilic solvents. The correct concentration preparation was monitored in the weighing record of the test item in each formulation process.
- Duration of treatment / exposure:
- Main groups (Groups 1 to 4)
Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 43 and 44 of study). They were treated for a total of 42 or 43 days. Dose volumes were adjusted once per week for each animalaccording to the last recorded body weight.
Females
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum (for approximately 42 days). Dose volumes were adjusted once per week for each animal according to the last recorded bodyweight. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Recovery groups (Groups 5 and 6)
Animals were dosed once a day, 7 days a week, for a minimum of 6 consecutive weeks. No treatment was given during the recovery period.
Positive Control group (Group 7)
Animals received a single dose approximately 24 hours before sacrifice. - Frequency of treatment:
- Once daily
- Details on study schedule:
- Dose levels (0, 62.5, 250 and 1000 mg/kg body weight/day) were selected in consultation with the Sponsor based on information from previous studies.
- Dose / conc.:
- 62.5 mg/kg bw/day (nominal)
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- Each main group comprised 10 male and 10 female rats (Groups 1 to 4). Two groups (control and high dose levels) included 5 animals per sex which were sacrificed after 2 weeks of recovery (Groups 5 and 6). For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male rats (Group 7).
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- The test item was administered orally by gavage. The oral route was selected as it is a possible route of exposure of the test item in man.
Dose levels of 62.5, 250 and 1000 mg/kg bodyweight/day were selected by the Sponsor based on information from previous studies. - Positive control:
- For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male rats (Group 7).
- Parental animals: Observations and examinations:
- Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
Clinical signs
Main and Recovery groups:
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded.Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions (15-45 minutes after treatment).
Observations of the cage tray:
Observations of the cage tray were performed during mating period for all main groups and were recorded daily.
Positive Control group:
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded.
Neurotoxicity assessment (removal of animals from the home cage and open arena)
Once before commencement of treatment and at least once per week from the start of treatment until termination, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena (for at least 3 minutes). The test included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded. Animals were examined in an open arena for a minimum of 3 minutes.
Grip strength and sensory reaction to stimuli
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reaction to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli); an assessment of grip strength was also performed. Measurements were performed using a computer generated random order (for the main groups). For males (main groups), the tests were performed on Day 40 of the study and for females on Day 3 post partum (main groups). For animals of the recovery groups, the tests were performed on Day 41 of the study (during treatment) and once during Week 2 of recovery (Day 10).
Motor activity assessment (MA)
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order (for the main groups). For males (main groups) the tests were performed on Day 41 of the study and for females on Day 3 post partum (main groups). For animals of the recovery groups, the tests were performed on Day 42 of the study (during treatment) and once during Week 2 of recovery (Day 13).
Body weight
Main groups: Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.
Recovery groups: Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.
Positive Control group: Animals were weighed on the day of allocation and on the day of dosing.
Food consumption
Main groups: The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.
Recovery groups: The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation.
Clinical pathology investigations
During the necropsy procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group under conditions of food deprivation for haematological, coagulation and clinical chemistry evaluations.
The blood samples collected were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– Heparin anticoagulant for biochemical tests
– Citrate anticoagulant for coagulation tests
The measurements performed on blood samples are listed below:
Haematology
– Haematocrit
– Haemoglobin
– Red blood cell count
– Reticulocyte count
– Mean red blood cell volume
– Mean corpuscular haemoglobin
– Mean corpuscular haemoglobin concentration
– White blood cell count
– Differential leucocyte count
Neutrophils
Lymphocites
Eosinophils
Basophils
Monocytes
Large unstained cells
– Platelets
Coagulation
– Prothrombin time
– Activated partial thromboplastin time
Clinical chemistry
– Alkaline phosphatase
– Alanine aminotransferase
– Aspartate aminotransferase
– Gamma-glutamyltransferase
– Urea
– Creatinine
– Glucose
– Triglycerides
– Bile acids
– Inorganic phosphorus
– Total bilirubin
– Total cholesterol
– Total protein
– Albumin
– Globulin
– A/G Ratio
– Sodium
– Potassium
– Calcium
– Chloride - Oestrous cyclicity (parental animals):
- Vaginal smears were taken daily in the morning starting from two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine the following:1. anomalies of the oestrous cycle2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
- Sperm parameters (parental animals):
- Parameters examined in [all/PF1/F2] male parental generations:Testis weight, epididymis weight, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS).The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also performed.
- Litter observations:
- A parturition check was performed from Day 20 to Day 25 post coitum. Female nos. 47 and 53 (Group 3) which did not give birth after 25 days of post coitum period were sacrificed shortly after (Day 26 post coitum). These animals were found not pregnant at necropsy. In addition, female no. 69 (Group 4) was sacrificed for humane reasons on Day 3 post coitum and it was not possible to evaluate pregnancy.
As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1 and 4 post partum. Pups killed or dying during the lactation period were weighed before the despatch to necropsy. Observations were performed once daily for all litters.
Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum. - Postmortem examinations (parental animals):
- Main and Recovery groups
One animal, sacrificed for humane reasons, was killed under carbon dioxide asphyxiation. Animals that had completed the scheduled test period and selected for blood collection, were killed by exsanguination under isofluorane anaesthesia. Animals that had completed the scheduled test period and not selected for blood collection, were killed by carbon dioxide asphyxiation.
Positive Control group
Positive Control group animals were killed under carbon dioxide asphyxiation.
Parental males (Main groups)
The males were killed after the mating of all females or after at least 28 days of treatment period.
Parental females (Main groups)
The females with live pups were killed on Day 4 post partum. The females which did not give birth 25 days after positive identification of mating were killed shortly after.
Males and females (Recovery groups)
Animals were killed after 2 weeks of recovery.
Positive Control group
Animals were killed approximately 24 hours after treatment.
Necropsy (Main and Recovery groups)
The clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed (excluding the animal sacrificed for humane reasons) and the required tissue samples preserved in fixative and processed for histopathological examination.
Females (Main groups)
All females were also examined for the following:
– external and internal abnormalities;
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals).
Uteri of apparently non-pregnant females or uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.
Organ weights (Main and Recovery groups)
From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed:
Adrenal glands
Brain (cerebrum, cerebellum, medulla/pons)
Epididymides
Heart
Kidneys
Liver
Ovaries with oviducts
Parathyroid glands
Prostate gland
Seminal vesicles with coagulating glands
Spleen
Testes
Thymus
Thyroid
Uterus including cervix
The ratios of organ weight to body weight were calculated for each animal.
Tissues fixed and preserved (Main and Recovery groups)
Samples of all the tissues listed below in were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).Tissues fixed and preserved (Main and Recovery groups)Samples of all the tissues listed below in were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).
Abnormalities
Adrenal glands
Bone marrow (from sternum)
Brain (cerebrum, cerebellum, medulla/pons)
Caecum
Clitoral gland
Colon
Duodenum
Epididymides
Heart
Ileum (including Peyer’s patches)
Jejunum
Kidneys
Liver
Lungs (including mainstem bronchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Nasal cavity
*Oesophagus
*Ovaries with oviducts
Parathyroid glands
Pituitary gland
Penis
Prostate gland
Rectum
Sciatic nerve
Seminal vesicles with coagulating glands
Spinal column
*Spinal cord (cervical, thoracic, lumbar)
Spleen
Stomach (forestomach and glandular)
Testes
Thymus (where present)
Thyroid
Trachea
Urinary bladder
Uterus including cervix
Vagina
*not examined as no signs of toxicity or target organ involvement were observed
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also performed.
The examination was restricted as detailed below:
i Tissues specified above from 5 males and 5 females (main groups) randomly selected (animals evaluated for clinical pathology) inthe control and high dose group killed at term.
ii Tissues specified above from all animals killed or dying during the treatment period.
iii All abnormalities in all main groups. - Postmortem examinations (offspring):
- Pups that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal.
All pups found dead in the cage were examined for external and internal abnormalities. All live pups sacrificed at termination were killed and examined for external abnormalities and sex confirmation by gonadal inspection. - Statistics:
- Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The nonparametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups was assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p < 0.05.
- Reproductive indices:
- Group mean values were calculated for all parameters. Data from nonpregnant females were excluded from group mean calculations.
Males
Copulatory Index (%) = no: of mated x100/ no: of males paired
Fertility Index (%) = no: of males which induced pregnancy x 100 / no: of paired
Females
Copulatory Index (%) = no: of mated x 100 / no: of paired
Fertility Index (%) = no: of pregnant females x 100 / no: of females paired - Offspring viability indices:
- Males and females
Copulatory Interval (Mean time to mating) = Mean number of days between pairing and mating
Females
Pre-birth loss (post-implantation loss) was calculated as a percentage from the formula:no: of visible implantations - total litter size at birth/ no: of visible implantations x 100
Pre-implantation loss was calculated as a percentage from the formula:no: of corpora lutea - no: of visible implantations/no: of corpora lutea x 100
Pup loss at birth was calculated as a percentage from the formula:Total litter size - live litter size/ Total litter size x 100
Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:Total litter size at birth - live litter size at Day 4/ Total litter size at birth x 100
Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No clinical signs of toxicological relevance or signs of neurotoxicity (weekly observations at removal from the cage and in an open arena, alterations in motor activity, grip strength and sensory reaction to stimuli) were observed during the study in males and females. Slight orange staining in the cage tray was observed during the mating phase in some cages of mid- and high dose animals.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One female (animal no. 69) of the high dose group, receiving 1000 mg/kg bodyweight/day was killed for humane reasons on Day 3 of the gestation phase. On the day of sacrifice, hunched posture and marked swelling of the fore and hind limbs were recorded. Macroscopic findings observed at post mortem examination in this animal were represented by swollen and oedematous consistency of hindlimbs and right forelimb, as well as unilateral pelvic dilatation.Marked arthritis of hindlimbs and one forelimb, observed at histopathology, could be considered as a factor contributory to the illness status of the animal sacrificed for humane reasons.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No differences in body weight were observed in treated animals compared to the control group.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No differences in food consumption were observed in treated animals compared to the control group.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No relevant changes were observed. The statistically significant differences between controls and treated males (mean corpuscular volume, neutrophils and eosinophils percentages), were of minimal severity and/or not dose-related, therefore considered of no toxicological significance.
Coagulation: No changes were recorded. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No relevant changes were observed. The statistically significant differences recorded (urea, glucose, chloride, phosphorus in males) were not dose-related,
therefore considered unrelated to treatment. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- Main and recovery groups
Observation of animals at removal from the cage and in an open arena did not reveal changes attributable to the test item.
No alterations in motor activity, grip strength and sensory reaction to stimuli were observed in any treatment group at the examination performed at the end of treatment.
At the end of recovery period, motor activity, grip strength and sensory reaction to stimuli were comparable between the control and treated group in both sexes. - 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):
- No microscopic observation was observed in treated animals that could be considered treatment-related.
A limited number of lesions, reported in control and/or treated animals, were considered to be an expression of spontaneous and/or incidental pathology, seen in this species and age of untreated animals.
In addition seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted. - Histopathological findings: neoplastic:
- no effects observed
- Description (incidence and severity):
- No microscopic observation was observed in treated animals that could be considered treatment-related.
- Other effects:
- not examined
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- Concerning reproductive parameters, no treatment-related anomalies were noted in the oestrus cycle of the treated females when compared to controls. Oestrus cycle did not show intergroup differences.
- Reproductive function: sperm measures:
- no effects observed
- Description (incidence and severity):
- Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- Concerning reproductive parameters, no treatment-related anomalies were noted in the oestrus cycle of the treated females when compared to controls. Oestrus cycle, pre-coital intervals, copulatory index and fertility index did not show intergroup differences.
All females mated. However, 2 females in the mid-dose group (nos. 47 and 53) were found not pregnant. One male of the control group (no. 6) did not mate.
The copulatory index was 100% for females of all dose groups and males of Groups 2, 3 and 4, while it was 90% for control males. The fertility indices were 80% in Group 3 and 100% in Groups and 1, 2, and 4 for both sexes. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Other: No effects of toxicological significance at highest dose tested: 1000 mg/kg bw/day
- Key result
- Critical effects observed:
- no
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Cold to touch, pale aspect, apparently no food intake (milk) and small appearance were the main clinical signs noted in control and treated pups.
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- Found dead pups were observed both in control and treated groups, without dose relation.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No significant differences in mean pup weights.
- 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
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Necropsy findings in deceased pups and in pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.
No milk in the stomach and autolysed abdominal/thoracic organs were generally observed in pups which died during the lactation period. - Histopathological findings:
- not examined
- Other effects:
- not examined
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOEL
- Generation:
- F1
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No effects of toxicological significance at highest dose tested: 1000 mg/kg bw/day
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Lowest effective dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Treatment related:
- no
- Relation to other toxic effects:
- not specified
- Dose response relationship:
- no
- Relevant for humans:
- no
- Conclusions:
- No treatment-related effects indicating systemic toxicity were observed in male or female animals at any of the dose levels investigated (0, 62.5, 250 and 1000 mg/kg body weight/day). No effects on sexual function and fertility or in developmental parameters and lactation were observed at any of the dose levels investigated.Based on the results of the present study, the NOEL (No Observed Effect Level) for reproductive and developmental toxicity was considered to be 1000 mg/kg bw/day for males and females.
- Executive summary:
The toxicity as well as any possible effects of Vat Black 25 when administered by oral route at dose levels of 62,5, 250 and 1000 mg/kg body weight/day, on male and female Sprague Dawley rats reproductive performance (such as gonadal function, mating behaviour, conception, development of conceptuses and parturition) and recovery from any treatment-related effects during a period of 2 weeks were investigated in this study.
One female of the high dose group, receiving 1000 mg/kg bodyweight/day, was killed for humane reasons on Day 3 of the gestation phase.
This death was considered to be unrelated to treatment.
No clinical signs of toxicological relevance or signs of neurotoxicity (weekly observations at removal from the cage and in an open arena, alterations in motor activity, grip strength and sensory reaction to stimuli) were observed during the study in males and females. Slight orange staining in the cage tray was observed during the mating phase in some cages of mid- and high dose animals.
No differences in body weight and food consumption were observed in treated animals compared to the control group.
No changes of toxicological significance were recorded at haematological, clinical chemistry investigations or at coagulation tests in animals sacrificed at the end of the study.
No treatment-related anomalies were noted in the oestrus cycle of the treated females when compared to controls. Copulatory and fertility indices did not show any treatment-related differences among treated and control groups.
Parturition, lactation, implantation, litter data and sex ratio did not show any changes of toxicological relevance.
Necropsy findings in pups did not reveal any treatment-related effect.
No relevant changes were detected at post mortem examination (terminal body weight, organ weights, macroscopic and microscopic examinations) in treated animals, when compared with controls.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within
the different stages and a regular layering in the germinal epithelium was described.
Based on the results of the present study, the NOEL (No Observed Effect Level) for general toxicity, reproductive and developmental toxicity was considered to be 1000 mg/kg bw/day for males and females.
OESTRUS CYCLE – BEFORE PAIRING – GROUP SUMMARY DATA
Animal Number |
Group 1 Oestrus Cycles |
Animal Number |
Group 2 Oestrus Cycles |
Animal Number |
Group 3 Oestrus Cycles |
Animal Number |
Group 4 Oestrus Cycles |
X0160001 X0160003 X0160005 X0160007 X0160009 X0160011 X0160013 X0160015 X0160017 X0150019 |
4 3 1 4 4 3 3 3 3 3 |
X0160021 X0160023 X0160025 X0160027 X0160029 X0160031 X0160033 X0160035 X0160037 X0160029 |
4 3 2 3 3 3 2 2 2 4 |
X0160041 X0160043 X0160045 X0160047 X0160049 X0160051 X0160053 X0160055 X0160057 X0160059 |
4 3 4 3 4 3 2 4 4 3 |
X0160061 X0160063 X0160065 X0160067 X0160069 X0160071 X0160073 X0160075 X0160077 X0160079 |
4 4 4 4 4 4 3 3 4 4 |
Means |
3.1 |
|
2.8 |
|
3.4 |
|
3.8 |
REPRODUCTIVE PARAMETERS OF ANIMALS – SUMMARY
MALES
Group |
1 |
2 |
3 |
4 |
Copulatory Index % |
90.0 |
100.0 |
100.0 |
100.0 |
Fertility Index % |
100.0 |
100.0 |
80.0 |
100.0 |
REPRODUCTIVE PARAMETERS OF ANIMALS – SUMMARY
FEMALES
Group |
1 |
2 |
3 |
4 |
Copulatory Index % |
100.0 |
100.0 |
100.0 |
100.0 |
Fertility Index % |
100.0 |
100.0 |
80.0 |
100.0 |
IMPLANTATION, PRE-IMPLANTATION LOSS DATA, PRE-BIRTH LOSS DATA AND GESTATION LENGTH OF FEMALES – GROUP MEAN DATA
Group |
|
Corpora Lutea |
Implantations |
Pre-Implantation loss % |
Total litter size at birth |
Pre-birth loss % |
Gestation length (days) |
1 |
Mean SD N |
18.30 2.87 10 |
17.40 2.46 10 |
4.61 4.28 10 |
16.30 1.70 110 |
5.74 6.95 0 |
22.10 0.32 10 |
2 |
Mean SD N |
15.70 4.14 10 |
15.30 4.32 10 |
3.70 6.84 10 |
14.60 4.33 110 |
4.39 6.64 0 |
22.20 0.42 10 |
3 |
Mean SD N |
16.38 2.67 8 |
16.38 2.67 8 |
0.00 0.00 8 |
15.88 2.53 8 |
2.88 4.41 8 |
22.38 0.52 8 |
4 |
Mean SD N |
15.11 1.17 9 |
15.11 1.17 9 |
0.00 0.00 9 |
14.22 1.20 9 |
5.71 6.91 9 |
22.00 0.00 9 |
Statistical analysis: Kruskall Wallis test
William’s test if group mean differences are different from control at p<0.05
* = mean value of group is significantly different from control
LITTER DATA AT BIRTH, ON DAY 1 AND ON DAY 4POST PARTUMOF FEMALES – GROUP MEAN DATA
Group |
|
At birth |
On Day 1post partum |
On Day 14post partum |
||||||
Total litter size |
Liver litter size |
Pup loss (%) |
Litter weight (g) |
Mean pup weight (g) |
Liver litter size |
Cumulative loss (%) |
Litter weight (g) |
Mean pup weight (g) |
||
1 |
Mean SD N |
16.30 1.70 10 |
16.20 1.81 10 |
0.67 2.12 10 |
106.16 18.56 10 |
6.87 0.70 10 |
13.40 4.88 10 |
16.29 29.78 10 |
129.70 50.81 10 |
9.20 1.84 10 |
2 |
Mean SD N |
14.60 4.33 10 |
14.40 4.27 10 |
1.26 2.66 10 |
103.16 26.81 10 |
7.29 0.88 10 |
14.20 4.10 10 |
2.41 3.15 10 |
138.17 33.24 10 |
10.04 1.35 10 |
3 |
Mean SD N |
15.88 2.53 8 |
14.13 5.11 8 |
12.18 27.18 8 |
97.63 38.48 8 |
7.24 0.94 8 |
13.25 5.26 8 |
17.36 29.71 8 |
133.74 51.32 8 |
10.30 1.35 8 |
4 |
Mean SD N |
14.22 1.20 9 |
14.22 1.20 9 |
0.00 0.00 9 |
99.81 10.15 9 |
7.21 0.61 9 |
13.44 1.74 9 |
5.41 9.44 9 |
132.79 20.00 9 |
9.88 0.72 9 |
Statistical analysis: Kruskall Wallis test
William’s test if group mean differences are different from control at p<0.05
* = mean value of group is significantly different from control
SEX RATIO OF PUPS – GROUP MEAN DATA
Group |
|
At birth |
On Day 4post partum |
||||||
M |
F |
Total |
%males |
M |
F |
Total |
%males |
||
1 |
Mean SD N |
7.00 1.70 10 |
9.30 2.67 10 |
16.30 1.70 10 |
43.60 12.14 10 |
6.00 2.67 10 |
7.40 3.27 10 |
13.40 4.88 10 |
41.28 18.56 10 |
2 |
Mean SD N |
7.70 2.26 10 |
6.90 3.63 10 |
14.60 4.33 10 |
56.81 19.68 10 |
7.40 2.12 10 |
6.80 3.61 10 |
14.20 4.10 10 |
56.31 19.89 10 |
3 |
Mean SD N |
8.00 1.85 8 |
7.88 3.64 8 |
15.88 2.53 8 |
52.05 15.49 8 |
6.63 3.07 8 |
6.63 3.70 8 |
13.25 5.26 8 |
50.71 13.38 8 |
4 |
Mean SD N |
7.00 1.50 9 |
7.22 1.79 9 |
14.22 1.20 9 |
49.43 10.92 9 |
6.67 1.66 9 |
6.78 1.79 9 |
13.44 1.74 9 |
49.68 10.89 9 |
Statistical analysis: Kruskall Wallis test
William’s test if group mean differences are different from control at p<0.05
* = mean value of group is significantly different from control
CLINICAL SIGNS OF PUPS – INDIVIDUAL DATA
Group 1
Animal Number |
Pup number |
Sign (Day (s)post partum) |
X0160001 |
All pups 6 1, 2, 5, 7+6, 9, 13, 16, 18 10, 14, 19 3, 4, 8, 11, 12, 15, 17 |
Cold to touch; Apparently no food intake (1-3); Smaller (3) Cold to touch; Apparently no food intake; Smaller (4) Found dead (4) Found dead (3) Missing (4) |
X0160003 |
9, 16 6 |
Smaller than others (1, 4); Cold to touch (1) Missing (1) |
X0160005 |
7 4 |
Smaller than others; Apparently no food intake (1); Found dead (2) Missing (3) |
X0160007 |
All pups |
N |
X0160009 |
12 |
Pale (0-3) |
X0160011 |
15 |
Found dead (2) |
X0160013 |
# 14 |
Found dead (0) Smaller than others (1-4); Apparently no food intake (4) |
X0160015 |
5, 10, 14, 15, 16 13 |
Found dead (1) Missing (1) |
X0160017 |
All pups |
N |
X0160019 |
9 |
Smaller than others (4) |
# = Pup dead prior to identification
CLINICAL SIGNS OF PUPS – INDIVIDUAL DATA
Group 2
Animal Number |
Pup number |
Sign (Day (s)post partum) |
X0160021 |
9 13 |
Pale (1-4) Smaller than others (4) |
X0160023 |
4 |
Humane Kill (1) |
X0160025 |
All pups |
N |
X0160027 |
All pups |
N |
X0160029 |
# |
Found dead (0) |
X0160031 |
All pups |
N |
X0160033 |
15 5 |
Smaller than others (1-4) Smaller than others (4) |
X0160035 |
All pups |
N |
X0160037 |
# |
Found dead (0) |
X0160039 |
All pups 4, 20 21 |
Smaller (0, 2, 3) Pale (2-4); Smaller than others (4) Missing (2) |
# = Pup dead prior to identification
CLINICAL SIGNS OF PUPS – INDIVIDUAL DATA
Group 3
Animal Number |
Pup number |
Sign (Day (s)post partum) |
X0160041 |
All pups |
N |
X0160043 |
8 14, 17 |
Smaller than others (1) Pale (1) |
X0160045 |
15 2 |
Missing (1) Missing (2) |
X0160049 |
7 |
Smaller than others (1-3) |
X0160051 |
17 |
Smaller than others (0-4) |
X0160055 |
#, # 16 3, 13, 15 10 |
Found dead (0) Smaller than others (1) Found dead (1) Found dead (2) |
X0160057 |
#, #, #, #, #, #, #, #, #, #, # #, # 2 |
Found dead (0) Cold to touch; Apparently no food intake (0) Missing (1) |
X0160059 |
# |
Found dead (0) |
# = Pup dead prior to identification
CLINICAL SIGNS OF PUPS – INDIVIDUAL DATA
Group 4
Animal Number |
Pup number |
Sign (Day (s)post partum) |
X0160061 |
All pups |
N |
X0160063 |
10 14 |
Pale (1) Pale (1-4); Smaller than others (4) |
X0160065 |
15 4, 13, 14 |
Smaller than others (0); Missing (1) Missing (2) |
X0160067 |
All pups |
N |
X0160071 |
11 |
Tail missing (0-4) |
X0160073 |
2 |
Missing (1) |
X0160075 |
All pups |
N |
X0160077 |
All pups |
N |
X0160079 |
All pups |
N |
# = Pup dead prior to identification
NECROPSY FINDINGS IN HUMANE KILL PUPS – INDIVIDUAL DATA
Group 2
Animal Number |
Day of sacrifice |
Sex |
Pup number |
Description |
X0160023 |
1 |
M |
4 |
Skin: cannibalised, dorsum |
NECROPSY FINDINGS IN DECEDENT PUPS – INDIVIDUAL DATA
Group 1
Animal Number |
Day found dead |
Sex |
Pup number |
Description |
X0160001 |
3 3 3 4 4 4 4 4 4 4 4 |
F F F M M M F F F F F |
10 14 19 1 2 5 7+6 9 13 16 18 |
Abdominal cavity: all organs autolysed Abdominal cavity: all organs autolysed Abdominal cavity: all organs autolysed Abdominal cavity: all organs autolysed Abdominal cavity: all organs autolysed Abdominal cavity: all organs autolysed Abdominal cavity: all organs autolysed Abdominal cavity: all organs autolysed Abdominal cavity: all organs autolysed Abdominal cavity: all organs autolysed Abdominal cavity: all organs autolysed |
X0160005 |
2 |
M |
7 |
Abdominal cavity: all organs autolysed |
X0160011 |
2 |
F |
15 |
N |
X0160013 |
0 |
M |
# |
Abdominal cavity: all organs autolysed No milk in stomach |
X0160015 |
1 1 1 1 1 |
M F F F F |
5 10 14 15 16 |
N N N N N |
N = No abnormalities detected
# = Dead prior the identification
NECROPSY FINDINGS IN DECEDENT PUPS – INDIVIDUAL DATA
Group 2
Animal Number |
Day found dead |
Sex |
Pup number |
Description |
X0160029 |
0 |
M |
# |
Abdominal cavity: all organs autolysed No milk in stomach |
X0160037 |
0 |
F |
# |
No milk in stomach |
# = Dead prior the identification
NECROPSY FINDINGS IN DECEDENT PUPS – INDIVIDUAL DATA
Group 3
Animal Number |
Day found dead |
Sex |
Pup number |
Description |
X0160055 |
0 0 1
1
1
2 |
F F M
F
F
F |
# # 3
13
15+1
10 |
No milk in stomach No milk in stomach Abdominal cavity: all organs autolysed No milk in stomach Abdominal cavity: all organs autolysed No milk in stomach Abdominal cavity: all organs autolysed No milk in stomach N |
X0160057 |
0
0
0
0
0
0
0
0
0
0
0
|
M
M
M
M
M
M
M
M
F
F
F |
#
#
#
#
#
#
#
#
#
#
# |
Abdominal cavity: all organs autolysed No milk in stomach Abdominal cavity: all organs autolysed No milk in stomach Abdominal cavity: all organs autolysed No milk in stomach Abdominal cavity: all organs autolysed No milk in stomach Abdominal cavity: all organs autolysed No milk in stomach Abdominal cavity: all organs autolysed No milk in stomach Abdominal cavity: all organs autolysed No milk in stomach Abdominal cavity: all organs autolysed No milk in stomach Abdominal cavity: all organs autolysed No milk in stomach Abdominal cavity: all organs autolysed No milk in stomach Abdominal cavity: all organs autolysed No milk in stomach |
X0160059 |
0 |
M |
# |
N |
N = No abnormalities detected
# = Dead prior the identification
NECROPSY FINDINGS IN DECEDENT PUPS – INDIVIDUAL DATA
Group 4
Animal Number |
Day found dead |
Sex |
Pup number |
Description |
X0160063 |
2 |
F |
10 |
N |
N = No abnormalities detected
NECROPSY FINDINGS IN PUPS SACRIFICED ON DAY 4POST PARTUM– INDIVIDUAL DATA
Group 1
Animal Number |
Sex |
Pup Number (s) |
Description |
X0160001 |
F |
6 |
No milk in stomach |
X0160003 |
M F |
1, 2, 3, 4, 5 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 |
N N |
X0160005 |
M F |
1, 2, 3, 5, 6, 8 9, 10, 11, 12, 13, 14, 15, 16, 17 |
N N |
X0160007 |
M F |
1, 2, 4, 5, 6, 7, 8, 9 3, 10, 11, 12, 13, 14 |
N N |
X0160009 |
M F |
1, 2, 3, 4, 5, 6 7, 8, 9, 10, 11, 12, 13, 14, 15 |
N N |
X0160011 |
M F |
1, 2, 3, 4 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17 |
N N |
X0160013 |
M F F |
1, 2, 3, 4, 5, 6, 7 8, 9, 10, 11, 12, 13 14 |
N N No milk in stomach |
X0160015 |
M F |
1, 2, 3, 4, 6, 7 8, 9, 11, 12 |
N N |
X0160017 |
M F |
1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15 |
N N |
X0150019 |
M F |
1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 |
N N |
N = No abnormalities detected
NECROPSY FINDINGS IN PUPS SACRIFICED ON DAY 4POST PARTUM– INDIVIDUAL DATA
Group 2
Animal Number |
Sex |
Pup Number (s) |
Description |
X0160021 |
M F |
1, 2, 3, 4, 5, 6, 7, 8 9, 10, 11, 12, 13 |
N N |
X0160023 |
M F |
1, 2, 3, 5, 6, 7, 8, 11 9, 10, 12, 13, 14, 15 |
N N |
X0160025 |
M F |
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17 |
N N |
X0160027 |
F |
1, 2, 3, 4 |
N |
X0160029 |
M F |
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15 |
N N |
X0160031 |
M F |
1, 2, 3, 4 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 |
N N |
X0160033 |
M F |
1, 2, 3, 4, 5, 6, 7, 8 9, 10, 11, 12, 13, 14, 15, 16 |
N N |
X0160035 |
M F |
1, 2, 3, 4, 5, 6, 7, 8 9, 10, 11, 12, 13, 14 |
N N |
X0160037 |
M F |
1, 2, 3, 4, 5, 6, 7, 8 9, 10, 11, 12, 13, 14, 15 |
N N |
X0160039 |
M F |
1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 |
N N |
N = No abnormalities detected
NECROPSY FINDINGS IN PUPS SACRIFICED ON DAY 4POST PARTUM– INDIVIDUAL DATA
Group 3
Animal Number |
Sex |
Pup Number (s) |
Description |
X0160041 |
M F |
1, 2, 3, 4, 5, 6, 7, 8, 12, 13 9, 10, 11, 14 |
N N |
X0160043 |
M F |
1, 2, 3, 4, 5, 6 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 |
N N |
X0160045 |
M F |
1, 3, 4, 5, 6, 7, 8 9, 10, 11, 12, 13, 14 |
N N |
X0160047 |
NP |
|
|
X0160049 |
M F |
1, 2, 3, 4, 5, 6, 7 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 |
N N |
X0160051 |
M F |
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 12, 13, 14, 15, 16, 17 |
N N |
X0160053 |
NP |
|
|
X0160055 |
M F |
1, 2, 4, 5, 6 7, 8, 9, 11, 12, 14, 16, 17+15+18 |
N N |
X0160057 |
M F |
1 3 |
N N |
X00160059 |
M F |
1, 2, 3, 4, 5, 6 7, 8, 9, 10, 11 |
N N |
N = No abnormalities detected
NP = Not pregnant
NECROPSY FINDINGS IN PUPS SACRIFICED ON DAY 4POST PARTUM– INDIVIDUAL DATA
Group 4
Animal Number |
Sex |
Pup Number (s) |
Description |
X0160061 |
M F |
1, 2, 3, 4, 5, 6, 7, 8 9, 10, 11, 12, 13, 14, 15 |
N N |
X0160063 |
M F |
1, 2, 3, 4, 5, 7, 8 9, 10, 11, 12, 13, 14, 15 |
N N |
X0160065 |
M F |
1, 2, 3, 5, 6 7, 8, 9, 10, 11, 12 |
N N |
X0160067 |
M F |
1, 2, 3, 4, 5, 6, 7, 8 9, 10, 11, 12, 13, 14, 15 |
N N |
X0160071 |
M M F |
1, 2, 3, 4, 5, 9+6 11 6, 7, 8, 10, 11, 12, 13 |
N Tail: not evident N |
X0160073 |
M F |
1, 3, 4, 5, 8 6, 7, 9, 10, 11, 12, 13 |
N N |
X0160075 |
M F |
1, 2, 3, 4, 5, 6, 7 8, 9, 10, 11, 12, 13, 14, 15, 16 |
N N |
X0160077 |
M F |
1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14 |
N N |
X0160079 |
M F |
1, 2, 3, 4 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 |
N N |
N = No abnormalities detected
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27 May 2015 to 10 March 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on species / strain selection:
- The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and because there is ample experience
and background data on this species and strain. - Sex:
- male
- Details on test animals or test system and environmental conditions:
- A total of 125 Sprague Dawley [Crl:CD(SD)BR] rats (65 males and 60 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, were ordered from and supplied by Charles River Italia S.p.A., Calco (Lecco), Italy.After arrival, on 21 May 2015, the weight range for each sex was determined (189-216 g for females, 226-250 g for males, slightly outside the range at order) and the animals were temporarily identified within the cage by means of a coloured mark on the tail.A health check was then performed by a veterinarian. An acclimatisation period of at least 14 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C +/- 2°C and 55% +/- 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
- Route of administration:
- oral: gavage
- Vehicle:
- The vehicle for the test item was sesame oil.
The vehicle for the positive control item was sterile water for injection.
Test item
The required amount of Vat Black 25 was dissolved/suspended in the vehicle. The formulations were prepared daily (concentrations of 12,5, 50 and 200mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
Positive Control item
The required amount of positive control item was dissolved in the vehicle. The formulation was prepared on the day of dosing at a concentration of0.2 mg/mL. - Details on exposure:
- The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
Analysis was not performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory as the test item is neither extractable in hydrophilic nor in lipophilic solvents. The correct concentration preparation was monitored in the weighing record of the test item in each formulation process.
Positive Control
Animals received a single dose approximately 24 hours before sacrifice. Mitomycin-C (positive control) was administered once by intraperitoneal injection at the dose volume of 10 mL/kg body weight. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal. - Duration of treatment / exposure:
- Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 43 and 44 of study). They were treated for a total of 42 or 43 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
- Frequency of treatment:
- Once a day
- Dose / conc.:
- 62.5 mg/kg bw/day
- Dose / conc.:
- 250 mg/kg bw/day
- Dose / conc.:
- 1 000 mg/kg bw/day
- No. of animals per sex per dose:
- Each main group comprised 10 male and 10 female rats (Groups 1 to 4). Two groups (control and high dose levels) included 5 animals per sex which were sacrificed after 2 weeks of recovery (Groups 5 and 6). For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male rats (Group 7).
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male rats (Group 7).The required amount of positive control item was dissolved in the vehicle (sterile water for injection). The formulation was prepared on the day of dosing at a concentration of 0.2 mg/mL. No documents on Mitomycin-C are included in the report. Determination of the stability and concentration of solutions of the positive control item was not undertaken.Mitomycin-C (positive control) was administered once by intraperitoneal injection at the dose volume of 10 mL/kg body weight. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.Positive Control group animals were killed under carbon dioxide asphyxiation.
- Tissues and cell types examined:
- Bone marrow from one femur of males only.
- Details of tissue and slide preparation:
- Extraction of bone marrow
Samples of bone marrow were collected approximately 24 and 48 hours after the 2 final treatments, from the same 5 males of the main groups randomly selected for clinical pathology investigation (see section 4.4). Samples of bone marrow were also collected approximately 24 hours after the single treatment from all males of Group 7 (Positive Control group). One femur of each animal was rapidly dissected out and cleaned of surrounding tissue. In order to extract the bone marrow, the bone was cut at the proximal end and irrigated with foetal calf serum using a syringe. The suspension of cells was aspirated, and this procedure was repeated several times.Preparation of the smearsThe suspension thus obtained was centrifuged at 1000 rpm for at least 5 minutes and the supernatant was completely removed. The cells of the sediment were resuspended and transferred onto clean microscope slides as smear preparations. They were air-dried and then fixed with methanol for 10 minutes. Subsequently, slides were stained with haematoxylin and eosin solutions. Finally, the slides were rinsed in distilled water and allowed to dry.Scoring of the slides and data analysisFor each animal, at least three slides were prepared. These slides were randomised and coded by staff not subsequently involved in the scoring.The adequate quality and the sufficient number of cells were evaluated before scoring. Scoring was performed using a microscope and high-power objective. Immature polychromatic erythrocytes (PCEs) stain a pink-purple colour (since they retain basic ribosomal material for approximately 24 hours after enucleation), and can be distinguished from the pink normochromatic erythrocytes (NCE).Erythrocytes lack nuclei, making micronuclei obvious when present; the criteria of Schmid (1976) were used to score micronuclei. At least four thousand polychromatic erythrocytes per animal were scored for the presence of micronuclei. At the same time the number of normochromatic erythrocytes was recorded, as well as the number of micronucleated NCE. The proportion of immature erythrocytes among total erythrocytes gives an indication of the toxicity of the treatment; a reduction in the proportion indicates inhibition of cell division. Finally, the incidence of micronucleated PCE provides an index of induced genetic damage. - Evaluation criteria:
- Acceptance criteria
The assay was considered valid if the following criteria were met:
– The incidence of micronucleated PCEs of the vehicle control group fell within the historical negative control range.
– The positive control item results fell within the historical control range and were significantly increased, at statistical analysis, when comparedwith the concurrent negative control.
– 5 males per group were available for slide analysisEvaluation of results
The test item was considered to induce micronuclei if a statistically significant increase in the micronucleus incidence of polychromatic erythrocytes(at p< 0.05) was observed in any treatment group and a dose-effect relationship was demonstrated. Where statistically significant increases in the incidence of micronucleated PCEs were observed, but all results were inside the distribution of negative control values within this laboratory, then historical control data were used to demonstrate that these increases did not have any biological significance. - Statistics:
- Only counts from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleus frequencies per 1000 cells), a modified chi-squared calculation was employed to compare treated and control groups. The degree of heterogeneity within each group was first calculated and where significant it was taken into account in the comparison between groups. If there was no significant within-group heterogeneity, the chi-squared test was used to compare treated groups with the controls. If there was significant within-groups heterogeneity, then that group was compared with the controls using a variance ratio (F) value calculated from the between-group and within-group chi-squared values.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Incidence of micronucleated cells
Following treatment with the test item, a slight increase in the number of micronucleated PCEs over the concurrent negative control was observed for animals from the low and high dose groups; however, the incidences of micronucleated PCEs were comparable to RTC historical control data for negative control animals. A marked increase in the frequency of micronucleated PCEs was observed in the positive control group.Bone marrow cell toxicityThe ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.
Analysis of resultsFollowing treatment with the test item, statistically significant increases in the incidence of micronucleated PCEs over the negative control value were observed for animals from the low and high dose levels (p<0.01). A significant dose effect relationship was found after a trend test evaluation (p<0.05). The dose-related and statistically significant increases of micronucleated PCEs could be attributable to the low incidence of micronucleated cells in the vehicle control group, which fell in the lower part of the distribution range of historical control data. In addition, the frequency of micronucleated immature erythrocytes observed at all dose levels was inside the distribution of historical negative control data (95% upper confidence limit = 2.0), therefore the observed increases were not considered biologically meaningful. - Conclusions:
- Interpretation of results (migrated information): negative
On the basis of the results obtained, it is concluded that Vat Black 25 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions. - Executive summary:
The purpose of this study was to provide information on toxic effects on male and female rats after repeated dosing with the test item, the micronucleus test was included in order to assess the ability of the test item to induce cytogenetic damage in rat bone marrow, as measured by the induction of micronuclei in polychromatic erythrocytes.
Experimental procedures were based on the following guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Incidence of micro nucleated cells
Following treatment with the test item, a slight increase in the number of micro nucleated PCEs over the concurrent negative control was observed for animals from the low and high dose groups; however, the incidences of micro nucleated PCEs were comparable to RTC historical control data for negative control animals.
A marked increase in the frequency of micro nucleated PCEs was observed in the positive control group.
Bone marrow cell toxicity
The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.
Analysis of results
Following treatment with the test item, statistically significant increases in the incidence of micro nucleated PCEs over the negative control value were observed for animals from the low and high dose levels (p<0.01). A significant dose effect relationship was found after a trend test evaluation (p<0.05). The dose-related and statistically significant increases of micro nucleated PCEs could be attributable to the low incidence of micro nucleated cells in the vehicle control group, which fell in the lower part of the distribution range of historical control data. In addition, the frequency of micro nucleated immature erythrocytes observed at all dose levels was inside the distribution of historical negative control data (95% upper confidence limit = 2.0), therefore the observed increases were not considered biologically meaningful.
Conclusions
On the basis of the results obtained, it is concluded that Vat Black 25 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.
Dose level (mg/kg/day) |
Incidence in micronucleated PCEs |
PCE/s(PCEs+NCEs) % Over the mean control value |
||
Mean |
SE |
Range |
||
0.00 62.5 250 1000 Mitomycin-C 2.00 mg/kg |
0.2 0.9 0.3 0.9 8.2 |
0.1 0.3 0.1 0.2 1.2 |
0.0 – 0.5 0.3 – 1.8 0.0 – 0.5 0.5 – 1.5 5.5 – 12.5 |
100 101 97 96 97 |
SUMMARY OF INCIDENCE OF MICRONUCLEATED CELLS
INCIDENCE OF MICRONUCLEATED CELLS/1000 CELLS |
|||||||||||||
Dose level mg/kg/day |
Scored cells |
NCE/PCE Ratio |
PCE/(PCE+NCE) Ratio |
% over the Mean control value |
Polychromatic |
Normochromatic |
|||||||
PCE |
NCE |
Mean |
SE |
Min |
Max |
Mean |
SE |
Min |
Max |
||||
0.00 62.5 250 1000 |
20000 20000 20000 20000 |
22374 21840 23814 24165 |
1.12 1.09 1.19 1.21 |
0.47 0.48 0.46 0.45 |
100 101 97 96 |
0.2 0.9 0.3 0.9 |
0.1 0.3 0.1 0.2 |
0.0 0.2 0.0 0.5 |
0.5 1.8 0.5 1.5 |
0.0 0.0 0.0 0.0 |
0.0 0.0 0.0 0.0 |
0.0 0.0 0.0 0.0 |
0.0 0.2 0.0 0.2 |
Mitomycin C 2.00 |
20000 |
23531 |
1.18 |
0.46 |
97 |
8.2 |
1.2 |
5.5 |
12.5 |
0.0 |
0.0 |
0.0 |
0.0 |
NCE/PCE ratio: The ratio of NCE/PCE calculated as the mean of the ratio values for the individual animals.
PCE/(PCE+NCE) ratio: The ratio of PCE/(PCE+NCE) calculated as the ratio of the total PCEs over the total erythrocytes scored
% over the mean control: Percentage of the PCE/(PCE+NCE) ratio of each treated group value over the negative control value
MEAN: The group mean incidence of micronucleated PCEs and NCEs
SE: The standard error of the mean incidence
MIN: Minimum value observed in an individual animal
MAX: Maximum value observed in an individual animal
HISTORICAL CONTROL DATA
INCIDENCES OF MICRONUCLEATED PCEs (%) (1991 – 2015) |
||
Male animals |
||
|
Vehicle controls |
Positive controls |
Mean |
0.7 |
16.4 |
SD (σn-1) |
0.70 |
8.41 |
n |
165 |
164 |
Upper confidence limit (95%) |
2.0 |
NC |
Maximum |
3.5 |
47.5 |
Minimum |
0.0 |
0.5 |
SD = standard deviation
n = number of animals
NC = not calculated
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- yes
- Remarks:
- See below under 'Priniciples of method...'
- Qualifier:
- according to guideline
- Guideline:
- other: OECD 474 adopted on September 2014
- Deviations:
- yes
- Remarks:
- See below under 'Priniciples of method...'
- Principles of method if other than guideline:
- Protocol deviations:
Uterus of the female humanely killed on Day 3 post coitum was not immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation
and pregnancy was therefore not established in this animal.
Animal no. 41 was not observed on Day 4 post partum and recovery males and females were not observed on Day 47.
Animals of the positive control group were not weighed prior to sacrifice.
Parturition check for animal no. 3 started on Day 19 of gestation instead of Day 20.
Slides for micronucleus test were not dried overnight as indicated in the study protocol but as necessary to obtain complete dehydration.
These deviations were not considered to have compromised the purpose or conduct of the study.
No other deviations occurred. - GLP compliance:
- yes
- Limit test:
- no
Test material
- Reference substance name:
- Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions
- EC Number:
- 944-232-9
- Molecular formula:
- Not available - UVCB substance
- IUPAC Name:
- Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions
- Test material form:
- solid: particulate/powder
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- A total of 125 Sprague Dawley [Crl:CD(SD)BR] rats (65 males and 60 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, were ordered from and supplied by Charles River Italia S.p.A., Calco (Lecco), Italy.After arrival, on 21 May 2015, the weight range for each sex was determined (189-216 g for females, 226-250 g for males, slightly outside the range at order) and the animals were temporarily identified within the cage by means of a coloured mark on the tail.A health check was then performed by a veterinarian. An acclimatisation period of at least 14 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C +/- 2°C and 55% +/- 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study.There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
Administration / exposure
- Route of administration:
- oral: gavage
- Details on route of administration:
- The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
- Vehicle:
- other: Sesame oil
- Details on oral exposure:
- The required amount of Vat Black 25 was dissolved/suspended in the vehicle. The formulations were prepared daily (concentrations of 12,5, 50 and 200 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
- Analytical verification of doses or concentrations:
- no
- Details on analytical verification of doses or concentrations:
- Analysis was not performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory as the test item is neither extractable in hydrophilic nor in lipophilic solvents. The correct concentration preparation was monitored in the weighing record of the test item in each formulation process.
- Duration of treatment / exposure:
- Main groups (Groups 1 to 4):
Males:
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 43 and 44 of study). They were treated for a total of 42 or 43 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females:
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum (for approximately 42 days). Dose volumes were adjusted once per week for each animal according to the last recorded body weight. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day1 post partum. Thereafter individual dose volumes remained constant.
Recovery groups (Groups 5 and 6):
Animals were dosed once a day, 7 days a week, for a minimum of 6 consecutive weeks. No treatment was given during the recovery period.
Positive Control group (Group 7):
Animals received a single dose of the positive control (for genotoxicity endpoint) approximately 24 hours before sacrifice. - Frequency of treatment:
- Once daily
Doses / concentrationsopen allclose all
- Dose / conc.:
- 62.5 mg/kg bw/day (nominal)
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- Each main group comprised 10 male and 10 female rats (Groups 1 to 4). Two groups (control and high dose levels) included 5 animals per sex which were sacrificed after 2 weeks of recovery (Groups 5 and 6). For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male rats (Group 7).
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Dose levels have been selected in consultation with the Sponsor based on information from previous studies.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: A recovery group was included to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase. In addition, the micronucleus test was included (5 male rats of the main groups) in order to assess the ability of the test item to induce cytogenetic damage in rat bone marrow, as measured by the induction of micronuclei in polychromatic erythrocytes.
- Post-exposure recovery period in satellite groups: 2 weeks - Positive control:
- Positive control treatments for genotoxicity endpoints used the well-known clastogen Mitomycin-C (Sigma-Aldrich, batch no. SLBH9906V, expiry date: January 2018).
Examinations
- Observations and examinations performed and frequency:
- Main and Recovery groups:
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded.
Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions (15-45 minutes after treatment).
Observations of the cage tray
Observations of the cage tray were performed during mating period for all main groups and were recorded daily.
Positive Control group
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded.
Neurotoxicity assessment (removal of animals from the home cage and open arena).
Once before commencement of treatment and at least once per week from the start of treatment until termination, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena (for at least 3 minutes). The test included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded.
Grip strength and sensory reaction to stimuli
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reaction to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli); an assessment of grip strength was also performed. Measurements were performed using a computer generated random order (for the main groups). For males (main groups), the tests were performed on Day 40 of the study and for females on Day 3 post partum (main groups). For animals of the recovery groups, the tests were performed on Day 41 of the study (during treatment) and once during Week 2 of recovery (Day 10).
Motor activity assessment (MA)
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order (for the main groups). For males (main groups) the tests were performed on Day 41 of the study and for females on Day 3 post partum (main groups). For animals of the recovery groups, the tests were performed on Day 42 of the study (during treatment) and once during Week 2 of recovery (Day 13).
Body weight
Main groups
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.
Recovery groups
Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.
Positive Control group
Animals were weighed on the day of allocation and on the day of dosing.
Food consumption
Main groups
The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.
Recovery groups
The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation.
Clinical pathology investigations
During the necropsy procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group under conditions of food deprivation for haematological, coagulation and clinical chemistry evaluations.
The blood samples collected were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– Heparin anticoagulant for biochemical tests
– Citrate anticoagulant for coagulation tests
The measurements performed on blood samples are listed below:
-Haematology
– Haematocrit
– Haemoglobin
– Red blood cell count
– Reticulocyte count
– Mean red blood cell volume
– Mean corpuscular haemoglobin
– Mean corpuscular haemoglobin concentration
– White blood cell count
– Differential leucocyte count
Neutrophils
Lymphocites
Eosinophils
Basophils
Monocytes
Large unstained cells
– Platelets
Coagulation
– Prothrombin time
– Activated partial thromboplastin time
Clinical chemistry
– Alkaline phosphatase
– Alanine aminotransferase
– Aspartate aminotransferase
– Gamma-glutamyltransferase
– Urea
– Creatinine
– Glucose
– Triglycerides
– Bile acids
– Inorganic phosphorus
– Total bilirubin
– Total cholesterol
– Total protein
– Albumin
– Globulin
– A/G Ratio
– Sodium
– Potassium
– Calcium
– Chloride - Sacrifice and pathology:
- Main and Recovery groups
One animal, sacrificed for humane reasons, was killed under carbon dioxide asphyxiation. Animals that had completed the scheduled test period and selected for blood collection, were killed by exsanguination under isofluorane anaesthesia. Animals that had completed the scheduled test period and not selected for blood collection, were killed by carbon dioxide asphyxiation.
Positive Control group
Positive Control group animals were killed under carbon dioxide asphyxiation.
Parental males (Main groups)
The males were killed after the mating of all females or after at least 28 days of treatment period.
Parental females (Main groups)
The females with live pups were killed on Day 4 post partum. The females which did not give birth 25 days after positive identification of mating werekilled shortly after.
Males and females (Recovery groups)
Animals were killed after 2 weeks of recovery.
Positive Control group
Animals were killed approximately 24 hours after treatment.
Necropsy (Main and Recovery groups)
The clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed (excluding the animal sacrificed for humane reasons) and the required tissue samples preserved in fixative and processed for histopathological examination.
Females (Main groups)
All females were also examined for the following:
– external and internal abnormalities;
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals).
Uteri of apparently non-pregnant females or uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.
Organ weights (Main and Recovery groups)
From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed:
Adrenal glands
Brain (cerebrum, cerebellum, medulla/pons)
Epididymides
Heart
Kidneys
Liver
Ovaries with oviducts
Parathyroid glands
Prostate gland
Seminal vesicles with coagulating glands
Spleen
Testes
Thymus
Thyroid
Uterus including cervix
The ratios of organ weight to body weight were calculated for each animal.
Tissues fixed and preserved (Main and Recovery groups)
Samples of all the tissues listed below in were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).
Abnormalities
Adrenal glands
Bone marrow (from sternum)
Brain (cerebrum, cerebellum, medulla/pons)
Caecum
Clitoral gland
Colon
Duodenum
Epididymides
Heart
Ileum (including Peyer’s patches)
Jejunum
Kidneys
Liver
Lungs (including mainstem bronchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Nasal cavity
*Oesophagus
*Ovaries with oviducts
Parathyroid glands
Pituitary gland
Penis
Prostate gland
Rectum
Sciatic nerve
Seminal vesicles with coagulating glands
Spinal column
*Spinal cord (cervical, thoracic, lumbar)
Spleen
Stomach (forestomach and glandular)
Testes
Thymus (where present)
Thyroid
Trachea
Urinary bladder
Uterus including cervix
Vagina
*not examined as no signs of toxicity or target organ involvement were observed.
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS).
The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also performed.
The examination was restricted as detailed below:
i Tissues specified above from 5 males and 5 females (main groups) randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term.
ii Tissues specified above from all animals killed or dying during the treatment period.
iii All abnormalities in all main groups. - Other examinations:
- No data
- Statistics:
- Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The nonparametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups was assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p < 0.05.
Results and discussion
Results of examinations
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No signs of toxicological relevance were observed during the study.
Slight orange staining in the cage tray was observed during the mating phase in some cages of mid- and high dose animals.
Observation of animals at removal from the cage and in an open arena did not reveal changes attributable to the test item.
No alterations in motor activity, grip strength and sensory reaction to stimuli were observed in any treatment group at the examination performed at the end of treatment.
At the end of recovery period, motor activity, grip strength and sensory reaction to stimuli were comparable between control and treated groups of both sexes. - Mortality:
- no mortality observed
- Description (incidence):
- One female of the high dose group, receiving 1000 mg/kg bodyweight/day was killed for humane reasons on Day 3 of the gestation phase. On the day of sacrifice, hunched posture and marked swelling of the fore and hind limbs were recorded.
Macroscopic findings observed at post mortem examination in this animal were represented by swollen and oedematous consistency of hindlimbs and right forelimb, as well as unilateral pelvic dilatation.
Marked arthritis of hindlimbs and one forelimb, observed at histopathology, could be considered as a factor contributory to the illness status of the animal sacrificed for humane reasons. Therefore, this death was considered to be unrelated to treatment. - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Throughout the study, no difference in body weights was recorded in animals of both sexes compared to the control group.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No effects on food consumption were observed in both sexes.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No relevant changes were observed. The statistically significant differences between controls and treated males (mean corpuscular volume, neutrophils and eosinophils percentages), were of minimal severity and/or not dose-related, therefore considered of no toxicological significance.
Coagulation: no changes were recorded. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No relevant changes were observed. The statistically significant differences recorded (urea, glucose, chloride, phosphorus in males) were not dose-related, therefore considered unrelated to treatment.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- Main and recovery groups
Observation of animals at removal from the cage and in an open arena did not reveal changes attributable to the test item.
No alterations in motor activity, grip strength and sensory reaction to stimuli were observed in any treatment group at the examination performed at the end of treatment.
At the end of recovery period, motor activity, grip strength and sensory reaction to stimuli were comparable between the control and treated group in both sexes. - Immunological findings:
- not specified
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Absolute and relative organ weights were comparable in all groups.
The following statistically significant difference was noted in the organ weights of the main groups:
– Decreased relative uterus weight in females receiving 250 and 1000mg/kg body weight/day (-30 % and -25 %, respectively). Absolute uterus weight was also decreased in all groups when compared to controls. This difference was without dose-dependency and not accompanied by histological findings. Therefore, it was considered not to be treatment-related. No toxicological significance was attributed to the other statistically significances observed in the weight of organs. - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no compound-related effects.
No relevant changes were noted in study animals sacrificed at the end of the treatment period or after 2 weeks of recovery period.
Changes such as pelvic dilatation, swollen liver and small size of thymus in the control and treated groups, both in final and recovery animals, were considered to be incidental. - Neuropathological findings:
- not specified
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- No microscopic observation was observed in treated animals that could be considered treatment-related.
A limited number of lesions, reported in control and/or treated animals, were considered to be an expression of spontaneous and/or incidental pathology, seen in this species and age of untreated animals.
In addition seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Details on results:
- No signs of genotoxicity were detected by measuring the micronuclei in polychromatic erythrocytes.
No treatment-related effects indicating systemic toxicity were observed in male or female animals at any of the dose levels investigated (0, 62.5, 250 and 1000 mg/kg body weight/day).
Effect levels
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No effects of toxicological significance at highest dose tested: 1000 mg/kg bw/day
Target system / organ toxicity
- Key result
- Critical effects observed:
- no
Any other information on results incl. tables
FATE OF FEMALES – GROUP INCIDENCE
Group |
1 |
2 |
3 |
4 |
Initial group size |
10 |
10 |
10 |
10 |
Not pregnant |
0 |
0 |
2 |
0 |
Unilateral implantation |
0 |
1 |
0 |
0 |
Humane killed |
0 |
0 |
0 |
1a |
With live pups on Day 4 post partum |
10 |
10 |
8 |
9 |
a= pregnancy not detected
CLINICAL SIGNS OF MALES – MAIN GROUPS – GROUP INCIDENCE
Interval: 1! – 30” Days Group Observation |
1 (10) |
2 (10) |
3 (10) |
4 (10) |
||||
|
a |
b |
a |
b |
a |
b |
a |
b |
No significant signs |
10 |
38.9 |
10 |
43.5 |
10 |
43.5 |
10 |
42.2 |
APPEARANCE Hair loss |
1 |
30.0 |
0 |
0.0 |
0 |
0.0 |
2 |
6.5 |
EYE – EAR – MOUTH Damaged ear |
1 |
16.0 |
0 |
0.0 |
0 |
0.0 |
0 |
0.0 |
Key: () = Number of animals alive at start of interval
a = Number of animal affected
b = Number of days with clinical sign/animal
Note: ! = Premating phase; “ = Mating phase
CLINICAL SIGNS OF FEMALES BEFORE PAIRING – MAIN GROUPS – GROUP INCIDENCE
Interval: 1 - 15 Days Group Observation |
1 (10) |
2 (10) |
3 (10) |
4 (10) |
||||
|
a |
b |
a |
b |
a |
b |
a |
b |
No significant signs |
10 |
15.0 |
10 |
15.0 |
10 |
15.0 |
10 |
15.0 |
Key: () = Number of animals alive at start of interval
a = Number of animal affected
b = Number of days with clinical sign/animal
CLINICAL SIGNS OF FEMALES –POST COITUMANDPOST PARTUMPERIODS - MAIN GROUPS – GROUP INCIDENCE
Interval: 0! – 4” Days Group Observation |
1 (10) |
2 (10) |
3 (10) |
4 (10) |
||||
|
a |
b |
a |
b |
a |
b |
a |
b |
No significant signs |
10 |
27.1 |
10 |
27.2 |
10 |
27.1 |
10 |
24.1 |
APPEARANCE Cyphosis Swollen |
0 0 |
0.0 0.0 |
0 0 |
0.0 0.0 |
0 0 |
0.0 0.0 |
1 1 |
1.0 1.0 |
Key: () = Number of animals alive at start of interval
a = Number of animal affected
b = Number of days with clinical sign/animal
Note: ! = Gestation phase phase; “ = Postpartum phase
CLINICAL OBSERVATIONS DURING TREATMENT – BEHAVIOURAL EXAMINATION – REMOVAL OF ANIMALS FROM THE HOME CAGE - MAIN GROUPS – GROUP INCIDENCE
MALES
Interval: 1! – 6” Weeks Group Observation |
1 (10) |
2 (10) |
3 (10) |
4 (10) |
||||
|
a |
b |
a |
b |
a |
b |
a |
b |
REMOVAL Removal easy |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
HANDLING REACTIVITY Handling reactivity normal |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
LACHRYMATION Lachrymation absent |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
PALPEBRAL CLOSURE Palpebral closure absent |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
SALIVATION Salivation absent |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
PILOERECTION Piloerection absent |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
Key: () = Number of animals alive at start of interval
a = Number of animals affected
b = Number of week with clinical sign/animal
Note: ! = Pretest phase; “ = Dosing phase
CLINICAL OBSERVATIONS DURING TREATMENT – BEHAVIOURAL EXAMINATION – REMOVAL OF ANIMALS FROM THE HOME CAGE - MAIN GROUPS – GROUP INCIDENCE
FEMALES
Interval: 1! – 6” Weeks Group Observation |
1 (10) |
2 (10) |
3 (10) |
4 (10) |
||||
|
a |
b |
a |
b |
a |
b |
a |
b |
REMOVAL Removal easy |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
HANDLING REACTIVITY Handling reactivity normal |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
LACHRYMATION Lachrymation absent |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
PALPEBRAL CLOSURE Palpebral closure absent |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
SALIVATION Salivation absent |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
PILOERECTION Piloerection absent |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
Key: () = Number of animals alive at start of interval
a = Number of animals affected
b = Number of week with clinical sign/animal
Note: ! = Pretest phase; “ = Dosing phase
CLINICAL OBSERVATIONS DURING TREATMENT – BEHAVIOURAL EXAMINATION – OPEN AREA – MAIN GROUPS – GROUP INCIDENCE
MALES
Interval: 1! – 6” Weeks Group Observation |
1 (10) |
2 (10) |
3 (10) |
4 (10) |
||||
|
a |
b |
a |
b |
a |
b |
a |
b |
REARING Rearing absent Rearing 1-3 Rearing 4-7 Rearing 8-10 Rearing 11-14 Rearing 15-20 Rearing 21-30 |
0 2 5 9 10 5 0 |
0.0 1.5 2.6 1.7 3.0 1.8 0.0 |
0 1 4 9 10 5 0 |
0.0 2.0 1.8 2.8 2.8 1.6 0.0 |
1 2 3 8 10 8 0 |
2.0 3.0 1.7 2.0 2.2 2.4 0.0 |
0 1 2 9 10 7 2 |
0.0 1.0 1.0 2.7 2.8 1.9 1.0 |
SPASMS Spasms absent |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
MYOCLONIA Myoclonia absent |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
GAIT Normal gait |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
MOBILITY IMPAIRMENT Mobility impairment absent |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
AROUSAL Arousal normal Arousal slow |
10 0 |
7.0 0.0 |
10 0 |
7.0 0.0 |
10 1 |
6.8 2.0 |
10 0 |
7.0 0.0 |
VOCALISATION Vocalisation absent |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
STEREOTYPIES Stereotypies absent |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
UNUSUAL RESPIRATION Unusual respiration absent |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
BIZARRE BEHAVIOUR Bizarre behaviour absent |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
URINATION Urination absent Urination 1-3 Urination 4-6 Urination 7-9 Urination more than 10 |
10 8 2 3 0 |
4.0 3.0 1.5 1.0 0.0 |
10 7 6 1 1 |
4.8 1.7 1.2 1.0 2.0 |
10 6 5 0 1 |
5.0 2.2 1.2 0.0 1.0 |
10 7 3 2 0 |
5.1 1.3 2.7 1.0 0.0 |
DEFECATION Defecation absent Defecation 1-3 Defecation 4-6 |
10 6 3 |
4.9 2.5 2.0 |
10 6 3 |
6.0 1.2 1.0 |
10 5 1 |
6.2 1.4 1.0 |
10 6 2 |
5.8 1.7 1.0 |
TREMORS Tremors absent |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
10 |
7.0 |
Key: () = Number of animals alive at start of interval
a = Number of animals affected
b = Number of weeks with clinical sign/animal
Note: ! = Pretest phase; “ = Dosing phase
CLINICAL OBSERVATIONS DURING TREATMENT – BEHAVIOURAL EXAMINATION – OPEN AREA – MAIN GROUPS – GROUP INCIDENCE
FEMALES
Interval: 1! – 6” Weeks Group Observation |
1 (10) |
2 (10) |
3 (10) |
4 (10) |
||||
|
a |
b |
a |
b |
a |
b |
a |
b |
REARING Rearing 11-14 Rearing 15-20 Rearing 21-30 Rearing more than 30 |
5 10 8 4 |
2.2 3.6 2.3 1.3 |
5 10 6 1 |
2.4 3.6 3.2 1.0 |
7 7 6 5 |
1.6 4.4 3.3 1.4 |
6 10 6 1 |
2.7 3.6 2.2 1.0 |
SPASMS Spasms absent |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
MYOCLONIA Myoclonia absent |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
GAIT Normal gait |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
MOBILITY IMPAIRMENT Mobility impairment absent |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
AROUSAL Arousal normal |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
VOCALISATION Vocalisation absent |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
STEREOTYPIES Stereotypies absent |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
UNUSUAL RESPIRATION Unusual respiration absent |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
BIZARRE BEHAVIOUR Bizarre behaviour absent |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
URINATION Urination absent Urination 1-3 Urination 4-6 Urination 7-9 Urination more than 10 |
10 7 3 1 1 |
5.1 1.7 1.7 1.0 1.0 |
10 6 3 0 0 |
5.6 1.5 1.0 0.0 0.0 |
10 5 5 2 0 |
5.2 1.6 1.4 1.0 0.0 |
10 9 4 0 0 |
4.0 2.1 1.8 0.0 0.0 |
DEFECATION Defecation absent Defecation 1-3 |
10 2 |
6.7 1.0 |
10 3 |
6.5 1.0 |
10 1 |
6.8 1.0 |
10 3 |
6.3 1.0 |
TREMORS Tremors absent |
10 |
6.9 |
10 |
6.8 |
10 |
6.9 |
10 |
6.6 |
Key: () = Number of animals alive at start of interval
a = Number of animals affected
b = Number of weeks with clinical sign/animal
Note: ! = Pretest phase; “ = Dosing phase
BODY WEIGHT (g) OF MALES – BEFORE PAIRING – MAIN GROUPS – GROUP MEAN DATA
Group(s) |
|
Day of Phase |
|||
1! |
1” |
8 |
15# |
||
1 |
(n) Mean SD |
10 291.27 8.04 |
10 351.93 14.40 |
10 391.82 19.31 |
10 418.66 24.10 |
2 |
(n) Mean SD |
10 290.91 6.82 |
10 343.76 11.97 |
10 375.38 16.31 |
10 400.39 20.38 |
3 |
(n) Mean SD |
10 290.21 8.48 |
10 344.05 15.02 |
10 378.62 15.50 |
10 404.02 17.39 |
4 |
(n) Mean SD |
10 291.32 7.25 |
10 351.16 12.67 |
10 387.61 16.39 |
10 416.80 17.10 |
Note: ! = Pretest phase; “ = Premating phase; # = Start of pairing phase
* = mean value of group is significantly different from control at p<0.05
** = mean value of group is significantly different from control at p<0.01
Statistical analysis:Dunnett’s test if group variances are homogenous
Modified t test is group variances are inhomogenous ($)
BODY WEIGHT (g) OF MALES – FROM PAIRING PERIOD TO SACRIFICE – MAIN GROUPS – GROUP MEAN DATA
Group(s) |
|
Day of Phase |
|||
8 |
15 |
22 |
29 |
||
1 |
(n) Mean SD |
10 440.52 28.74 |
10 463.91 35.21 |
10 484.62 32.37 |
10 491.88 43.22 |
2 |
(n) Mean SD |
10 424.16 21.21 |
10 445.27 27.57 |
10 467.04 31.46 |
10 474.21 35.30 |
3 |
(n) Mean SD |
10 424.73 19.19 |
10 449.58 19.40 |
10 470.60 23.64 |
10 479.52 34.21 |
4 |
(n) Mean SD |
10 436.14 16.77 |
10 463.39 22.11 |
10 484.57 21.84 |
10 492.04 28.45 |
Note: Data for Mating phase
* = mean value of group is significantly different from control at p<0.05
** = mean value of group is significantly different from control at p<0.01
Statistical analysis:Dunnett’s test if group variances are homogenous
Modified t test is group variances are inhomogenous ($)
BODY WEIGHT (g) OF FEMALES – BEFORE PAIRING – MAIN GROUPS – GROUP MEAN DATA
Group(s) |
|
Day of Phase |
|||
1! |
1” |
8 |
15# |
||
1 |
(n) Mean SD |
10 214.83 8.35 |
10 236.18 5.69 |
10 242.60 6.15 |
10 2.53.35 12.29 |
2 |
(n) Mean SD |
10 215.15 9.37 |
10 234.15 8.25 |
10 239.03 15.65 |
10 253.72 14.16 |
3 |
(n) Mean SD |
10 214.46 8.79 |
10 235.66 8.48 |
10 244.87 12.84 |
10 256.33 12.30 |
4 |
(n) Mean SD |
10 214.42 8.98 |
10 231.99 9.54 |
10 243.20 9.19 |
10 254.36 12.53 |
Note: ! = Pretest phase; “ = Premating phase; # = Start of pairing phase
* = mean value of group is significantly different from control at p<0.05
** = mean value of group is significantly different from control at p<0.01
Statistical analysis:Dunnett’s test if group variances are homogenous
Modified t test is group variances are inhomogenous ($)
BODY WEIGHT (g) OF PREGNANT FEMALES –POST COITUMANDPOST PARTUMPERIODS – MAIN GROUPS – GROUP MEAN DATA
Group(s) |
|
Day of Phase |
|||||
|
0! |
7 |
14 |
20 |
1” |
4 |
|
1 |
(n) Mean SD |
10 262.25 21.09 |
10 294.87 15.77 |
10 334.16 15.23 |
10 424.81 25.24 |
10 314.68 19.39 |
10 315.23 35.00 |
2 |
(n) Mean SD |
10 256.72 13.40 |
10 294.21 15.86 |
10 332.61 19.14 |
10 414.23 33.26 |
10 316.05 20.48 |
10 308.52 20.76 |
3 |
(n) Mean SD |
8 258.81 16.48 |
8 297.51 16.70 |
8 336.34 20.84 |
8 429.21 31.50 |
8 321.90 24.58 |
8 310.40 31.87 |
4 |
(n) Mean SD |
9 356.60 6.76 |
9 288.48 9.48 |
9 323.19 8.28 |
9 402.31 12.52 |
9 311.44 9.82 |
9 300.98 20.89 |
Note: ! = Gestation phase; “ =Post partumphase
* = mean value of group is significantly different from control at p<0.05
** = mean value of group is significantly different from control at p<0.01
Statistical analysis:Dunnett’s test if group variances are homogenous
Modified t test if group variances are inhomogenous ($)
MACROSCOPIC OBSERVATIONS – MAIN GROUP – UNSCHEDULED DEATH – GROUP INCIDENCE
Group: Number in group: |
--Females-- 4 1 |
Kidneys Pelvic dilation |
1 |
Forelimbs Abnormal shape Abnormal consistency |
1 1 |
Hindlimbs Abnormal shape Abnormal consistency |
1 1 |
MACROSCOPIC OBSERVATIONS – MAIN GROUPS – FINAL SACRIFICE – GROUP INCIDENCE
Group: Number in group: |
--Males-- |
--Females-- |
||||||
1 10 |
2 10 |
3 10 |
4 10 |
1 10 |
2 10 |
3 10 |
4 9 |
|
Adrenals Abnormal size |
1 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
Cervical nodes Abnormal colour |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
Kidneys Abnormal area (s) Pelvic dilation |
0 0 |
0 1 |
1 3 |
0 3 |
0 0 |
0 0 |
0 0 |
0 0 |
Liver Abnormal shape Abnormal size |
0 1 |
2 0 |
0 0 |
1 0 |
0 2 |
0 0 |
0 0 |
0 0 |
Ovaries Abnormal size |
|
|
|
|
1 |
0 |
0 |
0 |
Thymus Abnormal area (s) Abnormal colour Abnormal size |
0 0 0 |
1 0 0 |
0 1 0 |
1 0 0 |
0 0 0 |
0 0 1 |
0 0 0 |
0 0 2 |
Uterus Not pregnant Unilateral implantation |
|
|
|
|
0 0 |
0 1 |
2 0 |
0 0 |
Ears Abnormal area (s) |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Whole animal No abnormalities detected |
8 |
6 |
6 |
5 |
7 |
8 |
7 |
7 |
MICROSCOPIC OBSERVATIONS – UNSCHEDULED DEATH – GROUP INCIDENCE
Controls from group (s): 1
Tissues With Diagnoses |
Animal Sex: Dosage group: No. in group: |
--Animals Affected-- --Females-- 4 1 |
Kidneys HYDRONEPHROSIS |
Number examined: |
1 1 |
Lungs EMPHYSEMA INFLAMMATORY CELL FOCI |
Number examined: |
1 1 1 |
Forelimbs ARTHRITIS |
Number examined: |
1 1 |
Hindlimbs ARTHRITIS |
Number examined: |
1 1 |
MICROSCOPIC OBSERVATIONS – FINAL SACRIFICE – GROUP INCIDENCE
Controls from group (s): 1
Tissues With Diagnoses |
|
--Animals Affected-- |
|||||||
Animal Sex: |
--Males-- |
--Females-- |
|||||||
Dosage group: No. in group: |
Ctls 10 |
2 10 |
3 10 |
4 10 |
Ctls 10 |
2 10 |
3 10 |
4 10 |
|
Adrenals ACCESSORY NODULE CORTICAL CELL VACUOLIZATION CYTS/S |
Number examined: |
5 1 1 0 |
0 0 0 0 |
0 0 0 0 |
5 0 1 0 |
5 0 0 0 |
0 0 0 0 |
1 0 0 0 |
5 0 0 1 |
Cervical nodes CONGESTION |
Number examined: |
5 0 |
0 0 |
1 1 |
5 0 |
5 0 |
0 0 |
0 0 |
5 0 |
Heart INFLAMMATORY CELL FOCI |
Number examined: |
5 4 |
0 0 |
0 0 |
5 2 |
5 0 |
0 0 |
0 0 |
5 2 |
Kidneys INFLAMMATORY CELL FOCI NEPHROPATHY HYDRONEPHROSIS |
Number examined: |
5 1 0 0 |
1 0 0 1 |
4 1 2 1 |
5 0 1 2 |
5 0 1 0 |
0 0 0 0 |
0 0 0 0 |
5 0 0 0 |
Liver INFLAMMATORY CELL FOCI HEPATOCYTIC VACUOLATION CLEAR CELL CHANGE EXTRAMEDULLARY HAEMOPOIESIS |
Number examined: |
6 4 1 1 0 |
2 1 0 2 0 |
0 0 0 0 0 |
6 4 0 1 0 |
7 0 0 2 2 |
0 0 0 0 0 |
0 0 0 0 0 |
5 1 0 0 0 |
Lungs AGGREGATION OF ALVEOLAR MACROPHAGES |
Number examined: |
5 1 |
0 0 |
0 0 |
5 2 |
5 4 |
0 0 |
0 0 |
5 4 |
Prostate INFLAMMATORY CELL FOCI |
Number examined: |
5 1 |
0 0 |
0 0 |
5 2 |
|
|
|
|
Testes TUBULAR CELL DEGENERATION |
Number examined: |
5 1 |
0 0 |
0 0 |
5 0 |
|
|
|
|
Thymus ATROPHY CONGESTION |
Number examined |
5 0 0 |
1 0 0 |
1 1 1 |
5 0 0 |
5 0 0 |
1 1 0 |
0 0 0 |
6 1 0 |
Thyroid ECTOPIC THYMIC TISSUE |
Number examined: |
5 1 |
0 0 |
0 0 |
5 2 |
5 0 |
0 0 |
0 0 |
5 0 |
Ears CHRONIC INFLAMMATION |
Number examined: |
1 1 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
Note: Entries flagged with a – (minus) are significantly higher than control at the 0.05 level using the Kolmogorov-Smirnov one-tailed test.
Applicant's summary and conclusion
- Conclusions:
- Based on the results of the present study, the NOEL (No Observed Effect Level) for general toxicity, reproductive and developmental toxicity was considered to be 1000 mg/kg bw/day for males and females.
- Executive summary:
Repeated dose oral toxicity study performed in male & female Sprague-Dawley rats in accordance with OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test).
Male animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 43 and 44 of study). They were treated for a total of 42 or 43 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum (for approximately 42 days). Dose volumes were adjusted once per week for each animal according to the last recorded bodyweight. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Recovery group animals were dosed once a day, 7 days a week, for a minimum of 6 consecutive weeks. No treatment was given during the recovery period.
Each main group comprised 10 male and 10 female rats. Two groups (control and high dose levels) included 5 animals per sex which were sacrificed after 2 weeks of recovery.
One female of the high dose group, receiving 1000 mg/kg bodyweight/day, was killed for humane reasons on Day 3 of the gestation phase.
Marked arthritis of hind limbs and one forelimb, observed at histopathology, could be considered as a factor contributory to the illness status of the animal sacrificed for humane reasons. Therefore, this death was considered to be unrelated to treatment.
No clinical signs of toxicological relevance or signs of neurotoxicity (weekly observations at removal from the cage and in an open arena, alterations in motor activity, grip strength and sensory reaction to stimuli) were observed during the study in males and females. Slight orange staining in the cage tray was observed during the mating phase in some cages of mid- and high dose animals.
No differences in body weight and food consumption were observed in treated animals compared to the control group.
No changes of toxicological significance were recorded at haematological, clinical chemistry investigations or at coagulation tests in animals sacrificed at the end of the study.
No relevant changes were detected at post mortem examination (terminal body weight, organ weights, macroscopic and microscopic examinations) in treated animals, when compared with controls.
Based on the results of the present study, the NOEL (No Observed Effect Level) for general toxicity was considered to be 1000 mg/kg bw/day for males and females.
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