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EC number: - | CAS number: -
- 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
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP in line with accepted scientific principals although no formal methodology is referenced within the study report. Report considered fully adequate.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 009
- Report date:
- 2009
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: TOXI COOP Ltd. Cserkesz u. 90. 1103 Budapest, Hungary
- Age at study initiation: Young adult, 7 weeks
- Weight at study initiation: 240-263 g males; 178-211 g females
- Housing: 5 animals/cage
- Diet (e.g. ad libitum): ssniff SM R/M-Z+H autoclavable complete feed for rats and mice - breeding and maintenance; ad libitum
- Water (e.g. ad libitum): tap water as for human consumption from municipal supply; ad libitum
- Acclimation period: 6 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23.7
- Humidity (%): 36-58
- Air changes (per hr): 8-12
- Photoperiod (hrs dark / hrs light): 12 dark: 12 light
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 2.5 to 100 mg/mL in the Central Dispensary of LAB Research Ltd.
Formulations were prepared as appropriate for use within 72 hr (stored refrigerated), according to stability assessment results.
DIET PREPARATION
- Rate of preparation of diet (frequency):
Formulations were prepared as appropriate for use within 72 hr (stored refrigerated), according to stability assessment results.
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a
VEHICLE
- Justification for use and choice of vehicle (if other than water): Sterile water for injection, PhEUR
- Concentration in vehicle: Test material concentrations used were 2.5, 15 and 100 mg/mL
- Lot/batch no. (if required): 7810108 - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analysis of formulations (concentration, homogeneity) and assessment of test item
stability in this vehicle in the conditions employed on the study was performed in the
Analytical Laboratory of LAB Research Ltd. Stability tests indicated an up to 24 hour
stability at room temperature; in addition, under refrigeration conditions (5±3°C), at
concentrations from approximately 1 to 100 mg/mL in water, the solutions were stable
for up to 72 hours, with a recovery within the acceptable range of 100 ± 10% (103-
101 %). Concentration and homogeneity of formulations were evaluated by
spectrophotometry on duplicate samples collected from the top, middle and bottom of
the test item solutions, and one sample from the control, taken and analysed fresh on
the first and last week of treatment. Dose formulations were homogenous; the
measured concentrations varied between 99 to 106% of the nominal concentrations
(2.5, 15, 100 mg/mL). These results were considered suitable for the study purposes. - Duration of treatment / exposure:
- 28 days
- Frequency of treatment:
- The dosing solutions were administered daily for 28 days by oral gavage, using a bulb tipped gastric gavage needle attached to a syringe.
Day 0 was considered the first day of dose administration.
Doses / concentrationsopen allclose all
- Remarks:
- Doses / Concentrations:
25 mg/kg bw
Basis:
actual ingested
- Remarks:
- Doses / Concentrations:
150 mg/kg bw
Basis:
actual ingested
- Remarks:
- Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
- No. of animals per sex per dose:
- 10 animals per dose group (5m/5f)
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- The doses were determined on the basis of the results of a dose range finding study with the substance (LAB study code: 08/778-028P) in agreement with the Sponsor.
- Positive control:
- Not used
Examinations
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day, after treatment at approximately the same time. Detailed clinical
observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once weekly
thereafter.
BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight was recorded with precision of 1g at randomization, on the first day of treatment,
then weekly and before termination.
OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: Observation was performed on the skin, fur, eyes and mucous membranes, autonomic activity
(lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions). Sensory reactivity to different type of
stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted on all animals in the 4th
exposure week (Day 26).
HAEMATOLOGY:
- Time schedule for collection of blood:
Clinical pathology examinations (haematology, coagulation, and clinical biochemistry) were conducted on all animals pre-terminally.
- Anaesthetic used for blood collection: Yes (pentobarbital anaesthesia)
- Animals fasted: Yes, overnight period of food deprivation .
- How many animals: All
- Parameters checked:
Red Blood Cell (erythrocyte) count, White Blood Cell (leukocyte) count, Haemoglobin concentration, Haematocrit, Mean Corpuscular
(erythrocyte) Volume, Mean Corpuscular (erythrocyte) Haemoglobin, Mean Corpuscular (erythrocyte) Haemoglobin Concentration, Red Cell
(erythrocyte) volume, Platelet (thrombocyte) count, Mean Platelet Thrombocyte volume, Reticulocyte count (%), Neutrophil (%), Lymphocyte
(%), Monocyte (%), Basophil (%), Eosinophil (%), Large Unstained Cells (%), Partial Thromboplastin Time (sec), Prothrombin Time (sec).
CLINICAL CHEMISTRY: Yes
- How many animals: All
- Parameters checked:
Glucose Blood sugar concentration, Total Bilirubin concentration, Urea concentration, Cholesterol concentration, Creatinine concentration,
Phosphorous concentration, Sodium concentration, Potassium concentration, Calcium concentration, Chloride concentration, Total Protein
concentration, Albumin concentration, Alb/glob ration, Aspartate Aminotranferase activity, Alanine Aminotranferase activity, Alkaline
Phosphatase activity, Gamma Glutamyltranferase activity, Bile Acids.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 4th exposure week
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptivc), assessment of grip strength and motor activity was
conducted on all animals. General physical condition and behaviour of animals was tested. A modified Irwin test was be performed (Irwin, S.:
Comprehensive Observational Assessment: la. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of
the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).
The behaviour and the general condition of the test animals were normal during the study. There was no treatment-related effect on motor
activity or in the functional observation battery tests across groups of treated male or female animals and no findings indicative for
neurotoxicity were observed. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
Gross pathology consisted of an external examination, including identification of all clinically-recorded lesions, as well as a detailed internal
examination.
Gross pathology was performed on every experimental animal. Animals were euthanized by exsanguination under pentobarbital anaesthesia
at the end of the observation period (following the terminal clinical pathology blood collection).
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of
the tissues and organs were observed macroscopically.
The following organs were weighed and recorded: with precision of 0.01g, liver, kidneys, testes, epididymides, thymus, spleen, brain and heart; with precision of 0.001g, adrenals. Paired organs were weighed together. Absolute organ weights were measured, and relative organ
weights were calculated.
HISTOPATHOLOGY: Yes
Full histopathology was performed in Group 1 (control) and Group 4 (High dose), and all
organs and tissues with macroscopic findings from groups 2 and 3 (Low and Mid doses,
respectively). Organs or representative samples were embedded into paraffin. Slides
were stained with haematoxylin-eosin/phloxyne* and examined with light microscope.
* Due to a typographical error in the study plan, phloxyne was not indicated; however,
its use is a standard, suitable procedure for staining the microscopic slides. This
deviation is considered to have no impact on the outcome of the study and
interpretation of the results. - Statistics:
- Statistical analysis was performed on the numerical data as appropriate. The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test.
Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data were not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test. The mean daily food consumption, frequency of clinical observations and necropsy and histopathology findings were calculated.
Results and discussion
Results of examinations
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- no effects observed
- Details on results:
- CLINICAL SIGNS AND MORTALITY
No mortality occurred during the 28-day treatment period.
No toxicologically significant systemic clinical changes were noted following administration of INK BH11 M by oral gavage, daily for 28 days. Day 0 was regarded as the first day of treatment. Red or light red faeces and/or urine were observed in animals’ cages for both sexes, in the bedding, at all the dose levels tested from Day 1, or 2, respectively, as follows:
1000 mg/kg bw/day:
Red faeces were observed for 27/28 days in both the male and female animals, as of day 1 of treatment period. In addition, red urine occurred for 26/28 days in the male animals, or 22/28 days, in the females.
150 mg/kg bw/day:
Both male and female animals showed red faeces from day 2, for 26/28 days
25 mg/kg bw/day:
Light red faeces were observed for 18 to 23 days of 28 treatment days, in the male or female animals, respectively. These changes were ascribed to elimination of INK BH11 M or its metabolites through faeces and/or urine; moreover, in the absence of any clinical pathology alterations, they were not considered adverse effects. There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed towards the end of the treatment period. Minor changes were observed in the animals throughout all the dose groups, including controls when subjected to the modified Irwin test (functional observation battery). However, the variations noted demonstrated no treatment-related differences to the control, and were considered within the normal biological variation with respect to behaviour, reactions to different type of stimuli or manipulations. Incidences and extent of reactions observed in the treated animals were comparable to those observed in control animals, or within the historical control data, and are without any toxicological significance
BODY WEIGHT AND WEIGHT GAIN
No adverse effects were noted on the mean body weight and body weight gain values in the treated groups compared to control animals following daily administration of INK BH11 M at dose levels of up to and including 1000 mg/kg bw/day. Variations in the mean body weight values compared to control were noted, mainly in the female animals, occasionally with statistical significance (at 25 and 150 mg/kg bw/day) but without a dose related manner. These were considered due to high individual values in 3 control animals (151, 152 and 155), which led to relatively high mean body weight and body weight gain values in the control female group. In the absence of a dose or gender-related response, these changes were considered neither toxicologically significant, nor related to INK BH11 M administration, but expected variations in the population of Wistar rats
FOOD CONSUMPTION AND COMPOUND INTAKE
There were no test item related differences in the mean daily food consumption in any test item treated groups (25, 150, or 1000 mg/kg bw/day, male or female) when compared to the control
HAEMATOLOGY
There were no differences that were considered toxicologically significant between the control and test item-treated groups, or any adverse effects of INK BH11 M on haematology parameters in the male and female animals. Variations were noted on occasion in the mean values of different parameters, such as, but not limited to, platelets (PLT), basophile (BA%), neutrophile (NE%), monocite (MO%), eosinophil (EO%), or large unstained cells (LUC%) values at all the dose levels, in both sexes. However, these variations were not statistically significant, andevaluation of the mean and individual results in correlation with the control and historical haematology data did not reveal any test-item related cause of the changes noted, and/or no consistent dose or gender-related response was observed. Therefore, these differences observed between the control and treated groups were considered to be incidental or individual findings, which were not related to treatment and generally remained within the historical control ranges, or were with no toxicological significanceThere were no differences that were considered toxicologically significant between the control and test item-treated groups, or any adverse effects of INK BH11 M on haematology parameters in the male and female animals. Variations were noted on occasion in the mean values of different parameters, such as, but not limited to, platelets (PLT), basophile (BA%), neutrophile (NE%), monocite (MO%), eosinophil (EO%), or large unstained cells (LUC%) values at all the dose levels, in both sexes. However, these variations were not statistically significant, and evaluation of the mean and individual results in correlation with the control and historical haematology data did not reveal any test-item related cause of the changes noted, and/or no consistent dose or gender-related response was observed. Therefore, these differences observed between the control and treated groups were considered to be incidental or individual findings, which were not related to treatment and generally remained within the historical control ranges, or were with no toxicological significance
CLINICAL CHEMISTRY
Clinical chemistry evaluation revealed a significantly higher serum total bilirubin concentration in both sexes (males at 150 and 1000 mg/kg bw/day, females at 1000 mg/kg bw/day), exceeding the historical control ranges in the 1000 mg/kg bw/day female group, and with a trend to a dose response. However, there were no other clinical chemistry or histopathologic alterations that indicated changes in the liver. It is possible that the intense colour of the test item caused a spectral interference in determination of total bilirubin concentration. Statistically significant changes of aspartate aminotransferase activity (AST) and phosphorus (Phos.) mean values were noted in the treated animals compared to control. AST mean values were lower in male animals at all the dose levels tested, attaining statistical significance at 25 mg/kg bw/day (low dose); however, AST values were slightly increased in the female animals, with no statistical significance and remaining below the upper limit of the historical control range. Phosphorus mean value was statistically higher in the 1000 mg/kg bw/day female animals, but lower than control in the treated male animals. In summary, most of the observed variations in the clinical chemistry parameters, although they were occasionally statistically significant, were not dose-related, showed no consistent gender response and/or were within the normal historical control ranges. The only variation that may have a toxicological significance at 1000 mg/kg bw/day dose level in both sexes was the apparent total bilirubin increase, which may be due to spectral interference with the analytical method by the discoloured test item in the plasma. In the absence of any additional evidence of liver damage from other measurements of liver function or from liver histopathology, bilirubin changes in correlation with INK BH11 M administration is considered equivocal in the conditions of this study
NEUROBEHAVIOUR
The behaviour and the general condition of the test animals were normal during the study. There was no treatment-related effect on motor activity or in the functional observation battery tests across groups of treated male or female animals and no findings indicative of neurotoxicity were observed.
ORGAN WEIGHTS
The following organs were weighed and recorded: with precision of 0.01g, liver, kidneys, testes, epididymides, thymus, spleen, brain and heart; with precision of 0.001g, adrenals. Paired organs were weighed together. Absolute organ weights weremeasured, and relative organ weights were calculated
GROSS PATHOLOGY
Gross pathology was performed on every experimental animal euthanized byexsanguination under pentobarbital anaesthesia at the end of the observation period (following the terminal clinical pathology blood collection). Euthasol® 40 % batch no. 07E29 8, manufacturer AST Beheer B.V. Oudewater Netherlands, Produlab Pharma, Raamsdonksveer, stored at room temperature; expiry date, April 2009 was used for euthanasia. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size. The following organs/tissues were removed and preserved in 10% buffered formalin solution for histological processing: Gross lesions, lymph nodes (submandibular, mesenteric) sternum, skin and female mammary gland, salivary glands (submandibular), femur + bone marrow, spinal cord (cervical, lumbar, thoracic level), pituitary, thymus, trachea, lungs (with main stem bronchi), heart, thyroid + parathyroid, oesophagus, stomach, caecum, duodenum, ileum, jejunum, colon, rectum, urinary bladder, liver, pancreas, spleen, kidneys, adrenals, prostate, ovaries with oviduct*, uterus with cervix* and vagina, epididymes, brain (including cerebrum, cerebellum, pons and medulla oblongata), lachrymal gland with Harderian glands, seminal vesicle, muscle (quadriceps), sciatic nerve and aorta. The eyes with the optic nerve and the testes were preserved in modified Davidson fixative for histological processing. * cervix and oviducts were collected, although not listed in the study plan. This deviation has no impact on the outcome of the study and interpretation of the results.
HISTOPATHOLOGY: NON-NEOPLASTIC
In the absence of any additional evidence of liver damage from other measurements of liver function or from liver histopathology, bilirubin changes may be due to spectral interference with the analytical method by the coloured test item in the plasma, and a correlation with INK BH11 M administration is considered equivocal in the conditions of this study At necropsy, test item related red discoloration of the stomach, small intestine (duodenum, jejunum, ileum), colon, cecum and/or rectum was observed at 150 and 1000 mg/kg bw/day. Red digestive content was also noted at 150 and 1000 mg/kg bw/day. At microscopic evaluation, minimal focal/multifocal intracytoplasmic deposit of red pigment in the mesenteric and/or mandibular lymph nodes was noted at 1000 mg/kg bw/day. In addition, minimal focal intracytoplasmic deposit of red pigment in the Peyer’s patches of the ileum was recorded in animals treated at 150 and 1000 mg/kg bw/day.
Effect levels
open allclose all
- Dose descriptor:
- NOAEL
- Effect level:
- 150 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Basis for effect level:
- other: Histological findings
- Dose descriptor:
- NOEL
- Effect level:
- 25 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Basis for effect level:
- other: overall effects
Target system / organ toxicity
- Critical effects observed:
- not specified
Applicant's summary and conclusion
- Conclusions:
- Ink BH11 M administered daily by oral gavage for 28 days in Wistar rats did not lead to any toxicologically significant clinical adverse effects at dose levels of 25, 150, or 1000 mg/kg bw/day. Faeces of all animals and urin of high dose animals were coloured red or light red for up to 27 out of 28 days of treatment.
Significantly higher serum total bilirubin concentration was observed in both sexes exceeding historical control ranges. May be interference of test material with analytical method.
Red discoloration of GI tract. Deposite of red pigment in mesenteric and/or mandibular lymph nodes noted at 1000 mg/kg bw/day. Deposit of red pigment in Peyer's patches of ileum in animals treated at 150 and 1000 mg/kg bw/day doses. No adverse findings on organ weights.
Based on histological findings, NOAEL 150 mg/kg bw/day; NOEL 25 mg/kg bw/day - Executive summary:
In a 28 day repeat dose oral toxicity study, conducted to GLP and broadly in line with standard, accepted EU and OECD methods, INK BH11 M caused no mortality at 1000 mg/kg bw/day in CRL:(WI)BR rats. No toxicologically significant systemic clinical changes were noted following administration of INK BH11 M by oral gavage, daily for 28 days. There were no toxicologically significant changes in body weight, body weight gain or animal food consumption between the control and test item treated groups
The faeces of all animals administered up to, and including, 1000 mg/kg bw/day, and the urine of the high dose animals (1000 mg/kg bw/day) were colored red, or light red (faeces at 25 mg/kg bw/day) for up to 27 out of 28 days of treatment, in an apparent dose related manner. These changes were ascribed to elimination of INK BH11 M or its metabolites through faeces and/or urine; moreover, in the absence of any clinical pathology alterations, they were not considered to be an adverse effect.
However, based on histological findings, the no observed adverse effect level (NOAEL) for INK BH11 M is considered to be 150 mg/kg bw/day. The no observed effect level (NOEL) is considered to be 25 mg/kg bw/day.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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