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EC number: 632-619-2 | CAS number: 881685-58-1
- 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:
- chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21 Mar 2006 to 13 Jul 2008
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Referenceopen allclose all
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 008
- Report date:
- 2008
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 010
- Report date:
- 2010
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
- Version / remarks:
- 1981
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity)
- Version / remarks:
- 1998
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
- Version / remarks:
- 1988
- Qualifier:
- according to guideline
- Guideline:
- other: According to Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agrochemicals, 12 Nohsan No. 8147, Agricultural Production Bureau.
- Version / remarks:
- 24 Nov 2000
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- 632-619-2
- EC Number:
- 632-619-2
- Cas Number:
- 881685-58-1
- Molecular formula:
- C20 H23 F2 N3 O
- IUPAC Name:
- 632-619-2
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- HsdRCCHan
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 5-6 weeks old
- Weight at study initiation: Males 104 - 151 g and females 93 - 138 g
- Housing: Animals were housed, sexes separately, in multiple rat racks suitable for animals of this strain and the weight range expected during the course of this study. They were housed 5 per cage initially and in fours after they had been assigned to experimental groups.
- Acclimatisation period: approximately 1 week
- Diet Test and control diets based on CT1 diet, ad libitum
- Water Mains water, supplied by an automatic system, ad libitum
- Acclimation period: approximately 1 week prior to the start of the study.
ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes (hr) : At least 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 21 Mar 2006 To: 13 Jul 2008
Administration / exposure
- Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- DIET PREPARATION
- Mixing appropriate amounts with feed: The test and control diets were prepared (using RM1 diet), and transferred to the animal rooms as required. The diets were prepared in 60 kg batches from 1000 g premixes prepared by mixing the appropriate amount of test substance with milled diet. The amount of test substance required was made up to 60 kg diet.
- Test diets were stored at room temperature for up to 6 weeks. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Samples from all dietary levels (including controls) were taken prior to the start of the study at approximately three-monthly intervals throughout the study and analysed quantitatively for test substance.
- Prior to the start of the study the homogeneity of the test substance in RM1 diet was determined by analysing samples from the low and high dose levels and the chemical stability of the test substance in diet was determined for these same diets over a period of up to 42 days at room temperature.
- Samples were extracted with acetonitrile and aliquots of supernatant diluted with acetonitrile, as appropriate, to give sample solution concentrations within the range of the calibration standards used. Samples and standards were analysed by High Performance Liquid Chromatography (HPLC). - Duration of treatment / exposure:
- 104 weeks, with animals designated for the interim kill were exposed for 52 weeks.
- Frequency of treatment:
- Continuously
Doses / concentrationsopen allclose all
- Dose / conc.:
- 100 ppm
- Remarks:
- Group 2: Dietary equivalent to 5.5 and 6.9 mg/kg bw/day for males and females, respectively
- Dose / conc.:
- 500 ppm
- Remarks:
- Group 3: Dietary equivalent to 27.6 and 34.9 mg/kg bw/day for males and females, respectively
- Dose / conc.:
- 3 000 ppm
- Remarks:
- Group 4: Dietary equivalent to 173.5 and 232.8 mg/kg bw/day for males and females, respectively
- No. of animals per sex per dose:
- 52 in main study 104 weeks, and in addition 12 for interim sacrifice after week 52.
- Control animals:
- yes, plain diet
Examinations
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS:
- Time schedule: twice daily. Prior to the start of the study, all rats were examined to ensure that they were normal. Cage-side observations included recording any changes in clinical condition or behaviour.
DETAILED CLINICAL OBSERVATIONS:
- Time schedule: weekly, generally at the same time that the body weights were recorded. Any rats requiring euthanasia were killed and examined post mortem. Any rats found dead were examined post mortem as soon as possible after death.
BODY WEIGHT:
- Time schedule for examinations: The body weight of each rat was recorded immediately before feeding of the experimental diets commenced and then on the same day, where practicable, every subsequent week for weeks 2 - 15 of the study and then every 2 weeks until termination. All rats were weighed at termination.
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule: Weight of food given and food residues were recorded for the first 14 weeks of the study, week 16 and thereafter every fourth week.
- Food consumption was calculated as a mean value (g food/rat/day) for each cage.
- The food utilisation value per cage was calculated as the body weight gained by the rats in the cage per 100 g of food eaten for the first 12 weeks of the study.
- Dose rates (based on nominal dietary levels of test substance) were calculated in terms of mg substance/kg body weight.
FUNCTIONAL OBSERVATION BATTERY
- Time schedule for examinations: Detailed clinical assessments (during which each rat was removed from its cage and physically examined for changes in general health status) and quantitative assessments of landing foot splay, muscle weakness (fore- and hind-limb grip strength) and sensory perception (tail-flick test) were made in week 50 for all interim kill animals.
- The observations were made by one observer who was ‘blind’ with respect to the animal’s treatment, and recorded on a computer system by personnel not directly involved in the clinical observations. The presence and/or absence of all listed observations were recorded and the degree of condition noted (slight, moderate or extreme) where appropriate. The examination proceeded from the least to the most interactive test with observations recorded as follows:
- Assessment in the home cage: bizarre behaviour (circling, head flicking, head searching, walking backwards, rolling over sideways, paw flicking), vocalisation.
- Removal from the cage: approach response, response to touch (increased, decreased), vocalisation.
- In the standard arena: activity (increased, decreased), comatose, prostration, hunched posture, bizarre behaviour (circling, head flicking, head searching, walking backwards, rolling over sideways, paw flicking), convulsions (tonic, clonic), vocalisation, ataxia, tremors, reduced stability, abnormal gait, splayed gait, tiptoe gait, reduced limb function (fore, hind), upward curvature of the spine, downward curvature of the spine, piloerection, sides pinched in, ungroomed appearance, urinary incontinence, diarrhoea.
- Handling the animal: response to touch, convulsions, vocalisation, tremors, piloerection, skin colour (pale, hyperaemia, cyanosis), ungroomed appearance, hyperthermia, hypothermia, chromodacryorrhea, lachrymation, ptosis, enophthalmos, exophthalmos, miosis, mydriasis, stains around the mouth, stains around the nose, salivation, respiratory abnormalities (breathing rate, breathing depth, laboured breathing, gasping, irregular breathing, whistling, wheezing, croaking), thin appearance sides pinched in, dehydration, abdominal tone (increased, decreased), urinary incontinence, diarrhoea.
- Reflex tests: righting reflex (from supine position), response to sound (to finger click/clap), splay reflex (degree of splay when animal lifted by base of tail), visual placing response (animal is lifted by base of tail and slowly moved downwards towards the edge of arena), pupil response to light (after eye has been held closed for 10 seconds), palpebral membrane reflex (palpebral membrane touched with bristle and blink response observed), corneal reflex (hair is touched against cornea and blink reflex observed - only performed if palpebral reflex is absent), pinna reflex (bristle poked into ear canal), foot withdrawal reflex (to toe pinch).
- Quantitative measures: fore-limb and hind-limb grip strength, landing foot splay, time to tail flick.
- In addition, any other observations that may facilitate interpretation of the data were recorded.
OPHTHALMOSCOPIC EXAMINATION
- Time schedule for examinations: The eyes of all surviving main study animals from the control and high dose groups were examined during week 50 and the eyes of all surviving animals were examined in weeks 101 to 103 (prior to termination).
- The examination was carried out using an ophthalmoscope. The animals were examined after instillation of 0.5 % v/v tropicamide into the eyes to dilate the pupils. The eyes of animals designated for the main study were examined pre-experimentally, except for 4 control females, which were not examined in error. This error is considered not to have affected the integrity of the study.
HAEMATOLOGY AND CLINICAL CHEMISTRY: MICROSCOPY
- Time schedule for examinations: Blood was collected via the tail vein and samples were taken for haematology from pre-designated animals (n = 13 per group) in weeks 14, 27, 53 and 79. Blood was collected via the tail vein and samples were taken for clinical chemistry from a further designated thirteen male and thirteen female rats per group at weeks 14, 27, 53 and 79.
- Any designated rat which died or was killed before the scheduled time was replaced, as necessary, by an alternative animal where possible in order to maintain acceptable group sizes (minimum 10/sex/group). All surviving rats were bled by cardiac puncture at the interim kill in week 53 or at termination in week 105 for haematology and clinical chemistry.
HAEMATOLOGY
- The following measurements were made or determined on samples collected using EDTA as an anticoagulant: haemoglobin, mean cell haemoglobin concentration, haematocrit, platelet count, red blood cell count, total white cell count, mean cell volume, differential white cell count, mean cell haemoglobin and blood cell morphology.
- Clotting measurements consisting of prothrombin time and activated partial thromboplastin time were made on samples of blood collected in tubes containing sodium citrate as an anticoagulant. Blood cell morphology, including a differential white blood cell count was assessed by automated methods for all animals. Manual blood films were prepared and analysed as necessary.
CLINICAL CHEMISTRY (BLOOD)
- The following measurements were made on the plasma from blood samples collected into tubes containing lithium heparin as an anticoagulant: urea, alkaline phosphatase activity, creatinine aspartate, aminotransferase activity, glucose alanine aminotransferase activity, albumin, gamma-glutamyl transferase activity, total protein calcium, cholesterol, phosphorus (as phosphate), triglycerides, sodium, total bilirubin, potassium, creatine kinase activity and chloride.
CLINICAL CHEMISTRY (URINE)
- Time schedule for examinations: Individual urine samples were collected from a pre-designated thirteen males and thirteen females per group at weeks 13, 26, 52, 78 and 104. These were the same animals as those used for haematology analyses. Any designated rat which died or was killed before the scheduled time was replaced, as necessary, by an alternative animal where possible in order to maintain acceptable group sizes (minimum 10/sex/group).
- Samples were collected over a period of 16 - 18 hours during which the rats were housed individually in metabolism cages and denied access to food. The following parameters were measured or assessed and recorded on each urine sample: colour, appearance, volume, pH, specific gravity, protein, glucose, ketones, bilirubin and blood.
MOTOR ACTIVITY
- Locomotor activity was monitored by an automated activity recording apparatus. All interim kill animals were tested in week 50.
- Each observation period was divided into ten scans of five minutes duration. Treatment groups were counter balanced across test times and across devices. Motor activity was assessed in a separate room to minimise disturbances. During motor activity assessment, animals did not have access to food, water or items of environmental enrichment. - Sacrifice and pathology:
- GROSS PATHOLOGY
- With the exception of animals found dead, all rats were killed by over exposure to halothane Ph. Eur. vapour followed by exsanguination by cardiac puncture.
- From all animals at interim kill and those from scheduled termination, the following organs were removed, trimmed free of extraneous tissue and weighed: adrenal, glands liver, brain, ovaries, epididymides, spleen, heart testes, kidneys and uterus (with cervix). Paired organs were weighed together.
HISTOPATHOLOGY
- All animals were examined post mortem. This involved an external observation and a detailed internal examination of all organs and structures.
- The following tissues were examined in situ, removed and examined and fixed in an appropriate fixative: gross lesions including masses, oesophagus, adrenal gland, ovary, aorta, oviduct, brain (cerebrum, cerebellum and brainstem), pancreas, bone - femur (including knee joint) parathyroid gland, bone marrow – femur, Peyer’s patches, caecum, pharynx, cervix, pituitary gland, colon, prostate gland, duodenum, rectum, epididymis, salivary gland eyes (retina, optic nerve), seminal vesicle, ex-orbital lachrymal, gland skin, harderian gland, spinal cord (cervical, thoracic, lumbar) heart, spleen, ileum, sternum, jejunum, stomach, kidney, testis, larynx, thymus, liver, thyroid gland, lung, trachea, lymph node – cervical, urinary bladder, lymph node – mesenteric, uterus, mammary gland - (females only), vagina, nerve – sciatic, voluntary muscle and nose.
All tissues from all animals were submitted for histology. Tissues for histology were routinely processed, embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin.
MICROSCOPIC EXAMINATION
- All submitted tissues were examined by light microscopy. - Statistics:
- - All data were evaluated using the MIXED procedure in SAS (2004). Body weights were considered by analysis of covariance on initial (week 1) body weight,
separately for males and females. Food consumption and food utilisation during the periods weeks 1 - 4, 5 - 8, 9 - 12 and 1 - 12 were considered by analysis of variance, separately for males and females.
- Haematology data were considered by analysis of variance, separately for males and females.
- Motor activity measurements, time to tail-flick (in the sensory function test), landing foot splay and grip strengths were considered by analysis of variance, separately for males and females.
- Organ weights were considered by analysis of variance and analysis of covariance on final bodyweight, separately for males and females. Summary data are presented for organ to body weight ratios but these were not analysed statistically as the analysis of covariance provides a better method of allowing for differences in terminal body weights.
Results and discussion
Results of examinations
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were more males in the 3000 ppm group with observations of scabs and subcutaneous masses than in the control group. There were more incidences of scabs in females in the 3000 ppm group than in the control group, although the number of animals affected was similar. All other observations affected a small number of animals or were recorded in similar numbers of control and treated rats and are considered not to be related to treatment.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Survival was good with approximately 75% of males and 67% of females on the study surviving to scheduled termination after two years. There were no treatment related effects on survival.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body weights of animals receiving 3000 ppm test substance were reduced in comparison with control throughout the study. The maximum difference from control was 14 % in males and 30 % in females. Body weight gain in females at 3000 ppm was approximately 40% lower than control at the end of the study.
Body weights of males at 500 ppm groups were similar to control throughout the study. Body weights of females in the 500 ppm group were consistently lower than control. The effect was more pronounced during the second year of the study reaching a maximum of 12% below control for body weight and 17 % for body weight gain.
Body weights of males at 100 ppm groups were similar to control throughout the study. Body weights of females in the 100 ppm group were considered to be unaffected by treatment. During the second year of the study body weights in the 100 ppm females diverged from control attaining statistical significance towards the end of the study. However the body weights of the concurrent controls were consistently higher than historical control data, particularly during the second year of the study, whilst the growth curve of the 100 ppm group closely followed that typically seen in historical control curves. Therefore the body weights of females in the 100 ppm group were concluded to reflect the normal growth pattern of this strain of rat in this laboratory and the minor differences seen were considered to be unrelated to treatment. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Food consumption was reduced in rats in the 3000 ppm group from the start of the study to approximately week 11 in males and week 14 in females; thereafter values were similar to control. There were no consistent effects on food consumption in rats in the 100 or 500 ppm groups.
The efficiency of food utilisation from weeks 1 - 12 was reduced in both sexes in the 3000 ppm group. Food utilisation was slightly less efficient than control in females in the 500 ppm group and the difference was statistically significant overall for weeks 1 - 12. There were no effects on food utilisation in males in the 500 ppm group or either sex in the 100 ppm group. - Food efficiency:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Food utilisation for males fed 1000 ppm was statistically significantly less efficient than control between weeks 1 - 4, 5 - 8 and overall (weeks 1 - 13). The food utilisation for females fed 1000 ppm and both sexes at the lower dose levels was unaffected by treatment with test substance.
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In week 50 findings were similar in control rats and those receiving 3000 ppm. In weeks 101 to 103 there were more females with ophthalmoscopy findings in the 3000 ppm group than in the control group. For this reason the eyes of all animals from the 100 and 500 ppm groups were also examined. The number of males with findings was 21, 17, 13 and 23 in the control, 100, 500 and 3000 ppm groups respectively. The number of females with findings was 7, 5, 12 and 14 in the control, 100, 500 and 3000 ppm groups respectively. Although there were more females with findings in the 3000 ppm group than in the control group these comprised a variety of common findings and there was no evidence of any effect of treatment.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment related effects were confined to differences in some parameters in females in the 3000 ppm group only. These comprised reduced haemoglobin, haematocrit and red blood cell count and increased platelet counts compared to control. White blood cell parameters were not affected by treatment. Other differences from control e.g. lower mean cell volume and mean cell haemoglobin in males in the 500 and 3000 ppm groups, were small and did not show a consistent pattern and were considered to be unrelated to treatment. There were no consistent effects on clotting times, small differences seen were inconsistent with time, sex and between the two measures of activated partial thromboplastin and prothrombin time.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Plasma triglycerides were reduced in the 3000 ppm group. This was evident throughout the study in males and from week 53 in females. Plasma triglycerides were also reduced in females in the 500 ppm group in the second year of the study. Differences in females at 100 ppm were intermittent, were not present at the end of the study and are considered to be unrelated to treatment.
Plasma cholesterol was increased in main study females in the 3000 ppm group at most time points. There was no effect in males or in the lower dose groups.
Plasma bilirubin levels were generally reduced in females in the 500 and 3000 ppm groups from week 27. There were some instances of lower plasma bilirubin levels in males in the 3000 ppm group. The isolated statistically significantly lower bilirubin value in females in the 100 ppm group at week 79 is considered not to be related to treatment, as the value is similar to the control value at week 53 and there is no difference from control at week 105.
Gamma glutamyl transferase activity was increased in males in the 500 and 3000 ppm groups from week 53. In females in the 3000 ppm group there were some high individual gamma glutamyl transferase values, but the difference from control was statistically significant in week 105 only.
Alanine transaminase activity was increased in males in the 3000 ppm group due to some high individual values and the difference from control was statistically significant in weeks 53 and 105. Activities of alkaline phosphatase and aspartate amino transferase were generally lower than control in males and females in the 3000 ppm group. There were a few instances of statistically significantly lower values in the 500 ppm group.
Sodium and chloride ions were marginally increased in females in the 3000 ppm group.
Chloride ions were occasionally higher than control in males in the 3000 ppm group but showed no consistent pattern and are considered to be unrelated to treatment. There was an isolated, higher calcium value at week 105 in females. There was no effect on plasma urea in males. In females in the 3000 ppm group values were statistically significantly increased in weeks 53 and 105.
There were a small number of high plasma creatinine values in the 3000 ppm group and values were statistically significantly higher than control in females in weeks 53 and 79, but in the absence of a difference at week 105, these differences are considered unrelated to treatment. Small differences from control in other parameters did not show a consistent pattern and were considered to be unrelated to treatment. - Endocrine findings:
- not examined
- Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no effects on the urine parameters measured or assessed. Urine pH was lower than control in females in the 3000 ppm group in week 13, however as there were no differences from control at other time points, this is considered to be unrelated to treatment.
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Functional observation battery: There were no treatment-related effects on landing foot splay, on fore limb grip strength or time to tail-flick. Hind limb grip strength was reduced compared to control in females in the 3000 ppm group. There was no effect in males in the 3000 ppm group or either sex in the 100 or 500 ppm groups.
All groups showed a typical pattern of reducing activity over the monitoring period with no evidence of an effect of treatment. A higher level of activity was recorded for 36-40 minutes in females in the 3000 ppm group than in the control group and this higher activity was also reflected in the overall activity of these animals. Activity of males in the 3000 ppm group and both sexes in the 100 or 500 ppm groups was similar to control. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Interim kill:
Liver weights were increased in the 3000 ppm group, by 17% in males and by 14% in females. There were no significant differences from control in the 100 or 500 ppm groups.
Adrenal weights and heart weights were lower than control in females in the 3000 ppm group.
Brain weights were higher than control in males in the 3000 ppm group.
At interim kill no treatment related differences were evident on the weights of epididymides, kidneys, ovaries, spleen, testes or uterus with cervix.
Terminal kill:
Liver weights were increased in the 3000 ppm group by 18% in males and by 27% in females. Weights were also increased by 12% in females in the 500 ppm group. There were no significant differences from control in either sex in the 100 ppm group.
Adrenal weights were lower than control in females in the 3000 ppm group when abnormally high adrenal weights were excluded.
Brain weights were higher than control in females in the 3000 ppm group.
At terminal kill no treatment related differences were evident on the weights of epididymides, heart, kidneys, ovaries, spleen, testes or uterus with cervix. - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were a higher number of females in the 3000 ppm group with watery fluid in the abdominal cavity. These were all decedents (1, 0, 2, 5 in the control, 100, 500 and 3000 ppm groups respectively). At terminal kill there were also a higher number of females in the 3000 ppm group with pale spots or areas on the liver and with a liver mass. There were no treatment related macroscopic findings in males in the 3000 ppm group or either sex in the 100 or 500 ppm groups.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- 12 month kill:
Treatment related findings were confined to the liver. There was an increased incidence of hepatocyte vacuolation, hepatocyte hypertrophy and increased hepatocyte/Kupffer cell pigmentation in the livers of all males and females in the 3000 ppm group. These findings were also present, although the severity was less, in the majority of males and females in the 500 ppm group. Liver findings in males and females in the 100 ppm group were similar to control indicating that there was no treatment related effect at this dose level.
Main study:
Treatment related findings were seen in the liver, kidneys (females) and mesenteric lymph nodes. There were treatment-related increases in the incidence of several changes in the liver which were sometimes accompanied by an increase in severity.
At 100 ppm, liver effects were centrilobular minimal hepatocyte hypertrophy (both sexes) and minimal centrilobular hepatocyte pigmentation (females). In males there was also a slightly higher incidence of findings typically seen in aging rats which were minimal hyperplasia of the bile ducts, accompanied in some animals by minimal fibrosis. The findings at 100 ppm were predominantly observed in animals at week 105. The earliest incidence of bile duct hyperplasia was observed at week 80 and the earliest incidence of pigmentation in females was seen at week 72.
Brown pigment was seen in the kidney tubules of animals from treated and control groups but the incidence was significantly greater than controls in females in the 500 and 3000 ppm groups. There was an increased incidence of sinus erythrocytosis in the mesenteric lymph nodes of males in the 3000 ppm group.
An increased incidence of eosinophilic droplets was seen in the nasal epithelium of both sexes in
the 500 ppm group but in the absence of a similar increase in the 3000 ppm group, this is considered not to be related to treatment.
A few spontaneous findings showed a slightly lower incidence in some treated groups in comparison with controls. These findings included indentation of the ventral brain, chronic cardiomyopathy, progressive chronic nephropathy, epithelial mineralisation of the renal pelvis, dilation /secretion of the mammary gland ducts and diffuse hyperplasia of the thyroid C-cells. A lower incidence and/or severity of spontaneous changes are often associated with decreased weight gain and are not usually regarded as an adverse effect of treatment.
A variety of other spontaneous changes was noted with no evidence of an effect of treatment. The incidence and the spectrum of these findings are consistent with changes commonly encountered in rats of this age kept under laboratory conditions - Histopathological findings: neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- In the 3000 ppm group there were increased incidences of uterine endometrial adenocarcinoma and liver hepatocellular adenoma in females. A reduced incidence of mammary fibroadenoma was also seen in females in the 3000 ppm group. All findings were statistically significant using the Peto-trend test. Endometrial adenocarcinoma of the uterus was considered to have led to the death or early termination of five females in the 3000 ppm group.
There was a higher incidence of thyroid follicular cell adenoma observed in the 3000 ppm males (7/64 – 10.9 % compared to controls 1/64 – 1.56 %) that was statistically significant when analysed with the Peto-trend test, but was not significant using Fisher’s exact test.
The historical control incidence from 3 previous studies in this laboratory (PR1248, PR1321 and PR1324) ranged from 1.6 to 9.6 %. While the incidence in 3000 ppm males was marginally outside the laboratories historical control range, an incidence of 10.9 % is within the range of control values held in the RITA data base (Wistar rats), which shows a wide variation from 0 to 28 % (104 studies carried out between 1983 and 2004). In the period 1997 – 2004 there were 32 recorded studies. In seven of these studies the number of thyroid adenomas was greater than 10.9 %, demonstrating that 10.9% is within the previously recorded range for the wider control population (1997 – 12 %; 1998 (x2) – 12%; 1998 – 14 %; 1999 – 28.0 %; 2000 – 14.7 %, 2002 – 11.8 %). No treatment related neoplastic findings were seen at 100 or 500 ppm.
Effect levels
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 100 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- histopathology: non-neoplastic
- Remarks on result:
- other: Dietary equivalent to 5.5 and 6.9 mg/kg bw/day in males and females, respectively.
Target system / organ toxicity
open allclose all
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 3 000 ppm
- System:
- hepatobiliary
- Organ:
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- no
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 3 000 ppm
- System:
- female reproductive system
- Organ:
- uterus
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- no
Any other information on results incl. tables
Verification of the Test Diets
The mean concentrations for all batches of diet preparations analysed were within 10 % of the nominal concentration. The homogeneity of the test substance in diet preparations at concentrations of 100 ppm and 3000 ppm, (for a batch size of 60 kg), was determined and considered satisfactory, percentage deviations from the overall mean were within 2 %. The reanalysis of the test substance in diet preparations at concentrations of 100 ppm and 3000 ppm when stored at room temperature was shown to be satisfactory for 42 days, covering the period of dosing.
Table 1. Intergroup comparison of hind limb grip strength (g) in females.
Dietary concentration of test substance (ppm) |
0 |
100 |
500 |
3000 |
Week 50 |
686 |
578 |
558 |
489** |
** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)
Table 2. Intergroup comparison of adjusted body weight (g): selected weeks.
Week
|
Dietary Concentration of test substance (ppm) |
|||||||
Males |
Females |
|||||||
0 |
100 |
500 |
3000 |
0 |
100 |
500 |
3000 |
|
1 |
125.8 |
125.7 |
125.6 |
126.8 |
113.1 |
112.6 |
111.1 |
112.0 |
2 |
166.4 |
166.2 |
165.4 |
159.1** |
134.3 |
134.4 |
133.7 |
129.3** |
13 |
373.3 |
367.9 |
370.7 |
344.7** |
230.1 |
224.7* |
222.9** |
206.9** |
27 |
440.6 |
433.6 |
438.9 |
405.4** |
256.5 |
251.0 |
249.3* |
227.2** |
39 |
483.2 |
474.9 |
481.5 |
439.9** |
277.1 |
273.2 |
270.5 |
238.2** |
53 |
523.3 |
513.1 |
521.8 |
469.4** |
305.4 |
297.6 |
294.7* |
248.0** |
79 |
571.5 |
565.1 |
573.2 |
504.2** |
363.5 |
346.4* |
332.8** |
267.5** |
105 |
610.1 |
603.3 |
623.3 |
530.2** |
403.5 |
382.0* |
359.2** |
296.2** |
* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)
** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided
Table 3. Intergroup comparison of food utilisation (g body weight gained/100 g food consumed).
Week
|
Dietary Concentration of test substance (ppm) |
|||||||
Males |
Females |
|||||||
0 |
100 |
500 |
3000 |
0 |
100 |
500 |
3000 |
|
1-4 |
22.88 |
22 72 |
22.92 |
22.07** |
14.86 |
14.50 |
14.19 |
12.53** |
5-8 |
10.24 |
10.21 |
10.42 |
937** |
6.34 |
6.07 |
6.05 |
5.64** |
9-12 |
6.26 |
6.25 |
6.20 |
5.47** |
3.23 |
3.04 |
321 |
2.71** |
Overall (1-12) |
13.20 |
13.10 |
13.20 |
12.30** |
8.03 |
7.80 |
7.73* |
6.86** |
* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)
** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)
Table 4. Intergroup comparison of liver weights adjusted for body weight (g).
Organ/week |
Dietary Concentration of test substance (ppm) |
|||||||
Males |
Females |
|||||||
0 |
100 |
500 |
3000 |
0 |
100 |
500 |
3000 |
|
Liver, 53 |
15.1 |
14.3 |
15.2 |
17.7** |
9.0 |
8.9 |
9.6 |
10.3** |
Liver , 105 |
16.6 |
16.5 |
17.5* |
19.5** |
10.6 |
11.2 |
11.9** |
13.4** |
* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)
** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)
Table 5. Incidence of selected microscopic non-neoplastic findings in the liver at week.
Week
|
Dietary Concentration of test substance (ppm) |
|||||||
Males |
Females |
|||||||
0 |
100 |
500 |
3000 |
0 |
100 |
500 |
3000 |
|
Total number examined (decedents) |
52 (15) |
52 (14) |
52 (11) |
52 (12) |
52 (14) |
52 (17) |
52 (22) |
52 (19) |
Hypertrophy, hepatocellular, centrilobular |
0 (0) |
12 (0)** |
45 (6)** |
49 (11)** |
0 (0) |
22 (5)** |
49 (19)** |
50 (17)** |
Vacuolation, hepatocellular, centrilobular |
3 (2) |
7 (1) |
32 (6)** |
39 (6)** |
1 (1) |
0 (0) |
18 (4)** |
3 (0) |
Pigment, hepatocellular, centrilobular |
0 (0) |
0 (0) |
1 (0) |
32 (6)** |
3 (0) |
34 (12)** |
49 (19)** |
46 (16)** |
Hyperplasia, bile ducts |
3 (1) |
11 (2)* |
19 (2)** |
12 (1)** |
15 (4) |
12 (5) |
17 (9) |
23 (6) |
Fibrosis, bile ducts |
1 (1) |
5 (1) |
10 (0)** |
12 (1)** |
6 (1) |
6 (2) |
12 (5) |
11 (3) |
Altered hepatocytes, eosinophilic |
7 (1) |
1 (2) |
23 (3)** |
32 (3)** |
9 (2) |
15 (3) |
26 (11)** |
29 (7)** |
Table 6. Incidence of selected microscopic non-neoplastic findings in the female kidney at week 105.
|
Dietary Concentration of test substance (ppm) |
|||
Finding |
0 |
100 |
500 |
3000 |
Pigment, brown, tubules |
18/52 |
24/52 |
30/52* |
42/52** |
* Statistically significant difference from control incidence, p<0.05 (Fisher’s exact test)
** Statistically significant difference from control incidence, p<0.01 (Fisher’s exact test)
Table 7. Incidence of selected microscopic non-neoplastic findings in the male mesenteric lymph node at week 105.
|
Dietary Concentration of test substance (ppm) |
|||
Finding |
0 |
100 |
500 |
3000 |
Erythrocytosis, sinus |
3/52 |
8/52 |
8/51 |
14/52** |
** Statistically significant difference from control incidence, p<0.01 (Fisher’s exact test)
Table 8. Incidence of selected microscopic neoplastic findings at 52 & 105 weeks.
|
Dietary Concentration of test substance (ppm) |
|||
0 |
100 |
500 |
3000 |
|
Total number examined (12 at interim kill, 52 up to 105 weeks) |
64 |
64 |
64 |
64 |
Males: thyroid follicular cell adenoma Total incidence Incidence at interim kill |
1
0 |
4
0 |
2
0 |
7
1 |
Females: thyroid follicular cell adenoma Total incidence Incidence at interim kill |
5
0 |
1
0 |
3
0 |
5
2 |
Females: hepatocellulair adenoma Females: uterine endometrial adenocarcinoma |
1 1 |
1 2 |
1 3 |
11 15 |
Applicant's summary and conclusion
- Conclusions:
- Oral administration of test substance in the diet at dose levels of 100 ppm (5.5 and 6.9 mg/kg/day in males and females) there were minimal treatment related non-neoplastic changes in the liver that were considered not to be adverse. 5.5 mg/kg bw/day was considered to be the no observed adverse effect level (NOAEL).
- Executive summary:
In an OECD TG 453 study in compliance with GLP, groups of 64 male and 64 female HsdRCCHan: WIST rats were fed diets containing 0 (control), 100, 500 or 3000 ppm test substance for up to 2 years (mean dietary equivalent to 5.5, 27.6 and 173.5 mg/kg bw/day for males and 6.9, 34.9 and 232.8 mg/kg bw/day for females, respectively). Twelve males and twelve females from each group were designated for interim kill after 52 weeks. The surviving animals continued to termination after 104 weeks on test. Clinical observations, body weights and food consumption were measured and blood and urine samples were taken for clinical pathology. A functional observation battery of tests and locomotor activity monitoring were performed during week 50 on the interim kill animals. An ophthalmoscopic examination was performed on all main study animals pre study, during weeks 50 and during weeks 101 to 103. All animals, including any found dead or killed prematurely were examined post mortem and tissues were taken for subsequent histopathology examination. At the scheduled 53 or 105 week examinations post mortem, cardiac blood samples were taken for clinical pathology and selected organs were weighed.
There were no treatment related effects on survival. There were higher numbers of males in the 3000 ppm group with scabs and subcutaneous masses than in the control group. There was no evidence of any effect of treatment on ophthalmoscopy findings. Body weights of animals receiving 3000 ppm test substance were reduced throughout the study. The maximum difference from control was 14 % in males and 30 % in females for body weight and 17 % and 40 % respectively for body weight gain. Body weights of females in the 500 ppm group were consistently lower than control. The effect was more pronounced during the second year of the study reaching a maximum of 12 % below control for body weight and 17 % for body weight gain. Body weights of females in the 100 ppm group were considered to be unaffected by treatment. Food consumption and the efficiency of food utilisation were reduced in rats in the 3000 ppm group in the early stages of the study only. There were no treatment related effects on the functional observation clinical assessments, landing foot splay, motor activity, time to tail flick or fore limb grip strength. Hind limb grip strength was lower than control in females in the 3000 ppm group reflecting their lower body weights. There were some indications of a small effect on haemoglobin and related parameters in rats in the 3000 ppm group. Platelet counts were increased in females in the 3000 ppm group at most time points. White blood cell parameters were not affected by treatment and there were no consistent effects on clotting times. There were a number of blood clinical chemistry findings in the 3000 ppm group. Plasma triglycerides were reduced in both sexes, plasma cholesterol was increased and plasma bilirubin levels were generally reduced in females. There was some evidence of increases in gamma glutamyl transferase and alanine transaminase activities. Activities of alkaline phosphatase and aspartate amino transferase were generally lower than control. Sodium and chloride ions were generally increased in females. There were a few small changes in blood clinical chemistry findings in the 500 ppm group but none in the 100 ppm group. There were no treatment-related effects on the urine parameters measured or assessed. Liver weights were increased in the 3000 ppm group at interim and terminal kill and in females in the 500 ppm group at terminal kill only. There were changes in the liver of rats in the 500 and 3000 ppm groups (hepatocellular hypertrophy, pigment in centrilobular hepatocytes, eosinophilic foci of altered hepatocytes, vacuolation of centrilobular hepatocytes and hyperplasia and fibrosis of the bile ducts). Minimal treatment related changes at 100 ppm were hepatocellular hypertrophy in both sexes, bile duct hyperplasia in males and pigment in centrilobular hepatocytes in females. An increased incidence of brown pigment was seen in the kidney of females in the 500 and 3000 ppm groups. There was an increased incidence of sinus erythrocytosis in the mesenteric lymph nodes of males in the 3000 ppm group. There were increased incidences of uterine endometrial adenocarcinoma and liver hepatocellular adenoma and a reduced incidence of mammary fibroadenoma in females at 3000 ppm. No effect on tumour incidence was seen in males or at the 100 or 500 ppm dose levels. Uterine endometrial adenocarcinoma was considered to have led to the death or early termination of five females in the 3000 ppm group.
In conclusion, oral administration of test substance in the diet at dose levels up to 3000 ppm for up to two years led to a marked effect on body weight/body weight gain, particularly in females. There were increases in uterine endometrial adenocarcinoma and liver hepatocellular adenoma in females at this dose level. Treatment-related non-neoplastic findings were seen in the liver, kidney (females) and mesenteric lymph nodes (males). A dose of 500 ppm was the no effect level for neoplastic changes. At this dose the main effects were non-neoplastic changes in the liver and kidney (females) and a reduction in body weight/body weight gain in females. At a dose of 100 ppm (5.5 and 6.9 mg/kg/day in males and females) there were minimal treatment related non-neoplastic changes in the liver that were considered not to be adverse. This dose was considered to be the no observed adverse effect level (NOAEL).
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