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EC number: 266-037-1 | CAS number: 65997-01-5
- 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:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- No data
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
Data source
Reference
- Reference Type:
- publication
- Title:
- Unnamed
- Year:
- 1 998
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- GLP compliance:
- yes
Test material
- Reference substance name:
- Stigmast-5-en-3-β-ol
- EC Number:
- 201-480-6
- EC Name:
- Stigmast-5-en-3-β-ol
- Cas Number:
- 83-46-5
- Molecular formula:
- C29H50O
- Reference substance name:
- (24R)-ergost-5-en-3β-ol
- EC Number:
- 207-484-4
- EC Name:
- (24R)-ergost-5-en-3β-ol
- Cas Number:
- 474-62-4
- Molecular formula:
- C28H48O
- IUPAC Name:
- (3S,8S,9S,10R,13R,14S,17R)-17-[(2R,5R)-5,6-dimethylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol
- Reference substance name:
- Stigmasta-5,22-dien-3-β-ol
- EC Number:
- 201-482-7
- EC Name:
- Stigmasta-5,22-dien-3-β-ol
- Cas Number:
- 83-48-7
- Molecular formula:
- C29H48O
- IUPAC Name:
- (3S,8S,9S,10R,13R,14S,17R)-17-[(E,2R,5S)-5-ethyl-6-methylhept-3-en-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol
- Reference substance name:
- Linoleic acid
- EC Number:
- 200-470-9
- EC Name:
- Linoleic acid
- Cas Number:
- 60-33-3
- Molecular formula:
- C18H32O2
- IUPAC Name:
- octadeca-9,12-dienoic acid
Constituent 1
Constituent 2
Constituent 3
Constituent 4
- Specific details on test material used for the study:
- This study used sterol/fatty acid esters as test material, since this is the chemical form in which they are extracted from plants
Test animals
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Alpk:APfSD
- Details on species / strain selection:
- Strain used as the test lab had substantial background data available for it.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Rodent Breeding Unit of Zeneca Pharmaceuticals (Alderley Park, Macclesfield, IJK)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: Approximately 145-190g
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Up to 2 weeks
DETAILS OF FOOD AND WATER QUALITY:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: To:
Administration / exposure
- Route of administration:
- oral: feed
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): The phytosterol ester was added to the diet (modified form of the AIN (American lnstitute of Nutrition) diet) to give concentrations of 0.16, 1.6,.3.2 and 8.1% (w/w) equivalent to phytosterol ester concentrations of 0.1, 1.0, 2.0 and 5.0% (w/w), respectively. The dry ingredients were mixed in a mixer for approximately 1 minute. The phytosterol ester was then mixed with the maize oil component of the diet using a blender. The oil mixture was then added to the dry ingredients and the complete diet
mixed for 6 minutes.
- Storage temperature of food: A 20-day stability trial conducted at both ambient temperature (19-25°C) and refrigerator temperature (0-4°C) showed PE to be stable in the diet. It is not clear which of these temperatures was used for storage.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Prior to the start of the study the homogeneity and stability of phytosterol ester in the experimental diets was established. The homogeneity of three dietary phytosterol ester concentrations was determined (0.1, 1.0 and 10% w/w) to cover the range of dose levels anticipated for the feeding study. Each dietary dose level was determined by taking five separate samples for analysis from the following positions in the mixing bowl: top centre, middle centre, bottom centre, left centre, right centre). The diets were analysed on the day of preparation (from the homogeneity samples) which also constitute day 0 of the stability trial.
Four additional samples were taken from each diet; two were stored at ambient temperature (19-25°C) and two were stored refrigerated (0-4°C). A pot from each storage temperature was then analysed on day 13 or day 20 to determine the concentration of phytosterol ester. In addition, the actual achieved concentration of phytosterol ester used in the feeding study was determined by analysis of all dose levels for diets prepared for use in week 1, 7 and 13. In each case the diet samples were extracted with petroleum spirit and analysed by HPLC with UV detection at 210 nm and quantitation was achieved by comparison with standards of known concentrations.
Recovery data showed the method of analysis to be acceptable for levels of phytosterol ester in the diet in the range 0.1% to 10% (w/w) and indicated an error bar of ±20% for the 0.1% level and ±15% for the 1.0% and 10% levels. Homogeneity of mixing the diets was demonstrated, within experimental error, for diet samples prepared at 0.1%, 1.0% and 10% (w/w). A 20-day stability trial conducted at both ambient temperature (19 - 25°C) and refrigerated temperature (0-4°C) showed phytosterol esters to be stable in the diet. The actual concentrations of phytosterol esters in the three batches of diet prepared at week 1, 7 and 13 were within ±12% of the nominal concentrations and within the accepted experimental error of the analytical method used. - Duration of treatment / exposure:
- 90 days
- Frequency of treatment:
- Continuous in diet
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0.16 other: % (w/w) phytosterol ester
- Remarks:
- Equivalent to phytosterol concentration of 0.1% (w/w)
- Dose / conc.:
- 1.6 other: % (w/w) phytosterol ester
- Remarks:
- Equivalent to phytosterol concentration of 1.0% (w/w)
- Dose / conc.:
- 3.2 other: % (w/w) phytosterol ester
- Remarks:
- Equivalent to phytosterol concentration of 2.0% (w/w)
- Dose / conc.:
- 8.1 other: % (w/w) phytosterol ester
- Remarks:
- Equivalent to phytosterol concentration of 5.0 % (w/w)
Equivalent to phytosterol dose of 4100 mg/kg bw/day and phytosterol ester dose of 6600 mg/kg bw/day
- No. of animals per sex per dose:
- 20
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale:
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: No satellite groups
- Post-exposure recovery period in satellite groups: No post exposure group
Examinations
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On day 1 then weekly thereafter (at the same time as body weight measurements).
BODY WEIGHT: Yes
- Time schedule for examinations: On day 1 then weekly thereafter.
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: yes
WATER CONSUMPTION: Yes
- Time schedule for examinations: Continuous
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: An ophthalmoscopic examination of all the animals was conducted prior to the start of the study. The eyes of rats from the control and high dose groups were also examined during week 13. The examination was carried out using a Fison's binocular indirect ophthalmoscope after instillation of 0.5% (w/w) tropicamide into the eyes to dilate the pupils.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the 90 day feeding period.
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: Not specified
- How many animals: All animals
- Parameters checked in table [No.1] were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the 90 day feeding period.
- Animals fasted: Not specified
- How many animals: All animals
- Parameters checked in table [No.1] were examined.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2) - Statistics:
- Body weights were considered by analysis of covariance on initial (week 1) body weight, separately for males and females. Weekly food consumption, food utilisation and weekly water consumption were considered by analysis of variance, separately for males and females.
Haematology and clinical chemistry were considered by analysis of variance. Male and female data were analysed together and results examined to determine whether any differences between control and treated groups were consistent between sexes.
Organ weights were considered by analysis of variance and analysis of covariance on final body w eight, separately for males and females. All data were evaluated using the GLM procedure in SAS version 6.12 (1989). Least squares means for each group were calculated using the LSMEAN option in SAS PROC GLM. Analysis of variance and covariance allowed for the replicate structure of the study design. Unbiased estimates of differences from control were provided by the difference between each treatment qroup least-squares mean and the control group least-squares mean.
Differences from the control were tested statistically by comparing each treatment group least squares mean with the control group least squares mean using a two sided Student's t-test based on the error mean square in the analysis.
Results and discussion
Results of examinations
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Description (incidence):
- Three rats of the low dose group were killed for humane reasons, but this was not related to the test substance.
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Group mean food consumption for males receiving phytosterol ester at dietary levels of 1.6%, 3.2% and 8.1% was slightly higher than that of concurrent controls on occasions during the study. This was probably due to the lower energy content of the diets due to the phytosterol component and wasn't considered to be of any toxicological significance.
- Food efficiency:
- no effects observed
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Only minor statistically significant changes observed, which were not related to the test substance.
Slightly reduced platelet counts were observed for females at all doses, in comparison with concurrent controls. However, the magnitude of these changes was not sufficient to be considered biologically significant; for example, a 50-fold increase in dietary phytosterol levels from 0.16% to 8.1%, resulted in only a 3% reduction in platelet count. While slightly reduced platelet counts were also observed for males receiving phytosterol ester at dietary levels of 1.6% and
3.2%, group mean platelet count for males receiving phytosterol ester at the highest dietary level of 8.10% was not statistically different from that of controls.
Marginally reduced prothrombin time as observed in females at all doses and marginally increased APTT time was observed for males receiving phytosterol ester at dietary levels of 3.2% and 8.1%. These differences from control values were very small and were considered to be of no biological or toxicological significance. - Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- Only minor statistically significant changes observed, which were not related to the test substance.
Minor increases in plasma albumin, phosphorus, or magnesium levels and slight increases in several plasma or serum enzymes (including plasma alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, creatinine kinase and lactate dehydrogenase activities and serum 5'-nucleotidase activity) were observed for males and/or females at dietary phytosterol ester levels of 1.6%, 3.2% and 8.1%. However, in the absence of any significant organ weight changes
or histopathological findings in these animals, the changes were concluded not to be biologically significant. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- There were no statistically significant changes to organ weights in comparison with the control group.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- There were no macroscopic findings that could be attributed to treatment.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- There were no microscopic findings that could be attributed to treatment.
A slightly increased incidence of microlithiasis was observed in the kidney cortex of females of the high dose group. This is a common finding of variable incidence in the kidneys of female rats of this strain and was not considered to be an adverse effect. - Histopathological findings: neoplastic:
- not examined
Effect levels
open allclose all
- Dose descriptor:
- NOAEL
- Effect level:
- >= 8.1 other: %
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Dose descriptor:
- NOAEL
- Effect level:
- >= 6 600 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- other: phytosterol ester
- Dose descriptor:
- NOAEL
- Effect level:
- >= 4 100 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- other: phytosterol
Target system / organ toxicity
- Key result
- Critical effects observed:
- no
Applicant's summary and conclusion
- Conclusions:
- In a 90-day repeated dose toxicity study (reliability score 1) conducted using a protocol equivalent to OECD 408 and in compliance with GLP, administration of phytosterol in the diet of Wistar rats for 90 days did not lead to any adverse effects up to dietary concentrations of 8.1% phytosterol ester (equivalent to 6600 mg/kg bw/day; or 4100 mg/kg bw/day phytosterol).
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