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EC number: 406-040-9 | CAS number: 125643-61-0
- 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
Basic toxicokinetics
Administrative data
- Endpoint:
- basic toxicokinetics
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: OECD guideline study conducted according to GLP. However, data on the urinary deconjugation experiment with beta-glucuronidase/sulfatase at the high dose level were missing. This deviation did not affect the outcome of the study.
Cross-reference
- Reason / purpose for cross-reference:
- reference to same study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 006
- Report date:
- 2006
Materials and methods
- Objective of study:
- distribution
- excretion
- metabolism
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 417 (Toxicokinetics)
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- -
- EC Number:
- 406-040-9
- EC Name:
- -
- Cas Number:
- 125643-61-0
- Molecular formula:
- C24-26 H40-44 O3
- IUPAC Name:
- C7-9-(branched)-alkyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate
- Details on test material:
- Specific activity: 4.59 GBq/mmol; 124 mCi/mmol
Physical state: Liquid
Radiochemical purity: 96.0 - 99.8%
Solubility (20°C): Water: 0.5 +/- 0.7 µg/I; Methanol: >50 g/ml
The test material was stored below -15°C in the dark.
Non-radiolabelled test substance (supplied by Sponsor)
Purity: 98.8%
Storage: At ambient temperature in the dark.
Expiry date: 31 December 2010
Constituent 1
- Radiolabelling:
- yes
- Remarks:
- 14C- labeled phenolic ring
Test animals
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- Strain as stated in the report: CD-l (albino) [Crl:CD1(ICR)]
Sex: Male
Bodyweight at dosing: 25 - 40 g
Age at dosing: 6-8 weeks
Supplier: Charles River UK Ltd., Margate, Kent, UK
Acclimatization: at least 5 days before the intended date of dosing.
Housing: During acclimatization animals were housed in groups of up to four mice in solid bottomed plastic cages.
After dosing, the animals were housed in sub-groups of 3 in glass metabolism cages (Metabowls®).
Diet ad libitum: VRFl diet manufactured by Special Diet Services
Water ad libitum: tap water
ENVIRONMENTAL CONDITIONS
Room temperature: 21 +/- 2°C.
Relative humidity: 55 - 15%.
Light/dark cycle: alternating 12 hour light/dark.
Air changes: approximately 15 per hour.
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- other: 0.5% carboxymethylcellulose in 0.1% Tween 80
- Details on exposure:
- 14C-labelled test substance was stored in acetonitrile at a concentration of 495.91 µCi/ml (1.58 mg/ml) at or below -15°C. Dose formulations were freshly prepared on the day of administration. To confirm homogeneity and concentration of radioactivity in the dose formulations (Groups 1, 2 and 4) aliquots were taken during the dosing procedure and were diluted with acetonitrile. Subsequently aliquots were taken in triplicate for radioassay. Purity checks of the dose formulations were carried out at each dosing time and all were >95%.
- Duration and frequency of treatment / exposure:
- single gavage.
Doses / concentrations
- Remarks:
- Doses / Concentrations:
0.5 and 50 mg/kg bw in 10 ml vehicle/ kg bw.
- No. of animals per sex per dose / concentration:
- 4 subgroups of 3 males (=12 males) received 50 mg/kg bw (designated as Group2)
8 subgroups of 3 males (=24 males) splitted into two treated groups received 0.5 mg/kg bw (designated as Group 1 and 4, respectively) - Control animals:
- no
- Details on study design:
- Rationale for the selection of the administration route and dose levels
The oral route was chosen as these ia an expected route for human exposure to the test substance. Dose levels were selected upon consultation of the Regulatory Authority (BfR) at a level where no observable effects were expected (0.5 and 50 mg/kg bw in 10 ml/kg bw vehicle, respectively).
Dose preparation and administration
14C-labelled test substance was stored in acetonitrile at a concentration of 495.91 µCi/ml (1.58 mg/ml) at or below -15°C. Dose formulations were freshly prepared on the day of administration and homogeneity and concentration of radioactivity in the dose formulations were confirmed. The dose solutions were administered orally using a graduated syringe with a rubber gavage tube. The exact amount of formulation administered was determined gravimetrically by weighing the dose syringe when loaded and again after dosing.
Administration of oral doses at 0.5 mg/kg bw (Groups 1 and 4):
A nominal portion of 141.33 - 141.48 µCi of the 14C- labeled test substance was dispensed into a scintillation vial. The solvent was evaporated under nitrogen gas at 37°C and re-suspended in vehicle (9 ml) by use of ultrasonication. Once suspended the formulation was stirred continuously.
Administration of oral dose at 50 mg/kg bw (Group 2):
Non-radiolabelled test substance (43.1 mg), was dispensed into a scintillation vial and 600 µCi 14C-labelled test substance stock solution (nominal) was added. The solution was stirred thoroughly and the solvent was evaporated under nitrogen gas at 37°C and re-suspended in vehicle by ultrasonication. Once suspended the formulation was stirred continuously. The radiochemical purity of the formulation was determined after dose administration.
Measurement of radioactivity
Radioactivity was measured by liquid scintillation counting (LSC) with automatic quench correction. Radioactivity below twice the background level was considered to be below the limit of detection.
Aliquots of liquid samples were mixed with Ultima Gold scintillator (Packard BioScience) or Monoflow 4 for measurement of radioactivity.
Solid samples were combusted in oxygen using a sample oxidizer. The combustion products were absorbed into CO2 sorbent and mixed with Permafluor E+ scintillator. The efficiency of the oxidizer was found to be above 95%. Measurements of radioactivity were corrected for oxidizer efficiency. - Details on dosing and sampling:
- PHARMACOKINETIC STUDY
Excretion and tissue distribution after single oral doses (0.5 and 50 mg/kg bw; Group 1 and 2, respectively):
Following administration of radiolabeled test substance to four subgroups of three male mice, each subgroup was transferred into a glass metabolism cage.
Urine was collected into cooled receivers at time intervals of 0 - 6, 6 - 24 h, and subsequently at intervals of 24 h up to sacrifice after 168 h. Prior to dosing, pre-dose urine samples were collected for use as backgrounds.
Feces were collected separately into cooled receivers at 24 hr intervals up to 168 h. Cage washes were performed every 24 h throughout the sample collection period and retained for radioactivity measurement.
Blood samples were taken under isoflurane by cardiac puncture immediately prior to sacrifice at 168 h into heparinised tubes. Radioactivity of whole blood and plasma were measured.
At sacrifice the following tissues were removed for analysis of radioactivity:
Gastrointestinal tract with contents, Heart, Kidneys, Liver, Lungs, Muscles and Testes
Tissues were pooled for each subgroup of three mice and all samples were stored at or below -15°C prior to analysis. The residual carcass was retained. Radioactivity of tissues, carcass and feces was assessed after homogenization of the respective fraction.
Measurement of volatile radioactivity (0.5 mg/kg bw; Group 4):
Each subgroup of three mice were transferred to glass metabolism cages and 14CO2 of the expired air was trapped in a mixture of ethanolamine: 2-ethoxyethanol (1 : 3, v/v) during 24 hour intervals for up to 48 h. At 48 h the mice were sacrificed by cervical dislocation.
METABOLITE CHARACTERISATION STUDIES
Urine samples from the intervals 0 - 6 h, 6 - 24 h and 24 - 48 h from mice of group 1 and 2 (0.5 and 50 mg/kg bw, respectively) were pooled and duplicate aliquots were taken for radioassay. Urine pool concentrates were analyzed by reverse phase HPLC and normal phase TLC.
Fecal extracts from the 0 - 24 h interval were pooled (10% by weight), concentrated under nitrogen gas and duplicate aliquots were taken for radioassay. Fecal extracts were further analyzed by reverse phase HPLC and normal phase TLC.
PREPARATION OF URINE FOR MASS SPECTROMETRY ANALYSIS
To identify metabolites detected via HPLC/TLC a fresh 0 - 48 h urine pool was prepared from animals treated with 50 mg/kg bw (Group 2). Sub-samples of approximately 2 ml were freeze-dried and re-suspended in methanol. After centrifugation and further washing steps, the samples were analyzed by RP-HPLC. The major urinary metabolites were identified by mass spectrometry (ESI) coupled to a UPLC-UV-system at the Department of Mass Spectrometry for structural investigative work. Particular ions were selected for MS/MS analysis and the corresponding spectra were compared with that from the authentic test substance and the expected metabolite (Metilox acid) to confirm the structure of metabolites.
TREATMENT FOR CLEAVAGE OF CONJUGATES
Urine from sampling interval 0 - 48 h collected from groups 1 and 2, respectively was pooled and duplicate aliquots were taken for radioassay.
For the deconjugation experiments, portions of these pools were incubated with buffer and beta-glucuronidase/sulphatase at 37°C for 18h. Phenolphthalein glucuronic acid was added into a control sample to confirm beta-glucuronidase activity. Samples were analyzed by RP-HPLC and normal phase TLC.
Aliquots of the urine pools were further subjected to mild acid/base hydrolysis. Samples were acidified/basified and incubated at 37°C for 18 h. Upon neutralisation samples were analyzed by reverse phase HPLC and normal phase TLC.
Results and discussion
- Preliminary studies:
- No preliminary study was conducted.
Main ADME resultsopen allclose all
- Type:
- distribution
- Results:
- Upon sacrifice after 7 days, less than 0.3% radioactivity were detected in tissues at 0.5 and 50 mg/kg bw, respectively.
- Type:
- metabolism
- Results:
- A hydroxylated and a glucuronide conjugate of the hydroxylated metabolite, as well as the metabolite (14 - 18% and 0.4 - 1.1 % of dose, respectively) were detected at 0.5 and 50 mg/kg bw.
- Type:
- excretion
- Results:
- 85% to 93% of applied dose were excreted within 48 hours.
Toxicokinetic / pharmacokinetic studies
- Details on distribution in tissues:
- Following administration of 0.5 mg/kg bw radio-labeled test substance radioactivity in carcass, muscle, testes and plasma was below 0.001 µg equivalents/g wet weight. Radioactivity in the other tissues ranged from 0.01 (heart, kidneys, lungs) to a maximum of 0.02 % of administered dose in whole blood.
Following administration of 50 mg/kg bw radio-labeled test substance the tissue radioactivity was highest in whole blood and liver (0.293 and 0.128 µg equivalents/g wet weight, respectively). Radioactivity detected in kidney, lungs, heart and gastro intestine tract (including content) ranged from (0.083 to0.028 µg equivalents/g). All other tissue concentrations (carcass, muscle, plasma and testes) accounted for approximately 0.015 µg equivalents/g wet weight. The radioactivity detected in organs corresponded to 0.01 to 0.02% of the ingested dose.
Following administration of 0.5 mg/kg bw radio-labeled test substance to mice the mean excretion of radioactivity in the expired air during the 0 – 48 h intervall accounted for a total of 0.07% of dose.
- Details on excretion:
- The overall mean recovery following administration of 0.5 mg/kg bw radio-labeled test substance to mice accounted for 99.48% of the dose (Group 1). More than 85% of the given dose was excreted within 48 hours (44% via urine and 43% via feces). Urinary and fecal excretion during the 0 - 168 h interval accounted for 47.7 % and 43.8% of the dose and 7.94% of the dose were recovered in the cage-washes at the same time interval.
Radioactivity in tissues ranged from 0.01 to a maximum of 0.02 % of administered dose.
The overall mean recovery following administration of 50 mg/kg bw radio-labeled test substance accounted for 96.92% of the dose (Group 4).Approximately 93% of dose was excreted within 48 hours (38% via urine and 55%via feces). Urinary and fecal excretion during the 0 - 168 h interval accounted for 38.8% and 56.1% of the dose and 1.94% of the dose were recovered in the cage-washes at the same time interval
Radioactivity in tissues ranged from 0.01 to a maximum of 0.02 % of administered dose.
Following administration of 0.5 mg/kg bw radio-labeled test substance to mice the mean excretion of radioactivity in the expired air during the 0 – 48 h intervall accounted for a total of 0.07% of dose
Metabolite characterisation studies
- Metabolites identified:
- yes
- Details on metabolites:
- A total of 3 metabolites in the freshly prepared urine pools of animals treated with 50 mg/kg bw were identified via molecular weight and fragmentation pattern using a mass spectrometer operating in the negative electrospray mode (ESI LC/MS). Metabolites in the other urine and fecal fractions were correlated to these identified metabolites via their retention times.
Any other information on results incl. tables
Details on metabolites:
A total of 3 metabolites in the freshly prepared urine pools of animals treated with 50 mg/kg bw were identified via molecular weight and fragmentation pattern using a mass spectrometer operating in the negative electrospray mode (ESI LC/MS). Metabolites in the other urine and fecal fractions were correlated to these identified metabolites via their retention times.
In the feces collected 0-24 h after treatment the unchanged test substance was the major component (31.6% of applied dose). The main metabolite accounted for 7.4% while all other metabolites accounted for less than 5.6 % of dose.
Table 1: Radioactivity in selected tissues 7 days after single oral dose of radio-labeled test substance in µg equivalents/g.
Tissue |
Dose (mg/kg bw) |
|
0.5 (group 1) |
50 (Group 2) |
|
Carcass |
0.000 +/- 0.000 |
0.005 +/- 0.005 |
GIT |
0.001 +/- 0.000 |
0.028 +/- 0.012 |
Heart |
0.001 +/- 0.000 |
0.071 +/- 0.014 |
Kidney |
0.001 +/- 0.000 |
0.083 +/- 0.015 |
Liver |
0.002 +/- 0.001 |
0.128 +/- 0.025 |
Lungs |
0.001 +/- 0.001 |
0.081 +/- 0.027 |
Muscle |
0.000 +/- 0.000 |
0.013 +/- 0.015 |
Testes |
0.000 +/- 0.000 |
0.008 +/- 0.001 |
Whole blood |
0.003 +/- 0.001 |
0.293 +/- 0.093 |
Plasma |
0.000 +/- 0.000 |
0.015 +/- 0.004 |
GIT: Gastro intestinal tract (with contents) |
Deviations from the protocol:
No data on the urinary deconjugation experiment with beta-glucuronidase/sulfatase at 50 mg/kg bw were given.
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information): no bioaccumulation potential based on study results
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