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EC number: 231-885-3 | CAS number: 7775-00-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
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- 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
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- Nanomaterial catalytic activity
- Endpoint summary
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- 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
Endpoint summary
Administrative data
Link to relevant study record(s)
- Endpoint:
- basic toxicokinetics in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 07 Feb 2011 to 27 Apr 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Objective of study:
- metabolism
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The objective of this study was to compare the metabolism in vitro of the test substances using cryopreserved hepatocytes from mouse, rat, rabbit and human. Incubations were conducted with cryopreserved hepatocytes in triplicate for each species at three test substance concentrations (1, 10 and 100 µM) over three incubation times (0, 1 and 4 h). HPLC-UV and LC-MS methodologies were used to determine the metabolite profiles generated during the hepatocyte incubations with test substance. LDH leakage was used to monitor cell viability after exposure of the test substance.
- GLP compliance:
- yes
- Radiolabelling:
- no
- Details on test animals or test system and environmental conditions:
- Source: All cryopreserved hepatocytes were obtained from Celsis In Vitro Technologies.
Strains: Mouse CD-1, Rat Sprague-Dawley, Rabbit New Zealand White, Human N/A. - Route of administration:
- other: in vitro application in liquid medium
- Vehicle:
- ethanol
- Details on exposure:
- For each species, components were mixed together in the following order, to give a total incubation volume of 1.0 mL:
- Foetal calf serum (100 μL)
- Test substance (10 μL of solution in ethanol),
- Supplemented Williams’ Medium E
- Hepatocyte suspension (mouse 0.3 × 10^6 viable cells, other species 1 × 10^6 viable cells). - Duration and frequency of treatment / exposure:
- 0, 1 and 4 h
- Dose / conc.:
- 1 other: µM
- Dose / conc.:
- 10 other: µM
- Dose / conc.:
- 100 other: µM
- Details on study design:
- INCUBATION CONDITIONS
The hepatocytes were incubated for 0, 1 and 4 h in polyethylene vials in an orbital shaking water bath (set at 60 rpm and 37°C), under an atmosphere of humidified 95% oxygen : 5% carbon dioxide. Reactions were terminated after the appropriate incubation time by placing the incubation vessels on ice followed immediately by addition of chilled acetonitrile (1 mL) and vortex mixing. Samples were then homogenised by ultrasonic disruption (approximately 20 s) using an ultrasonic probe. The resulting samples were stored briefly at approximately -70°C (to ensure rapid freezing) followed by storage at approximately -20°C pending processing for analysis.
CONTROL INCUBATIONS
On each occasion, control incubations in the absence of hepatocytes were conducted in parallel at each substrate concentration for 4 h only. Additionally, control incubates were conducted with hepatocytes in the absence of test substance for 4 h only. Positive control incubations for metabolic activity were conducted with 7-ethoxy[14C]coumarin (7-EC) as substrate.
NUMBER OF REPLICATES:
Incubations were conducted in triplicate, with the exception of the control hepatocytes in the absence of test substance which were conducted singly.
MEASUREMENT OF LDH LEAKAGE
Incubations with test substance were conducted in parallel to those not containing any test substance, so as to assess whether the inclusion of test substance in the incubation medium had any effect on the leakage of LDH from the hepatocytes. The incubations without test substance comprised of duplicate samples containing 0.3 ×10^6 viable cells/mL (mouse) or 1 x 10^6 viable cells/mL (rat, rabbit, human) in supplemented Williams’ Medium E in the presence of 1% (v/v) ethanol. These were directly compared to samples containing the same number of viable hepatocytes, supplemented Williams’ Medium E and test substance (at final concentration of 100 μM) and 1% (v/v) ethanol. Aliquots (0.5 mL) of the incubation volume were removed at 0, 1 and 4 h and centrifuged at 3,500 rpm (1,068 x g) for 1 min. The supernatant was carefully separated from the cell pellet. The cell pellets from all samples were lysed in 5 mL final volume of 1% (v/v) Triton X-100 in PBS. All pellets and supernatants were stored at approximately 4°C until assay.
The rate of change of absorbance at 340 nm was determined over 1 min in a Shimadzu UV-1700 series spectrophotometer with temperature controlled cell chamber. The assay was performed in a 1 mL, 1 cm light path cuvette at 37°C and consisted of the following components (all pre-warmed to 37-38 °C prior to use): 100 μL cell medium or cell lysate diluted appropriately (1:4, v/v) with PBS, 800 μL NADH in PBS (200 μg/mL) and 100 μL pyruvate in PBS (150 μg/mL). The activity of LDH was measured in LDH units. The magnitude of enzyme leakage into the supernatant was expressed as a percentage of the total cellular LDH activity.
METABOLITE PROFILING
HPLC system: Waters Acquity UPLC (including binary pump, autosampler and UV detector)
Mass spectrometer: Waters Q-Tof micro
HPLC conditions:
- Column: Phenomenex Luna C8(2), 3 μm, 150 × 4.6 mm
- Guard column: Phenomenex Luna C8, 4 × 2 mm
- Column oven temperature: 40°C
- Mobile phase A: 0.005% (v/v) formic acid in purified water
- Mobile phase B: Acetonitrile
- Flow rate: 0.7 mL/min
- UV detection: 220 nm
- Gradient (time : % eluent B): 0 min: 50%, 10 min: 90%, 12 min: 100%, 12.1 min: 50%
- MS settings: split ration 1:9, ionisation: negative, desolvation gas: nitrogen 450 L/h, cone gas: argon 50 L/h, scan rate: 1/s - Metabolites identified:
- yes
- Details on metabolites:
- METABOLITE IDENTIFICATION
A total of eight separate metabolites of the substance were observed and identified, labelled C1 to C8. Interspecies differences in the metabolite profiles were typically small at each incubation time.
There are 8 metabolites detected. See for the chemical structures the illustration attached). These are further described in text at the section additional information on results.
In summary, Cyclemax is mostly conjugated. There are two non-conjugated metabolites. The first is Cyclemax propyl acid, where the aldehyde is oxidised into the acid mainly present at the start of the incubationand present in all species (C8). The second acid is the Cyclemax methyl acid (iso-propyl benzoic acid), present at high dosing >=10 umol in low concentrations in rat, mouse and rabbit but not in humans, potentially formed when the conjugation system becomes saturated. - Executive summary:
In vitro metabolism of the test substance using cryopreserved hepatocytes from mouse, rat, rabbit and human was assessed in a GLP study. Incubations were conducted with cryopreserved hepatocytes in triplicate for each species at three test substance concentrations (1, 10 and 100 µM) over three incubation times (0, 1 and 4 h). LC-MS methodology was used to determine the metabolic profiles generated during the hepatocyte incubations with test substance. The hepatocytes used in the study were shown to be metabolically viable over the incubation periods used and negative controls without substance and without cells were included. It was not possible to detect the unchanged test substances by mass spectrometry and it was considered likely that if present some intermediate metabolites would also have been undetected. It was therefore not possible to derive the actual percentage of total material represented by each metabolite observed, although the relative percentage of the total peak area detected within each component was calculated, with the assumption made that each component was detected with equal intensity. A total of eight components (labelled C1 to C8) were detected following hepatocyte incubations with the test substance, with similar results obtained for each species. Metabolite C6 (glucuronide conjugate of the alcohol) was the largest metabolite in most of the 1 h and 4 h hepatocyte incubations, although the intermediate (alcohol) was not observed. Metabolite C8 (acid) was observed widely and was the second largest component in most mouse, rabbit and human hepatocyte incubations. It was also the only component detected in the 0 h (control) incubations, although levels were low. Metabolite C5 (glucuronide conjugate) was also detected in most incubations and tended to be the second largest component in rat hepatocyte incubations. Most of the remaining metabolites were present at low levels and/or in a limited number of incubations, although metabolite C4 (hydroxylated substance) was detected in most incubations.
Reference
METABOLITE IDENTIFICATION
A total of eight separate metabolites of the substance were observed and identified, labelled C1 to C8. Interspecies differences in the metabolite profiles were typically small at each incubation time.
A total of eight components (labelled C1 to C8) were detected following hepatocyte incubations with the test substance, with similar results obtained for each species.
- Metabolite C6 (glucuronide conjugate of the alcohol) was the largest metabolite in most of the 1 h and 4 h hepatocyte incubations, although the intermediate (alcohol) was not observed.
- Metabolite C8 (the aldehyde turned into acid) was observed widely and was the second largest component in most mouse, rabbit and human hepatocyte incubations. It was also the only component detected in the 0 h (control) incubations, although levels were low.
- Metabolite C5 (glucuronide conjugate) was also detected in most incubations and tended to be the second largest component in rat hepatocyte incubations.
- Most of the remaining metabolites were present at low levels and/or in a limited number of incubations
- Although metabolite C4 (hydroxylated substance) was detected in most incubations.
- Metabolite C1 eluted with a typical retention time of 3.14 min. It was detected in hepatocyte incubations from the mouse (plus one human profile, 100μM, 4 h incubation) and levels were variable (not detected to <20% of total peak area). The molecular ion [M-H]- was 369 and the molecular weight was 370. The metabolite was identified as the glucuronide conjugate of the hydroxylated alcohol, although the position of the hydroxylation could not be confirmed.
- Metabolite C2 was detected in some profiles from all species, but typically only at low levels. The typical retention time was 3.10 min (similar to C1) and the molecular ion [M-H]- was 207, giving a molecular weight of 208. This component was identified as a hydroxylated acid, although it was not possible to determine the position of the hydroxylation.
- Metabolite C3 eluted with a typical retention time of 3.64 min and was observed at variable levels in some mouse profiles (plus one rabbit profile, 10μM, 1 h incubation). The molecular ion [M+HCl+H]- was 375, which indicated a molecular weight of 340, and C3 was identified as a hexose conjugate of conjugate of the alcohol.
- - Metabolite C4 was detected at variable levels in some hepatocyte incubations at 1 and 4 h from all species, typically at higher substance concentrations. The typical retention time was 4.10 min, the molecular ion [M-H]- was 207 and the molecular weight was 208. This component was identified as a hydroxylated acid (similar to C2), although it was not possible to determine the position of the hydroxylation.
- Metabolite C5 eluted with a typical retention time of 4.29 min and was observed in some hepatocyte incubations at 1 and 4 h from all species, generally at higher substance concentrations. Variable levels of metabolite C5 were observed in the profiles. The molecular ion [M-H]- was 351, which indicated a molecular weight of 352, and C5 was identified as the glucuronide conjugate of the substance.
- Metabolite C6 had a typical retention time of 4.76 min and was observed in the majority of 1 h and 4 h hepatocyte incubations from all species. It was the largest metabolite in most 1 h and 4 h incubations. The molecular ion [M-H]- was 353, giving a molecular weight of 354, and the metabolite was identified as the glucuronide conjugate of the alcohol.
- Metabolite C7 eluted with a typical retention time of 5.18 min. It was typically observed as a minor metabolite (<5% of total peak area) in 4 h mouse, rat and rabbit hepatocyte incubations at a substance concentration of 100μM. It was not detected in any human hepatocyte incubations. The molecular ion [M-H]- was 163, which indicated a molecular weight of 164. The identity of C7 was determined as 4-isopropylbenzoic acid, a finding that was supported by analysis of the reference substance.
- Metabolite C8 was detected in several hepatocyte incubations from all species. It was the only component in half of the 0 h incubations and was present at variable levels in a number of 1 h and 4 h incubations. The molecular ion [M-H]- was 191, the molecular weight was 192 and C8 was identified as an acid.
MOUSE HEPATOCYTES
A total of eight metabolites were observed and identified by LC-MS following incubations of the substance with mouse cryopreserved hepatocytes. These were C1 (glucuronide conjugate of the hydroxylated alcohol), C2 and C4 (hydroxylated acid), C3 (hexose conjugate of an alcohol), C5 (glucuronide conjugate), C6 (glucuronide conjugate of the alcohol), C7 (4-isopropylbenzoic acid) and C8 (acid). No metabolites were observed in 0 h (control), 1 h and 4 h hepatocyte incubations of the substance at a concentration of 1μM. Similarly, no metabolites were detected in the control 0 h incubations at 10μM and only C8 was present in the control 100μM incubations.
At a concentration of 10μM, the largest component following the 1 h incubations was metabolite C6 (49.9% of total peak area), followed by C8 (18.0%) and C5 (10.5%). Metabolites C1, C3 and C4 were also observed (each between approximately 6% and 8%). For the 100μM incubations, metabolite C6 (63.6%) was also the largest component, followed by C8 (17.0%), C3 and C5 (each between 6% and 7%). Other metabolites detected were C1, C4 and C7 (each <5%).
The profiles and levels of metabolites (in terms of total peak area) remained similar following the 4 h hepatocyte incubations. For the 10μM concentration, C6 (47.1%) was the largest component, followed by C4 (18.9%), C1 (13.9%), C5 (13.0%) and C8 (7.1%). The largest component at 100μM was also C6 (62.6%), which was followed by C8 (12.1%), C5 (10.7%) and C4 (7.4%), with C1, C2, C3 and C7 also observed (each <5%).
RAT HEPATOCYTES
Six individual metabolites were observed following incubations of the substance with rat cryopreserved hepatocytes; C2 and C4 (hydroxylated acid), C5 (glucuronide conjugate), C6 (glucuronide conjugate of the alcohol), C7 (4-isopropylbenzoic acid) and C8 (acid).
Similar to the mouse, no metabolites were observed in the profiles of the control 0 h rat hepatocyte incubations at substance concentrations of 1 and 10μM, whilst the only component detected at the 100μM concentration was metabolite C8. Metabolite C6 was the only component observed in the profiles following hepatocyte incubations for 1 h at a substance concentration of 1μM. This metabolite was also the largest component (61.6% and 63.4%, respectively) for incubations at substance concentrations of 10 and 100μM. The second largest component was C5 (20.5% and 18.9%,respectively), followed by C8 (16.5% and 16.0%, respectively), with small quantities (<5%) attributed to C4 and C2 (100μM only).
The profiles were also similar for the 4 h hepatocyte incubations at 1, 10 and 100μM. The only component observed in the profiles following hepatocyte incubations for 1 h at a substance concentration of 1μM was metabolite C6. For 10 and 100μM incubations, the largest component was also C6 (60.9% and 66.3%, respectively), whilst the second largest component was C5 (22.0% and 20.6%, respectively). For the 10μM incubations, metabolites C4 and C8 each represented between 8% and 9%, whilst for the 100μM incubations, metabolite C8 accounted for 5.8% with small quantities (<5%) associated with C2, C4 and C7.
RABBIT HEPATOCYTES
The same six individual metabolites were observed following incubations of the substance with rabbit cryopreserved hepatocytes as were observed for rat incubations; C2 and C4 (hydroxylated acid), C5 (glucuronide conjugate), C6 (glucuronide conjugate of the alcohol), C7 (4-isopropylbenzoic acid) and C8 (acid). Only a single metabolite, C8, was observed in the control 0 h rabbit hepatocyte incubations at substance concentrations of 1, 10 and 100μM. For the 1 h hepatocyte incubations at a substance concentration of 1μM, no metabolites were observed. Metabolite C8 (44.3% of the total peak area) was the largest component in the 10μM incubation, followed by C6 (34.1%) and C3 (21.6%). For the 100μM incubation, C8 (63.7%) was also the largest component, followed by C6 (25.6%), with C2, C4 and C5 also detected (each <5%).
Metabolite C6 was the only component detected in the 4 h hepatocyte incubations at a concentration of 1μM and this was the largest component in the incubations at 10 and 100μM concentrations (62.8% and 44.6%, respectively). The second largest component in the 10μM incubations was C8 (17.5%), followed by C2 (10.9%) and C5 (8.8%). For the 100μM incubations, metabolite C8 (35.9%) was the second largest component, followed by C2 (7.3%) and C4 (6.3%), with small quantities (each <5%) associated with C5 and C7.
HUMAN HEPATOCYTES
A total of six metabolites were observed and identified by LC-MS following incubations of the substance with human cryopreserved hepatocytes. These were C1 (glucuronide conjugate of hydroxylated alcohol), C2 and C4 (hydroxylated acid), C5 (glucuronide conjugate), C6 (glucuronide conjugate of the alcohol) and C8 (acid). Metabolites C3 (hexose conjugate of alcohol) and C7 (4-isopropylbenzoic acid) were not detected in human hepatocytes incubations.
Consistent with the mouse and rat incubations, no metabolites were observed in the profiles of the control 0 h human hepatocyte incubations at substance concentrations of 1 and 10μM, whilst only metabolite C8 was detected in the profiles for the 100μM incubations. Similarly, no metabolites were observed in the hepatocyte incubations for 1 h at a substance concentration of 1μM. However, metabolites were observed in 1 h incubations at the higher concentrations. For the 10μM incubation, metabolite C6 (73.5%) was the largest in terms of total peak area, followed by C8 (17.3%), C4 (6.2%) and C5 (3.0%). The profile for the 100μM incubation was similar, with the largest component being C6 (68.5%), followed by C8 (21.2%) and C4 (7.2%).Metabolites C2 and C5 were also detected (each <5%).
Following a 4 h incubation of human hepatocytes at a substance concentration of 1μM, only a single metabolite, C6, was observed. At the 10μM concentration, the largest metabolite was also C6 (80.8%), followed by C8 (8.8%), C4 (6.9%) and C5 (3.6%). Metabolite C6 (64.6%) was again the largest component for the 100μM incubation, followed by C4 and C8 (each approximately 14%), with C1, C2 and C5 also detected (each <5%).
LDH leakage
The substance did not significantly increase the leakage of LDH from the hepatocytes under the conditions of the test.
Metabolite quantification
It was not possible to detect the unchanged test substances by mass spectrometry and it was considered likely that if present some intermediate metabolites would also have been undetected. It was therefore not possible to derive the actual percentage of total material represented by each metabolite observed, although the relative percentage of the total peak area detected within each component was calculated, with the assumption made that each component was detected with equal intensity.
Description of key information
In vitro metabolism in cryopreserved hepatocytes
Introduction: Some effects were seen on male reproductive organs in a 14 -day study. The metabolite profiling was conducted to see whether there were species differences and /or metabolic pathways were overloaded.
Methods: In vitro metabolism of the test substance using cryopreserved hepatocytes from mouse, rat, rabbit and human was assessed in a GLP study. Incubations were conducted with cryopreserved hepatocytes in triplicate for each species at three test substance concentrations (1, 10 and 100 µM) over three incubation times (0, 1 and 4 h). LC-MS methodology was used to determine the metabolic profiles generated during the hepatocyte incubations with test substance. The hepatocytes used in the study were shown to be metabolically viable over the incubation periods used and negative controls without substance and without cells were included. It was not possible to detect the unchanged test substances by mass spectrometry and it was considered likely that if present some intermediate metabolites would also have been undetected. It was therefore not possible to derive the actual percentage of total material represented by each metabolite observed, although the relative percentage of the total peak area detected within each component was calculated, with the assumption made that each component was detected with equal intensity.
Results: A total of eight separate metabolites of the substance were observed and identified, labelled C1 to C8. Interspecies differences in the metabolite profiles were typically small at each incubation time.
There are 8 metabolites detected. See for the chemical structures the illustration attached). These are further described in text at the section additional information on results.
In summary, Cyclemax is mostly conjugated. There are two non-conjugated metabolites. The first is Cyclemax propyl acid, where the aldehyde is oxidised into the acid mainly present at the start of the incubationand present in all species (C8). The second acid is the Cyclemax methyl acid (iso-propyl benzoic acid), present at high dosing >=10 umol in low concentrations in rat, mouse and rabbit but not in humans, potentially formed when the conjugation system becomes saturated.
Key value for chemical safety assessment
Additional information
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