Registration Dossier
Registration Dossier
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EC number: 815-966-6 | CAS number: 915972-17-7
Specific investigations: other studies
Administrative data
Link to relevant study record(s)
- Endpoint:
- specific investigations: other studies
- Remarks:
- D2 tissue bioassay
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- From: September 29, 2015 To: October 07, 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- D2 tissue bioassay
- GLP compliance:
- no
- Type of method:
- in vitro
- Endpoint addressed:
- other: vascular
- Specific details on test material used for the study:
- Lot number: 080722
Expiration date of the lot/batch: n.a. - Analytical verification of doses or concentrations:
- no
- Remarks:
- Control
- Remarks:
- Solvent (DMSO)
- Remarks:
- Test substance
- Remarks:
- Test substance
- Remarks:
- Test substance
- Remarks:
- +(-)Sulpiride
- Remarks:
- (-)Sulpiride
- Remarks:
- Test substance + (-)Sulpiride (3.0E-06)
- Remarks:
- Test substance + (-)Sulpiride (3.0E-06)
- Remarks:
- Test substance + (-)Sulpiride (3.0E-05)
- Details on study design:
- DOPAMINE D2 RECEPTOR
Rings of rabbit ear artery were suspended in 20-mL organ baths filled with an oxygenated (95 % 02 and 5 % C02) and pre-warmed (3rC) physiological salt solution of the following composition (in mM): NaCI 118.0, KCI 4.7, MgS04 1.2, CaCI2 2.5, KH2P04 1.2, NaHC03 25 and glucose 11.0 (pH 7.4).
Yohimbine (1 µM) and propranolol (1 µM) were also present throughout tl1e experiments to block the a2- and ß-adrenergic receptors, respectively.
The tissues were connected to force transducers for isometric tension recordings. They were stretched to a resting tension of 1 g then allowed to equilibrate for 30 min during which time they were washed repeatedly and the tension readjusted. Thereafter, they were stimulated electrically with 10-s train pulses (minimal intensity required to trigger maximal contractions, 1 ms duration, 5 Hz), delivered at 2-min intervals by a constant current stimulator.
The experiments were carried out using semi-automated isolated organ systems possessing eight organ baths, with multichannel data acquisition.
The parameter measured is the maximum change in the amplitude of electrically-evoked twitch contractions induced by each compound concentration.
EVALUATION OF THE SPECIFICITY OF THE AGONIST ACTIVITY
The test substance was tested at 3 concentrations in duplicate after a 20-minute pre-treatment with solvent or (-)sulpiride at 3.0E-06 M.
The tissues are exposed to a submaximal concentration of the reference agonist (-)quinpirole (0.3 µM) to verify responsiveness and to obtain a control response.
Following washings and recovery of twitch contractions, the tissues are exposeci to (-)sulpiride (3 µM) or solvent for 20 min, then increasing concentrations of the test compound or the same agonist. The different concentrations are added cumulatively and each left in contact with the tissues until a stable response is obtained or for a maximurn of 20 min.
ln the case of solvent pre-treatment, the reference antagonist (-)sulpiride (3 µM) was tested at the end of the experiment against the highest concentration of the compound to confirm the involvement of the D2 receptor in this response. - Positive control:
- ln each experiment and if applicable, the respective reference compound was tested concurrently with the test compounds, and the data were compared with historical va!ues determined at Eurofins. The experiment was accepted in accordance with Eurofins validation Standard Operating Procedure.
Reference agonist: quinpirole
Reference antagonist: (-) sulpiride - Details on results:
- In the field-stimulated rabbit ear artery, the D2 receptor agonist (-)quinpirole induced a concentration-dependent decrease in the twitch contraction amplitude which was inhibited by the antagonist (-)sulpiride.
Solvent or (-)Sulpiride (3.0E-06M) did not affect the twitch contraction amplitude.
After a 20-minute pre-treatment with solvent, the test substance induced a concentration-dependent decrease in the twitch contraction amplitude which was not blocked with further addition of (-)sulpiride at 3.0E-06 M.
After a 20-minute pre-treatment with (-)sulpiride at 3.0E-06 M, the test substance induced a concentration-dependent decrease in the twitch contraction amplitude that was right shifted when compared their effect after solvent pre-treatment.
These results confirm the non-reversal of their effect with (-)sulpiride when added at the end of the experiment. The results indicate the importance of the (-)sulpiride addition protocole to reveal the specificity of the test substance response since addition of (-)sulpiride 20 minutes before the test substance decreases its effect.
The test substance behaves as agonists at the D2 receptor in the field-stimulated rabbit ear artery assay. - Endpoint:
- chemobiokinetics general studies
- Remarks:
- Binding assays
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- From: August 13, 2015 To: September 01, 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- IC50 or EC50 determination
- GLP compliance:
- no
- Endpoint addressed:
- basic toxicokinetics
- Specific details on test material used for the study:
- Lot number: 080722
Expiration date of the lot/batch: n.a. - Remarks:
- Test substance
- Remarks:
- Test substance
- Remarks:
- Test substance
- Remarks:
- Test substance
- Remarks:
- Test substance
- Remarks:
- Test substance
- Remarks:
- Test substance
- Remarks:
- Test substance
- Details on study design:
- The test were conducted in human recombinant (HEK-293 cells).
The test substance was tested at several concentrations for IC50 or EC50 determination.
Compound binding was calculated as a % inhibition of the binding of a radioactively labeled ligand specific for each target.
INTERPRETATION OF RESULTS
Results showing an inhibition (or stimulation for assays run in basal conditions) higher than 50% are considered to represent significant effects of the test compounds. 50% is the most common cut-off value for further investigation (determination of IC50 or EC50 values from concentration-response curves) that we would recommend.
Results showing an inhibition (or stimulation) between 25% and 50% are indicative of weak to moderate effects (in most assays, they should be confirmed by further testing as they are within a range where more inter-experimental variability can occur).
Results showing an inhibition (or stimulation) lower than 25% are not considered significant and mostly attributable to variability of the signal around the control level.
Low to moderate negative values have no real meaning and are attributable to variability of the signal around the control level. High negative values (≥ 50 %) that are sometimes obtained with high concentrations of test compounds are generally attributable to nonspecific effects of the test compounds in the assays. On rare occasion they could suggest an allosteric effect of the test compound. - Positive control:
- In each experiment and if applicable, the respective reference compound was tested concurrently with the test substance, and the data were compared with historical values determined at Eurofins.
Reference compounds:
Agonist radioligand: 7-OH-DPAT
IC50: 1.6E-09 M
Kl: 6.4E-10 M
nH: 0.9
Antagonist radioligand: butaclamol
IC50: 3.9E-09 M
Kl: 9.8E-10 M
nH: 1.3 - Details on results:
- The test substance showed little capability as orthosteric agonist or antagonist to receptors tested (D2s and D2L).
- Endpoint:
- chemobiokinetics general studies
- Remarks:
- Binding, tissue, enzyme and uptake and cellular and nuclear receptor functional assays.
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- From: June 09, 2015 To: July 13, 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- Binding, tissue, enzyme and uptake, cellular and nuclear receptor functional assays.
- GLP compliance:
- no
- Type of method:
- in vitro
- Specific details on test material used for the study:
- Lot number: 080722
Expiration date of the lot/batch: n.a. - Remarks:
- Test substance
- Details on study design:
- BINDING ASSAY
Transporters: dopamine transporter (h) (antagonist radioligand)
Source:human recombinant (CHO cells)
Ligand: [3H]BTCP
Concentration: 4 nM
Kd: 4.5 nM
Non specific: BTCP (10 µM)
Incubation: 120 min, 4 °C
Detection method: scintillation counting
CELLULAR AND NUCLEAR RECPTOR FUNCTIONAL ASSAYS
Receptors: D1 (h) (agonist effect)
Source: human recombinant (CHO cells)
Stimulus: none (10 µM dopamine for control)
Receptors: D1(h) (antagonist effect)
Source: human recombibnant (CHO cells)
Stimulus: dopamine (300 nM)
Incubation: 30 min, RT
Measured component: cAMP
Detection method: HTRF
ENZYME AND UPTAKE ASSAYS
Transporters: dopamine uptae
Souruce: rat striatum synaptosomes
Substrate/stimulus/tracer: [3H]DA (0.2 µCi/mL)
Incubation: 15 min, 37 °C
Measured component: [3H]DA incorporation into synaptosomes
Detection method: scintillation counting
TISSUE BIOASSAYS
Dopamine D1 Receptor
Rings of rabbit splenic artery were suspended in 20-mL organ baths filled with an oxygenated (95 % O2 and 5 % CO2) and pre-warmed (37 °C) physiological salt solution of the following composition (in mM): NaCl 118.0, KCl 4.7, MgSO4 1.2, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25 and glucose 11.0 (pH 7.4).
Benextramine (1 μM), propranolol (1 μM), (-)sulpiride (0.1 μM), pyrilamine (1 μM), atropine (1 μM), methysergide (1 μM) and indomethacin (3 μM) were also present throughout the experiments to block the α-adrenergic, β-adrenergic, dopamine D2, histamine H1, muscarinic and 5-HT2 receptors and to prevent prostanoid release, respectively.
The tissues were connected to force transducers for isometric tension recordings. They were stretched to a resting tension of 1 g then allowed to equilibrate for 60 min during which time they were washed repeatedly and the tension readjusted.
The experiments were carried out using semi-automated isolated organ systems possessing eight organ baths, with multichannel data acquisition.
The parameter measured is the maximum change in tension induced by each compound concentration.
Dopamine D2 Receptor
Rings of rabbit ear artery were suspended in 20-mL organ baths filled with an oxygenated (95 % O2 and 5 % CO2) and pre-warmed (37 °C) physiological salt solution of the following composition (in mM): NaCl 118.0, KCl 4.7, MgSO4 1.2, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25 and glucose 11.0 (pH 7.4).
Yohimbine (1 μM) and propranolol (1 μM) were also present throughout the experiments to block the α2- and β-adrenergic receptors, respectively.
The tissues were connected to force transducers for isometric tension recordings. They were stretched to a resting tension of 1 g then allowed to equilibrate for 30 min during which time they were washed repeatedly and the tension readjusted. Thereafter, they were stimulated electrically with 10-s train pulses (minimal intensity required to trigger maximal contractions, 1 ms duration, 5 Hz), delivered at 2-min intervals by a constant current stimulator.
The experiments were carried out using semi-automated isolated organ systems possessing eight organ baths, with multichannel data acquisition.
The parameter measured is the maximum change in the amplitude of electrically-evoked twitch contractions induced by each compound concentration. - Examinations:
- INTERPRETATION OF RESULTS
Results showing an inhibition (or stimulation for assays run in basal conditions) higher than 50 % are considered to represent significant effects of the test compounds. 50 % is the most common cut-off value for further investigation (determination of IC50 or EC50 values from concentration-response curves) that is recommended.
Results showing an inhibition (or stimulation) between 25 % and 50 % are indicative of weak to moderate effects (in most assays, they should be confirmed by further testing as they are within a range where more inter-experimental variability can occur).
Results showing an inhibition (or stimulation) lower than 25 % are not considered significant and mostly attributable to variability of the signal around the control level.
Low to moderate negative values have no real meaning and are attributable to variability of the signal around the control level. High negative values (≥ 50 %) that are sometimes obtained with high concentrations of test compounds are generally attributable to non-specific effects of the test compounds in the assays. On rare occasion they could suggest an allosteric effect of the test compound. - Positive control:
- In each experiment and if applicable, the respective reference compound was tested concurrently with the test compounds, and the data were compared with historical values determined at Eurofins.
- Details on results:
- The compound showed little capability as orthosteric agonists or antagonists to the radioligand receptors tested (D1, Dopamine transporter). There was no activity in the dopamine uptake assay.
In the D1 tissue bioassay, the test substance did not show agonist or antagonist activity.
In the D2 tissue bioassay, the test substance showed significant dose dependent activity that was not blocked with the addition of a dopamine antagonist (-)sulpiride.
The test compound did not show an antagonist effect at the D2 receptor. - Endpoint:
- endocrine system modulation
- Remarks:
- Estrogen receptor transcriptional activation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- From: July 20, 2015 To: December 07, 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OPPTS (OCSPP) 890.1300
- Version / remarks:
- 2009
- Deviations:
- no
- GLP compliance:
- yes
- Type of method:
- in vitro
- Endpoint addressed:
- other: endocrine potential
- Specific details on test material used for the study:
- Lot number: 080722
Description: yellow powder
Expiration date of the lot/batch: November 01, 2015 - Details on study design:
- The hERα-HeLa-9903 cell line is derived from a human cervical tumor and has two stably inserted constructs: (i) the hERα expression construct (encoding the full-length human receptor) and (ii) a firefly luciferase reporter construct bearing five tandem repeats of a vitellogenin estrogenresponsive element driven by a mouse metallothionein (MT) promoter TATA element.
The stably transfected hERα-HeLa-9903 cell line were used for the assay. The cells used in the study were appropriately labeled and were identified by cell type and passage number. Only hERα-HeLa-9903 cells that test negative for mycoplasma were used and certification was included in the final report as Appendix 3. The hERα-HeLa-9903 cells used had gone through ≤40 passages and ≤3 months in culture when used in the assays. The hERα-HeLa-9903 cells were grown for more than one passage from the frozen stock before use.
In view of the short-term nature of studies of this type, no analyses of stability, homogeneity or achieved concentration(s) were carried out on preparations of the test substance or reference control substances, either before or after the treatment phase. This is not considered to have affected the integrity of the study. For the reference control substances, stability is demonstrated by an appropriate response in the assay system. No edge effects were observed.
Preliminary assessments of cytotoxicity and precipitation were conducted in order to identify a suitable top concentration of the test substance for use in the transcriptional activation assays.
The reference chemicals (17β-Estradiol, 17α-Estradiol, Corticosterone and 17α-Methyltestosterone) and the test substance were dissolved in DMSO then serially diluted as appropriate in DMSO before further dilution in medium to prepare 2x concentrated stock solutions. When added to the cell culture plates, the stock solutions would yield the final serial concentrations for the reference chemicals and as determined for in the preliminary range finding assays for the test substance (see below).
The final concentration of DMSO in the medium was held constant at 0.1 % (v/v).
17β-Estradiol - 10E-15, 10E-14, 10E-13, 10E-12, 10E-11, 10E-10, 10E-9 and 10E-8 M
17α-Estradiol - 10E-13, 10E-12, 10E-11, 10E-10, 10E-9, 10E-8, 10E-7 and 10E-6 M
Corticosterone - 10E-11, 10E-10, 10E-9, 10E-8, 10E-7, 10E-6, 10E-5 and 10E-4 M
17α-Methyltestosterone - 10E-12, 10E-11, 10E-10, 10E-9, 10E-8, 10E-7, 10E-6 and 10E-5 M
After the three hour (minimum) post-seeding incubation, the plates were removed from the incubator and the media was aspirated. 75 μL of fresh media, followed by 75 μL of the 2x concentrated stock solutions were added to wells containing ~1 X 104 cells/well for a final volume of 150 μL/well.
After adding the reference /test substance, the plates were incubated in a 5 % CO2 incubator at approximately 37 °C for 20 – 24 hours.
Three independent runs of the transcriptional activation assay were conducted due to half log concentrations being run for all compounds instead of whole log concentrations. All concentrations were tested in replicates of 6/plate. In addition, for each concentration, 2 replicates/plate were prepared that incorporated the hERα antagonist ICI 182,780. Replicates incorporating the hERα antagonist allow for the identification of non-specific (i.e., non-hERα-mediated) induction of the luciferase gene. The duration of exposure was 24 h. A complete concentration response curve for each of 4 reference compounds (17β-estradiol, 17α-estradiol, corticosterone and 17α-methyltestosterone) was run each time the transcriptional activation assay was performed.
All concentrations were tested in replicates of 6/plate. In addition, for each concentration, 2 replicates/plate were prepared that incorporated the hERα antagonist ICI 182,780. Replicates incorporating a hERα antagonist allow for the identification of non-specific (i.e., non-hERα-mediated) induction of the luciferase gene as true hERα-mediated induction is inhibited by addition of an antagonist whereas non-specific induction is not. - Positive control:
- The positive controls were 17β-estradiol and 17α-estradiol. The weak positive control was 17α-methyltestosterone. The negative control compound was corticosterone. ICI 182,780 was also used to block the estrogenic activity. All of the control substances were assessed each run of the assay.
- Details on results:
- CONCENTRATION RANGE FOR THE TEST SUBSTANCE
The suitable top concentration of the test substance for use in the transcriptional activation assays was 10E-5 M, as higher concentrations exhibited problems with solubility (test substance precipitation observed) and cytotoxicity. There was no cytotoxicity (≥20 % reduction in cell viability) observed with the test substance or the controls in any valid run of the assay.
TRANSCRIPTIONAL ACTIVATION ASSAY
In both valid independent runs of the assay, the mean luciferase activity of the PC (1 nM 17β- estradiol) was greater than 4-fold that of the mean luciferase activity of the VC on each plate. In addition, in both independent runs of the assay the LogPC50, LogPC10, LogEC50 and Hill slope values for the 4 reference compounds (17β-estradiol, 17α-estradiol, 17α-methyltestosterone and corticosterone) were within the acceptable ranges, with the some minor exceptions. However, these deviations from the ranges suggested in the OPPTS guideline were minor and not considered to impact the interpretation of results as the assay response with 17β-estradiol, 17α-estradiol, 17α-methyltestosterone and corticosterone were characteristic of a strong estrogen, a weak estrogen, a weak agonist, and a negative compound, respectively, and the data are within Cyprotex historic values and within values cited elsewhere (Schapaugh et al, 2015).
Therefore, both independent runs of the assay were considered to have met the assay acceptance criteria and were considered to be valid.
TRANSCRIPTIONAL ACTIVATION ASSAY RESULTS
In the first valid independent run of the transcriptional activation assay, the test substance I resulted in an increase in luciferase activity of 0.3 ± 0.3% maximal induction at 10-8 M. In the second valid independent run, the test substance resulted in an increase in luciferase activity of 0.8 ± 0.9% maximal induction at 10-10 M.
The test substance was not an agonist of human estrogen receptor alpha (hERα) in the HeLa-9903 model system. - Endpoint:
- endocrine system modulation
- Remarks:
- Estrogen Receptor Binding
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- From: July 20, 2015 To: December 07, 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OPPTS (OCSPP) 890.1250
- Version / remarks:
- 2009
- Deviations:
- no
- GLP compliance:
- yes
- Type of method:
- in vitro
- Endpoint addressed:
- other: Estrogen Receptor Binding
- Specific details on test material used for the study:
- Lot number: 080722
Description: yellow powder
Expiration date of the lot/batch: November 01, 2015 - Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- female
- Details on study design:
- TEST SUBSTANCE PREPARATION
The test substance was prepared in the assay buffer, TEDG+PI, cooled on ice, and pH adjusted to ~7.4 with a small amount of sodium hydroxide. Serial dilutions of the test substances were prepared in TEDG+PI to yield the final concentrations.
POSITIVE, WEAK POSITIVE AND NEGATIVE CONTROL PREPARATION
Octyltriethoxysilane was the negative control. A 25 mM stock was prepared in DMSO and serially diluted as described for the test substances. The concentration range for the negative control was 1 x 1E-10 to 1 x 1E-03 M with DMSO kept at approximately 4 %.
The weak positive control was 19-norethindrone. A 10 mM stock was prepared in DMSO and serially diluted as described in the table on the following page. The concentration range tested for the weak positive control was from 3.16 x 1E-09 to 1 x 1E-04 M with DMSO kept at approximately 4 %.
The positive control, 17β-estradiol, strongly binds ERs and was included to ensure that the run was properly performed and to allow an assessment of variability in the conduct of the assay across time. Final concentrations of unlabeled 17β-estradiol ranged from 1 x 1E-11 to 1 x 1E-07 M. Fresh 25 μM 17β-estradiol stock was prepared and serial dilutions of the reference standard were performed in DMSO (final concentration of 4 %).
The dose concentrations of the test substances were not verified using analytical methods.
SOLUBILITY/PRECIPITATION ASSAY
The limit of test substance solubility was determined visually.
RAT UTERINE CYTOSOL
As per the guideline (OPPTS 890.1250) uteri from 100 Sprague-Dawley female rats (purchased from Charles River, 85 to 100 days of age at time of kill) ovariectomized seven days prior to being humanely killed were used to prepare the cytosol. The cytosol was prepared, and validated for use in this study and was not be older than 90 days. The cytosol was identified by the isolation date.
STOCK SOLUTION PREPARATION
TEG Buffer (20 mM Tris, 3 mM EDTA, 20 % glycerol, pH ~ 7.4 [cooled to ~4 °C before adjusting to pH 7.4] was prepared and stored at ~4 °C up to 3 months. The 60 % hydroxyapatite (HAP) slurry was prepared one day before use. The HAP was gently mixed with ~3X volume of TEDG+PI buffer in a graduated cylinder, and refrigerated for approximately 2 hours at ~4 °C. The HAP was then washed twice. After the second wash, the HAP sluffy settled overnight. The next day (day of use), the volume of HAP was noted. The supernatant was removed and the HAP was resuspended to a final volume of 60 % HAP in ice cold TEDG+PI. The HAP slurry was well-suspended and ice-cold when used in the separation procedure.
WORKING ASSAY BUFFER PREPARATION
On the day of assay, the Working Assay Buffer, or TEDG+PI buffer (10 mM Tris, 1.5 mM EDTA, 1 mM DTT, 0.5 % (v/v) Protease Inhibitor (PMSF), 10 % glycerol, pH ~7.4) was prepared using the 2X TEG buffer.
[3H]-17β-ESTRADIOL PREPARATION
The specific activity (SA) was adjusted for decay over time prior to performing dilutions. A 50 nM diluted stock of the [3H]-17β-estradiol was prepared so that 10 μL in a total volume of 500 μL per assay tube resulted in a final concentration of 1 nM. The 50 nM [3H]-17β-estradiol was kept on ice until test substances, 17β-estradiol, octyltriethoxysilane, and 19-norethindrone dilutions, as well as the assay tubes, were prepared).
ASSAY PREPARATIONS
Twelve (12) x 75 mm siliconized tubes were used for the assay. A master mixture of radioligand and buffer was prepared. Trace tubes were also required (tubes containing buffer and [3H]-17β-estradiol but no protein to determine total activity).
INDIVIDUAL TUBES
For the ER assay tubes, 380 μL of the master mixture above was added and kept on ice. For the trace tubes, 50 μL (1 nM [3H]-17β-estradiol) final was added directly to 10 mL of scintillation fluid in scintillation vials and counted immediately. The test substance, 17β-estradiol, 19-norethindrone, and octyltriethoxysilane were prepared and 20 μL was added per corresponding ER assay tube. After all chemicals were added to the tubes, 100 μL of cytosol was added to each tube (approximately 0.1 mg/mL or 10 μg/assay tube) for a final volume of 500 μL. The tubes and contents were kept in an ice bath prior to the addition of the cytosol. The ER assay tubes were vortexed after additions and incubated on a rotator at ~4 °C for 18 hours 42 min for valid run 1, 18 hours 24 min for valid run 2 and 20 hours 20 min for valid run 3.
SEPARATION OF BOUND [3H]-17β-ESTRADIOL FROM FREE [3H]-17β-ESTRADIOL
The ER assay tubes were removed from the rotator and placed in an ice-water bath. A repeating pipette was used to add approximately 250 μL of ice cold HAP slurry (60 % in TEDG+PI) to each assay tube. The tubes were vortexed for approximately 10 seconds at approximately 5 minute intervals for a total of approximately 15 minutes with tubes remaining in the ice-water bath between vortexing. Following the vortexing step, approximately 2 mL of the cold (~4 °C) TEDG+PI buffer was added, quickly vortexed, and centrifuged at ~4°C for approximately 10 minutes at 1000 x g. After centrifugation, the supernatant containing the free [3H]-17β-estradiol was immediately decanted and discarded. The HAP pellet contained the estrogen receptor bound [3H]-17β-estradiol. Approximately 2 mL of ice-cold TEDG+PI buffer was added to each tube and vortexed to resuspend the pellet. The tubes were centrifuged again at ~4 °C for approximately 10 minutes at approximately 1000 x g. The supernatant was quickly decanted and discarded. The wash and centrifugation steps were repeated once more. After the final wash, the supernatant was decanted. The assay tubes were allowed to drain briefly.
EXTRACTION AND QUANTIFICATION OF [3H]-17β-ESTRADIOL BOUND TO ER
Approximately 1.5 mL of absolute ethanol was added to each assay tube. The tubes were allowed to sit at room temperature for approximately 15 to 20 minutes, vortexing for approximately 10 seconds at approximately 5-minute intervals. The assay tubes were centrifuged for approximately 10 minutes at approximately 1000 x g. An approximately 1 mL aliquot was pipetted, taking care to avoid the centrifuged pellet, into a 20 mL scintillation vial containing approximately 10 mL of scintillation cocktail (Perkin Elmer Opti Fluor). The vial was capped and shaken. The vials were placed in a scintillation counter and each vial was counted for at least one minute with quench correction for determination of disintegrations per minute (DPM) per vial.
Standards (3H, 14C and background) were used to verify accurate counting, and the liquid scintillation analyzer has an enhanced Instrument Performance Assessment (IPA) for monitoring efficiencies, backgrounds, E2/B and Chi-square values for 3H and 14C over the life of the instrument. - Positive control:
- The positive controls were 17β-estradiol. The weak positive control was 19-norethindrone. The negative control compound was octyltriethoxysilane. ICI 182,780 was also used to block the estrogenic activity. All of the control substances were assessed each run of the assay.
- Details on results:
- CONCENTRATION RANGE FOR THE TEST SUBSTANCE
To identify a suitable top concentration for use in the binding assays, preliminary assessments of precipitation were conducted. Precipitation was observed for 1e-3 M of the test substance in the first run, but was not observed in runs 2 or 3. Therefore, the suitable top concentration ofthe test substance for use in the binding assays was 1e-3 M.
BINDING ASSAY ACCEPTANCE CRITERIA
All three independent runs of the assay were considered to have met the assay acceptance criteria and were considered to be valid.
BINDING ASSAY
In the first valid independent run the mean specific binding for the test substance was >93 % at every concentration tested with the exception of 10-3 M where the binding was 58.1 %, classifying the test substance as “equivocal” for this run. The mean specific binding for for the test substance was >92 % at every concentration tested, classifying the test substance as “non-interacting” for this run. The mean specific binding for the test substance was >92 % at every concentration tested, classifying the test substance as “non-interacting” for this run. The weak positive control 19-norethindrone had a LogIC50 of –5.4 M, and the LogIC50 of 17β-estradiol was -9.2 M, resulting in an RBA for 19-norethindrone of 0.0158 % and a log(RBA) of -1.8.
In the second valid independent run the mean specific binding for the test substance was >97 % at every concentration tested with the exception of 10-3 M where the binding was 78.6 %, classifying the test substance as “non-interacting” for this run. The mean specific binding for the test substance was >96 % at every concentration tested, classifying the test substance as “non-interacting” for this run. The mean specific binding for the test substance was >95 % at every concentration tested, classifying the test substacne as “non-interacting” for this run. The weak positive control 19-norethindrone had a LogIC50 of –5.4 M, and the LogIC50 of 17β- estradiol was -9.2 M, resulting in an RBA for 19-norethindrone of 0.0160 % and a log(RBA) of -1.8.
Finally, in the third valid independent run the mean specific binding for the test substance was >95 % at every concentration tested with the exception of 10-3 M where the binding was 72.7 %, classifying the test substance as “equivocal” for this run. The mean specific binding for the test substance was >94 % at every concentration tested, classifying the test substance as “non-interacting” for this run. The mean specific binding for the test substance was >97 % at every concentration tested, classifying the test substance as “non-interacting” for this run. The weak positive control 19-norethindrone had a LogIC50 of –5.4 M, and the LogIC50 of 17β- estradiol was -9.2 M, resulting in an RBA for 19-norethindrone of 0.0076 % and a log(RBA) of - 2.1.
The mean relative binding affinity, or RBA (calculated by dividing the LogIC50 of the control/test substance by the LogIC50 of the positive control 17β-estradiol) was 0.0131 % for 19-norethindrone (or a log(RBA) of -1.9).
The test substance was classified as “equivocal” in the first and third valid independent runs, and as “noninteracting” in the second valid independent run and thus has a final classification of “equivocal.” A result of “equivocal” should not be taken as a positive or a potential positive result, rather, it means the result is ambiguous, or cannot be properly identified due to limitations of the assay. - Endpoint:
- specific investigations: other studies
- Remarks:
- Induction of CYP1A1 and CYP1B1
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- From: April 27, 2015 To: December 03, 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The aim of this study was to investigate a possible mode of action for uterine tumour formation by assessing the levels of hepatic and uterine CYP1A1 and CYP1B1 following 14 days of test substance administration at a concentration of 3000 ppm in RM1 powder diet to 8-9 weeks old female Fischer 344 rats.
Liver was processed for isolation of microsomes and for analysis of CYP1Al and CYP1B1 enzymatic activity. Liver and uteri were processed for Taqman@ analysis of CYP1Al and CYP1B1 mRNA expression. - GLP compliance:
- no
- Type of method:
- in vivo
- Specific details on test material used for the study:
- Lot number: 080722
Expiration date of the lot/batch: November 01, 2015 - Species:
- rat
- Strain:
- Fischer 344
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Harlan UK Limited, Shaw's Farm, Blackthom, Bicester, Oxon, England, OX25 1 TP
- Age at study initiation: 9 -10 weeks
- Weight at study initiation: 146.71 - 147.18 g
- Housing: 5 rats/cage
- Diet: RM1 powdered diet (Special Diet Services Ltd., Stepfield, Witham, Essex, UK), homogeneous, concentration: 99.1% of target concentration, ad libitum
- Water: local supply, ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature: 19 -23 °C
- Humidity: 40 -70 %
- Air changes: 14 -15 air changes/hour
- Photoperiod: 12 hours light/12 hours darkness
IN-LIFE DATES: From: April 27, 2015 To: December 21, 2015 - Route of administration:
- oral: feed
- Details on analytical verification of doses or concentrations:
- Based on analyiical results it was concluded that test substance is stable in diet over a period of 32 days at ambient temperature. Sampling for analysis was performed as fol!ows: Immediately after preparation, triplicate approximately 2 g aliquots were taken from the top, middle and bottom ofthe diet prepared at a nominal 3000 ppm test substance. Samples were retained in glass vials, stored in the dark at room temperature, and analysed for achieved concentration and homogeneity. Rats were fed RM 1 powdered diet ortest diet ad libitum throughout the study.
- Duration of treatment / exposure:
- 14 days
- Dose / conc.:
- 3 000 ppm (nominal)
- No. of animals per sex per dose:
- 5 females per dose
- Control animals:
- yes, plain diet
- Examinations:
- Bodyweights
The bodyweight of each rat was recorded at the start of the study. The animals were weighed at least weekly. All animals were weighed prior to termination.
Clinical observations
Prior to the start of the study, all rats were observed to ensure they were physically normal and exhihited normal activity. Each rat was ohserved at least once daily during the study.
Food consumption
Food consumption was measured weekly. Diet jars were weighed and refilled as necessary. Any evidence of excessive diet spillage was noted.
Intercurrent deaths
No animals required euthanasia during the Study.
Terminal procedures
On the day of termination the rats were weighed, then transferred to the post martern room. Individual bodyweights were recorded. The rats were killed by exposure to a rising concentration of CO2. Food was not withdrawn prior to termination.
Removal of blood
Venous blood was taken post mortem via cardiac puncture and dispensed into lithium/heparin coated tubes. The tubes were mixed on a roll er für 10 min then cooled on ice. Red blood cells were removed by centrifugation (2,000 rpm for 10 min at 8 - 10 °C), then the supernatant (plasma) was transferred to a second tube and stored at approximately -70 °C for possible future use.
Liver Processing
Each liver was weighed. lndividual liver weights were recorded. Two pieces of liver (5 mm3) were removed from the left lobe for TaqMan analysis. These liver pieces were placed in the same cryovial, flash frozen in liquid nitrogen and then stored at approximately -70 °C until required for analysis of CYP1A1 and CYP1B1 gene expression.
Approximately 2 grams of liver were cut into chunks, snap frozen in liquid nitrogen and stored at -70 °C for possible future use.
The remaining liver was weighed and scissor-minced in ice-cold 1.15 % (v.dv) KCl prior to processing. Microsomes were isolated and stored at approximately -70 °C until required for enzyme activity assays and protein deteminations.
Uterus processing
Uteri were removed and weighed. Individual uteri weights were recorded. Four pieces of uterus (approximately 5 mm3) were removed for TaqMan analysis. These uterus pieces were placed
in the same cryovial, flash frozen in liquid nitrogen and then stored at approximately -70 °C until required, for anaiysis of CYP1A1 and CYP1B1 gene expression.
Biochemical Measurements
Protein determination
The protein concentration of the liver microsomes was determined in aqueous solutions using a modification of the method of Lowry et al., (1951) and bovine serum albumin standards.
Cytochrome P450 activity assays
• Microsomal ethoxyresorufin-0-deethylation (EROD) was used as a marker für CYP1A activity and was measured (Burke et al.. 1985).
• Microsomal estradiol hydroxylation was used as a marker for CYP1A/CYP1B activity, and was measured by the formation of 2-hydroxy- and 4-hydroxy-estradiol and determined by UV-HPLC.
Taqman analysis
RNA was extracted from rat liver. cDNA was synthesized from all available RNA samples. TaqMan analysis was performed on all available samples using primers specific for CYP1A1, and CYP1B1. Rat ß-actin was used as the internal standard. Data were analyzed by generation of generation of threshold cycle (CT) and delta CT values for all genes.
STATISTICAL ANALYSIS
Statistical camparisons between test substance-treated rats and their control group were undertaken for all numerical data sets using a 2-tailed Student's t-test. - Details on results:
- DIETARY ANALYSIS
- The diet was homogeneaus and the nominal 3000 ppm test substance diet achieved concentration was 2972.3 ppm (99.1% of target).
CLINICAL SIGNS AND MORTALITY
- No treatment related clinical signs of toxicity were observed throughout the study. No mortality was observed in this study.
FOOD CONSUMPTION AND WATER CONSUMPTION
- The average food consumption was reduced in treated rats (66.3 g/kg bw/d versus 82.6 g/kg bw/d).
- No test-substance influence on water consumption was observed.
BODY WEIGHT, ORGAN WEIGHTS AND ORGAN/BODY WEIGHT RATIOS
- Body weights and body weight changes: no significant differences between treated and control groups.
- Liver weights: not significantly increased in treated rats; liver/body weight ratio: significantly higher in treated rats (1.1-fold increase, p<0.05).
- Uteri weights and uterus/body weight ratio of treated animals were significantly decreased (33%).
BIOCHEMICAL RESULTS
- Hepatic microsomal ethoxyresorufin-O-deethylation (EROD) activity was significantly increased in treated rats (1.6-fold).
- Estradiol-2 -hydroxylation was significantly increased int reated rats (1.7-fold).
- No formation of 4 -hydroxyestradiol was observed.
- Liver: CYP1A1 mRNA expression was significanlty increased (3.9-fold) but not CYP1B1 mRNA expression.
- Uterus: CYP1A1 mRNA expression was significantly increased (57-fold) but not CYP1B1 mRNA expression.
Referenceopen allclose all
Agonist effect
the results are expressed as a percent of the control response to (-)Quinpirole (decrease in twitch contraction amplitude (mean values; n=2)
Compound |
Test concentration [M] |
% of control (-)quinpirole response |
||
1st |
2nd |
Mean |
||
Control |
- |
0 |
0 |
0 |
Solvent (DMSO) |
- |
0 |
3 |
2 |
Test substance |
1.0E-07 |
15 |
18 |
17 |
Test substance |
1.0E-06 |
41 |
53 |
47 |
Test substance |
1.0E-05 |
81 |
80 |
81 |
+(-)Sulpiride |
3.0E-06 |
90 |
82 |
86 |
Control |
|
0 |
0 |
0 |
(-)Sulpiride |
3.0E-06 |
0 |
-3 |
-2 |
Test substance + (-)Sulpiride (3.0E-06) |
1.0E-07 |
7 |
0 |
4 |
Test substance + (-)Sulpiride (3.0E-06) |
1.0E-06 |
25 |
14 |
20 |
Test substance + (-)Sulpiride (3.0E-06) |
1.0E-05 |
55 |
55 |
55 |
BINDING ASSAYS
Compound |
Test concentration [M] |
% Inhibition of control specific binding |
||
1st |
2nd |
Mean |
||
Test substance |
1.0E-05 |
7.8 |
3.3 |
5.5 |
Reference compound
BTCP: IC50: 1.3E-08 M; Ki: 6.7E-09 M; nH: 1.3
CELLULAR AND NUCLEAR RECEPTOR FUNCTIONAL ASSAYS
Test substance |
Test concentration [M] |
% of control agonist response |
||
1st |
2nd |
Mean |
||
Agonist effect |
1.0E-05 |
-0.4 |
2.5 |
1.1 |
Antagonist effect |
1.0E-05 |
-10.9 |
-25.7 |
-18.3 |
Reference compound
Agonist effect: Dopamine: EC50: 4.4E-08 M; nH: n.a.
Antagonist effect: SCH 23390: IC50: 4.0E-09 M; Ka: 6.4E-10 M; nH: n.a.
ENZYME AND UPTAKE ASSAYS
Compound |
Test concentration [M] |
% inhibition of control values |
||
1st |
2nd |
Mean |
||
Test substance |
1.0E-05 |
8.7 |
6.2 |
7.5 |
Reference compound
Dopamine uptake: GBR 12909: IC50: 2.2E-09M; nH: n.a.
TISSUE BIOASSAY
Agonist effect |
Test concentration [M] |
% of control SKF 82958 response |
||
1st |
2nd |
Mean |
||
Test substance |
1.0E-07 |
0 |
0 |
0 |
Test substance |
1.0E-06 |
0 |
0 |
0 |
Test substance |
1.0E-05 |
17 |
23 |
20 |
+ R(+)SCH 23390 |
1.0E-05 |
-18 |
-18 |
-18 |
SKF 82958 |
1.0E-07 |
13 |
14 |
14 |
SKF 82958 |
3.0E-07 |
69 |
64 |
67 |
SKF 82958 |
1.0E-06 |
106 |
98 |
102 |
Antagonist effect |
||||
Test substance |
1.0E-07 |
100 |
100 |
100 |
Test substance |
1.0E-06 |
102 |
103 |
103 |
Test substance |
1.0E-05 |
104 |
106 |
105 |
+ R(+)SCH 23390 |
1.0E-06 |
84 |
88 |
86 |
+ R(+)SCH 23390 |
3.0E-06 |
50 |
45 |
48 |
+ R(+)SCH 23390 |
1.0E-05 |
-4 |
-6 |
-5 |
The results are expressed as a percent of the control response to SKF 82958 (relaxation) (mean values; n=2)
Agonist effect |
Test concentration [M] |
% of control (-)Quinpirole response |
||
1st |
2nd |
Mean |
||
Test substance |
1.0E-07 |
12 |
21 |
17 |
Test substance |
1.0E-06 |
43 |
51 |
47 |
Test substance |
1.0E-05 |
81 |
80 |
81 |
+ (-)Sulpiride |
3.0E-06 |
73 |
74 |
74 |
(-)Quinpirole |
3.0E-08 |
12 |
24 |
18 |
(-)Quinpirole |
1.0E-07 |
58 |
69 |
64 |
(-)Quinpirole |
3.0E-07 |
101 |
99 |
100 |
Antagonist effect |
||||
Test substance |
1.0E-07 |
100 |
100 |
100 |
Test substance |
1.0E-06 |
98 |
95 |
97 |
Test substance |
1.0E-05 |
95 |
87 |
91 |
(-)Sulpiride |
3.0E-08 |
85 |
85 |
85 |
(-)Sulpiride |
3.0E-07 |
42 |
51 |
47 |
(-)Sulpiride |
3.0E-06 |
1 |
-1 |
0 |
The results are expressed as a percent of the control response to (-)Quinpirole (decrease in twitch contraction amplitude) (mean values; n=2)
Description of key information
Additional information
Multiple mechanistic studies were conducted to elucidate the mode of action for formation of uterine adenocarcinomas in F344 rats at high doses of Afidopyropen. These studies included studies that support the purported mode of action of the test substance acting as an effective dopamine agonist that ultimately results in an increased incidence of uterine adenocarcinomas (studies with dopamine receptors, studies measuring prolactin concentrations).
Dopamine related mechanistic studies
WoE: D2 tissue binding assay 2015/1204930
- Field stimulated rabbit ear artery; D2; agonist effect; standard protocol: test substance showed D2 agonist-like effects that were not reversed by (-)Sulpiride
- Field stimulated rabbit ear artery; D2; agonist effect, modified protocol; pre-incubation with (-)Sulpiride: concentration-dependent decrease in the twitch contraction amplitude; confirmatory of D2 agonist effect
WoE: Binding assay 2015/1204929
Radioligand binding:
- dopamine receptor (D1h) agonist and antagonist effect: no activity
- dopamine transporter (h): no activity
Dopamine uptake
- Functional assay using synaptosomes from rat striatum: no activity
Tissue bioassay
- Rabbit splenic artery; D1 agonist and antagonist effect: no activity
D2 tissue bioassay
- Field stimulated rabbit ear artery; D2; agonist effect: test substance showed D2 agonist-like effects that wer enot reversed by (-)Sulpiride
- Field stimulated rabbit ear artery; D2; antagonist effect: no activity
WoE: Fuctional assay 2015/1117539
Radioligand binding
- Dopamine receptor (D2h) agonist and antagonist effect: no activity
There were also studies for alternative modes of action that did not ultimately prove to be relevant for the test substance (such as estrogen receptor agonism, enzyme induction, estrogen transcriptional activation).
WoE: Estrogen receptor transcriptional activation 2015/1245767
The test substance is not an agonist of human estrogen receptor alpha (hERα) in the HeLa-9903 model system.
WoE: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol: 2015/1245768
The test substance was classified as 'equivocal', i.e. the result is ambiguous, or cannot be properly identified due to limitations of the assay.
WoE: Induction of CYP1A1 and CYP1B1 2015/1183794
This study investigated a possible mode of action for uterine tumour formation by assessing the levels of hepatic and uterine CYP1Al and CYP1B1 following 14 days test substance administration at a concentration of 3000 ppm in RM1 powder diet to 8-9 weeks old female Fischer 344 rats. Treatment of female F344 rats with the test substance via the diet at 3000 ppm resulted in small increases in liver weight and liver/bodyweight ratio. These changes were accompanied by small increases in CYP1A1 mRNA expression, EROD activity and the 2-hydroxylation of estradiol. No effects were observed on the 4-hydroxylation of estradiol or CYP1B1 mRNA expression. In the uterus, the test substance decreased uterine weight, while a moderate induction of CYP1A1 mRNA was observed. There were no increases in CYP1B1 mRNA expression.
WoE: Effect on prolactin levels in 28d rat oral study
The objective of the study was to determine treatment-related effects on prolactin concentrations after 4 weeks oral administration of the test substance via diet in comparison to the positive control Bromocriptine mesylate by gavage. The prolactin concentration in plasma on the circadian peak in the afternoon as well as after drug-induced (Metoclopramide) prolactin release from endogenous storages was determined. The analyses of prolactin concentration was performed considering the estrous cycle status of each animal.
The intent of this study and the doses used was to duplicate the dose levels used in the high dose rat cancer study (2014/1215781) and observe the effects on prolactin concentrations.
The test substance was administered orally via the diet to groups of 20 female Fischer F344 rats at concentrations of 0 (test group 0), 300 (test group 1), 1000 (test group 2) and 3000 ppm (test group 3) over a period of 4 weeks. Bromocriptine mesylate was administered orally by gavage to a group of 20 female Fischer F344 rats at a dose of 10 mg/kg bw/d (test group 4) over a period of 4 weeks. Two strategies were used to assess prolactin concentration. First, plasma samples were taken from all animals before (study day -7) and after a specific duration of exposure (in the afternoon of study day 24 and in the morning of study day 28). Second, animals only in the selected estrus cycle stage proestrous (or estrous) representing the stages with the highest plasma concentration of prolactin) were sampled and analyzed.
Clinical observations, food consumption and body weight were determined regularly during acclimatization period and at least weekly during administration period. Estrous cycle was evaluated on days -7 and -8 and from day 0 onwards. Prolactin concentration analysis was conducted on study day -7, 24 and 28 from all animals as well as from animals in the estrous cycle stages proestrus (and estrus) during parts of the administration period. After the administration period, all rats were sacrificed and assessed by gross pathology, followed by histopathological examinations of seven selected organs as well as all gross lesions.
At the 3000 ppm treatment, clinical examinations showed a decreased number of estrous cycles (0.15 in comparison to 3.7 in control) and a decreased number of animals with estrous cycle (3 out of 20). Clinical pathology showed decreased prolactin values in proestrus between study days 0 and 4 as well as between days 15 and 22. Decreased prolactin values were also observed after stimulation with metoclopramide compared to controls in rats of metestrus. Various pathology findings were present including decreased absolute and relative weight of ovaries, decreased absolute and relative uterus weight, decreased absolute and relative pituitary gland weight, decreased absolute and relative adrenal gland weight, diffuse atrophy of ovaries, diffuse atrophy of the uterus, diffuse atrophy of the cervix and diffuse atrophy of the vagina.
At the 1000 ppm treatment, clinical examinations showed no treatment-related adverse effects. Clinical Pathology showed decreased prolactin values in proestrus between study days 0 and 4 as well as between days 15 and 22. Decreased prolactin values were also observed after stimulation with metoclopramide compared to controls in rats of metestrus. There were no treatment-related, adverse effects on pathology.
At the 300 ppm Afidopyropen treatment there were no treatment-related adverse effects in any parameter.
In the treatment with Bromocriptine mesylate – positive control at 10 mg/kg bw/d, clinical examinations showed a slight body weight loss from day 0 to 7 as well as significantly increased number of estrous cycles and significantly decreased length of estrous cycles. Clinical pathology showed decreased prolactin values in proestrus between study days 0 and 3 as well as between days 16 and 22 and in estrus between study days 18 and 22. Decreased prolactin concentration was observed in estrus and metestrus in the afternoon of study day 24. Increased prolactin concentration in estrus and metestrus in the morning of study day 28 before metoclopramide stimulation. Decreased prolactin concentration values were observed after stimulation with metoclopramide compared to controls in rats of estrus and metestrus. Pathology showed an increased absolute (+49%) and relative (+47%) weights of ovaries as well as decreased absolute (-10%) and relative (-11%) weights of the pituitary gland.
Under the conditions of this study, with the administration of the test substance orally via the diet and Bromocriptine mesylate via gavage to female Fischer F344 rats for 4 weeks the NOEL (no observed effect level) was 100 ppm (test substance intake 18.2 mg/kg bw/d) based on prolactin plasma level alterations.
Both the test substance and Bromocriptine mesylate, caused a decrease of prolactin plasma levels with or without metoclopramide induced release.
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