Tricarbonyl(methylcyclopentadienyl)manganese

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-08-01 to 2015-01-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented, scientifically sound study, conducted simliar to OECD 417 under GLP principles and following appropriate design and guideline requirements for in vitro metabolism studies.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2015

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Principles of method if other than guideline:

The objective of the study was to investigate and compare the metabolic stability and metabolite profiles of mmt in rat, monkey, and human liver microsomes in vitro. Although the study report does not specifically reference the guideline, the methodology, design, and documentation follow the key aspects required by the guideline for metabolism studies using an in vitro model.

GLP compliance:

yes

Remarks:

Study not required to be conducted under GLP; however, study was conducted following applicable BRI internal standard SOPs established to meet GLP regulations. In addition, the study data and report underwent QC review by the BRI Study Director

Test material

Test material information

Constituent 1

Reference substance name:

Tricarbonyl(methylcyclopentadienyl)manganese

EC Number:

235-166-5

EC Name:

Tricarbonyl(methylcyclopentadienyl)manganese

Cas Number:

12108-13-3

Molecular formula:

C9H7MnO3

IUPAC Name:

tricarbonyl(methyl-η5-cyclopentadienyl)manganese

Test material form:

other: Liquid stored at room temperature

Details on test material:

- Name of test material (as cited in study report): HiTEC 3000 (methylcyclopentadienyl manganese tricarbonyl; mmt)
- Molecular weight (if other than submission substance): 218.10
- Physical state: Light to dark clear amber liquid
- Analytical purity: 90%
- Impurities (identity and concentrations): None identified
- Composition of test material, percentage of components: 24.4% Manganese (Mn) content
- Purity test date: Certificate of Analysis 2011-04-30
- Lot/batch No.: 0411 HO 009
- Expiration date of the lot/batch: 2021-04-30
- Storage condition of test material: Room temperature protected from light
-Stability under storage conditions: Stable
- Other: All procedures of handling mmt were performed under incandescent light

Radiolabelling:

no

Test animals

Species:

other: Human, rat and monkey liver microsomes

Strain:

other: Human: CYP M-class donors; Rat: Sprague-Dawley; Monkey: Cynomolgus

Sex:

male/female

Details on test animals or test system and environmental conditions:

Human liver microsomes pooled from at least 10 mixed-gender donors, rat liver microsomes pooled from at least 10 mixed-gender Sprague-Dawley rat donors and cynomolgus monkey liver microsomes pooled from at least 10 mixed-gender donors were used.

-In Vitro CYP M-class 50-donor mixed gender pooled human liver microsomes: BRI ID = STM-2039; Supplier = Celsis IVT; Supplier Lot = LPS
-Pooled female cynomolgus monkey liver microsomes: BRI ID = STM-2176; Supplier = XenoTech, LLC; Supplier Lot = 1110090
-Pooled male cynomolgus monkey liver microsomes: BRI ID = STM-2177; Supplier = XenoTech, LLC; Supplier Lot = 1110335
-Pooled female rat liver microsomes: BRI ID = STM-2136; Supplier = XenoTech, LLC; Supplier Lot = 0810451
-Pooled female rat liver microsomes: BRI ID = STM-2137; Supplier = XenoTech, LLC; Supplier Lot = 1210376

Liver microsomes from each of the three species were thawed at 37°C immediately before use and placed on wet ice until use within an hour. Protein concentrations were adjusted to 20 mg/mL with 250 mM sucrose before being spiked into the incubation mixture.

Administration / exposure

Route of administration:

other: incubation

Vehicle:

ethanol

Remarks:

anhydrous

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Preparation of the test article dose solutions was conducted under incandescent light. The test article, mmt, was diluted with anhydrous ethyl alcohol to provide dose solutions at 1005 mM and 201 mM, which corresponds to 201x target incubation concentrations of 5 mM and 1 mM, respectively.

The positive control stock solution was prepared at 15 mM testosterone in acetonitrile, corresponding to 200x target incubation concentration of 75 μM.

VEHICLE-Test Article (mmt in ethanol)
- Justification for use and choice of vehicle: No information
- Concentration in vehicle: 1 mM and 5 mM
- Lot/batch no.: 11092
- Purity: No information

VEHICLE-Positive Control (testosterone in acetonitrile)
- Justification for use and choice of vehicle: No information
- Concentration in vehicle: 75 uM
- Lot/batch no.: 50528
- Purity: HPLC Grade

Duration and frequency of treatment / exposure:

The reaction mixtures were incubated on an orbital shaker set at 100 rpm for 0, 0.5, 1, 2, 3 and 5 hours at 37°C.

Doses / concentrations

Remarks:

Doses / Concentrations:
mmt solutions in ethanol: 1 mM and 5 mM

Deactivated microsome controls were prepared by boiling the microsomal mixture for 5 min, cooled down to room temperature before adding the test article dose solution.

The solvent control was prepared in the same manner as for active microsome incubation but using 250 mM sucrose in place of microsomes.

Liver microsome incubation for mmt, the positive controls and the vehicle control (n = 2) were carried out in amber microcentrifuge vials at a final microsomal protein concentration of 5 mg/mL in a reaction buffer consisting of 62.2 mM potassium phosphate buffer (pH 7.4), 6.0 mM NADP+, 15.3 mM glucose 6-phosphate, 15.3 mM magnesium chloride and 2.8 units/mL of glucose 6-phosphate dehydrogenase.

Time zero samples were prepared by mixing with equal volume of ice-cold methanol before adding the test article dose solution.

No. of animals per sex per dose / concentration:

Human, monkey, and rat liver microsomes were incubated independently with mmt at 1 mM and 5 mM for 0, 0.5, 1, 2, 3 and 5 hours in duplicate.

Control animals:

other: See Any Other Information on Materials and Methods for details. Both concurrent, vehicle and concurrent, no treatment were used.

Positive control reference chemical:

Positive controls were liver microsomes treated with a P450 isoform specific substrate. Testosterone was evaluated as a positive control for CYP3A metabolic activity based on the formation of 6-beta-hydroxytestosterone monitored by the LC/MS/MS assay.

Details on study design:

At each of the time points, samples were removed from incubation and placed in a freezer set and maintained at nominal -80°C for at least 10 min before addition of an equal volume of ice-cold methanol under incandescent light to stop the reaction.

All samples were then vortex mixed and stored at nominal -80°C until LC/UV quantitation of parent mmt or LC/MS and LC/MS/MS analysis of mmt metabolites and degradation products was performed.

At time of analysis, incubated samples were equilibrated to room temperature, vortex mixed, then centrifuged at room temperature at 12,000 rpm for 5 minutes. Under incandescent light, 100 microliters supernatant was transferred to insert in amber LC vials, then crimp capped for LC/UV/MS, LC/MS or LC/MS/MS analysis.

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: liver microsomes
- Time and frequency of sampling: 0, 0.5, 1, 2, 3 and 5 hours
- Method type(s) for identification:
1) A reverse phase liquid chromatography coupled with ultraviolet spectrometer and tandem mass spectrometer (LC/UV/MS), operated in UV scan and ESI negative ionization scan mode, was employed for quantitation of parent (mmt) and for estimation of two major metabolites observed in
a pilot study as well as other potential metabolite(s) and degradation product(s) in rat, monkey and human liver microsome samples.
2) LC/MS electrospray scan was performed separately in negative and positive ion mode using a relatively long and slow solvent gradient, both along
with UV scanning, on samples collected from the last time point (T = 5 hr) against time zero (T = 0 hr) samples for metabolite and degradation
product profiling.
3) Under the same chromatographic conditions, LC/MS/MS analysis was performed on the potential metabolites observed.

Statistics:

Statistical analysis on the assay batch data was performed using Microsoft Excel 2007 and included the following calculations: % Bias, Mean % Bias, % CV, % Difference, Linear Calibration and Outlier t-test.

Quantification of test article metabolites will be performed using a Waters Model Quattro-Micro LC/MS/MS system controlled with MassLynx software (version 4.0).

Results and discussion

Preliminary studies:

In the pilot study (BRI Study AFT-2013-004), two major metabolites were identified, the carboxylcyclopentadienyl manganese tricarbonyl (mmt-acid) and the hydroxymethylcyclopentadienyl manganese tricarbonyl (mmt-alcohol). These two metabolites were further evaluated in this in vitro study using liver microsomes.

Main ADME results

Type:

metabolism

Results:

See Details on Metabolites and Any Other Information on Results below.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Not applicable

Details on distribution in tissues:

Not applicable

Details on excretion:

Not applicable

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Both of the two known metabolites, mmt-alcohol and mmt-acid as molecular ions of [M-H]- at m/z 233.1 and m/z 247.2 respectively in LC/MS, were observed in liver microsomal samples from all three species tested.

At a concentration of mmt of 1 mM, the formation of mmt-alcohol was in the order of monkey ≥human > rat while the formation of mmt-acid was in the order of rat = monkey > human.

At a concentration of mmt of 5 mM, the formation of mmt-alcohol stayed with monkey ≥ human > rat while the formation of mmt-acid was in the order of rat > monkey > human.

Two peaks of different retention times (RRT = 0.90 and 0.93) were observed during metabolite profiling using a relatively slow gradient to have the same m/z 233.1 corresponding to molecular ion [M-H]- of mmt-alcohol. These two peaks displayed distinct fragmentation patterns in LC/MS/MS analysis, suggesting they were isomers of two different identities. They could be from alkyl hydroxylation or cyclic aliphatic hydroxylation and their actual chemical structure remains to be identified.

At the retention time of mmt-acid, additional ions/peaks, m/z 203.1 and m/z 221.1, were observed in negative mode in liver microsomal samples from all three species, and both ions displayed similar characteristic fragmentation pattern as mmt-acid. The ion of m/z 203 but not 221.1 was also observed as a fragment of mmt-acid. The abundance m/z 203.1 was almost in the same pattern as that of mmt-acid in all three species, suggesting that it could be from in source fragmentation of mmt-acid. In contrast, the abundance pattern m/z 221.1 was different from mmt-acid and could be of different identity.

In addition, several other putative / potential metabolites and degradation products of mmt were observed with ESI LC/MS analysis. Under negative ESI LC/MS, ions/ peaks of m/z 154.8 and m/z 420.5 were observed in active but none or negligible in deactivated liver microsomes of all three species, m/z 342.3 and m/z 198.2 in active human liver microsomes, and m/z 263.5 in monkey liver microsomes. In contrast, the abundance of m/z 156.2 was higher in deactivated than in active liver microsomes of all three species, suggesting its formation via degradation.

Under positive ESI LC/MS, ions/peaks of m/z 327.6, 295.5 and 281.4 were observed in active but none or negligible in deactivated liver microsomes of all three species, m/z 321.5 in active human and monkey liver microsomes only. The abundance of pseudo-molecular ions of m/z 182.0 and 195.3 were higher in deactivated than in active liver microsomes of all three species. An ion/peak of m/z 331.5 was also detected at higher abundance in deactivated liver microsomes of all three species, much lower in active rat liver microsomes and none or negligible in active human and monkey liver microsomes.

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

Not applicable

Any other information on results incl. tables

Testosterone was used as a positive control of the metabolic reaction with significant formation of corresponding metabolite 6β-hydroxytestosterone, demonstrating validity of the in vitro assay conditions.

In active liver microsomes at 1 mM mmt, the formation of mmt-alcohol was in the order of monkey ≥human > rat while the formation of mmt-acid was in the order of rat = monkey > human. At mmt of 5 mM, the formation of mmt-alcohol stayed with monkey ≥ human > rat while the formation of mmt-acid was in the order of rat > monkey > human.   Species difference and similarity was observed in metabolite formation. The formation pattern of mmt-alcohol was very similar between monkey and human while the formation pattern of mmt-acid was similar between monkey and rat but different from human.

For all three species investigated, LC/UV assay data indicated that mmt disappearance occurred in all test systems investigated, including active and deactivated liver microsomes as well as the solvent control during the 5-hour incubation at both the low and high mmt concentrations. At both concentrations, mmt disappearance followed first order kinetics in active liver microsomes from all three species. At the low mmt concentration, rates of mmt disappearance were higher than those at high concentrations in all corresponding test systems tested.

At 1 mM, the rates of mmt disappearance in the active liver microsomes were comparable to that in the solvent control but much higher than those in the deactivated liver microsomes. At 1 mM, the remaining mmt at 5 hours was 10.6%, 16.4% and 24.2% in active liver microsomes from human, monkey and rat respectively, comparable to the 20.2% observed in the solvent control, but much lower than 41.3%, 52.7% and 46.7% observed in their corresponding deactivated liver microsomes.The results indicate that mmt degradation occurred during the incubation, and the higher rate of mmt disappearance in solvent control than in deactivated microsomes could be due to the presence of protein that protected mmt from degradation. In active liver microsomes, the mmt disappearance is likely the result of enzyme-mediated metabolism in addition to degradation.

At 5 mM, comparable levels of mmt remaining in the active and deactivated liver microsomes as well as in the solvent control were observed (ranged from 62.3% to 76.6%), suggesting saturation of the metabolism process.

Apparent intrinsic clearance of mmt at 1 mM in human, monkey and rat were 1.2, 1.5 and 1.0 μL/mg/mg microsomal protein, respectively. Apparent intrinsic clearance of mmt at 5 mM in human, monkey and rat were 0.29, 0.28 and 0.29 μL/mg/mg microsomal protein, respectively.

Applicant's summary and conclusion

Conclusions:

In this in-vitro metabolism study using rat, monkey and human liver microsomes, the LC/UV assay data indicate that mmt disappearance occurred in all test systems investigated, including active and deactivated liver microsomes as well as the solvent control during the 5-hour incubation at both the 1 uM and 5uM mmt concentrations. The available mmt after 5 hours was lowest in active human liver microsomes, followed by the activated monkey and rat microsomes The deactivated microsomes showed a higher available level of mmt after 5 hours, however even in this series, the deactivated human microsomes showed the lowest level of mmt amongst human, monkey and rat microsomes. mmt disappearance followed first order kinetics in active liver microsomes from all three species at both concentrations. The two known metabolites, mmt-alcohol and mmt-acid, were both observed in the rat, monkey, and human liver microsomal samples. Species difference and similarity was observed in metabolite formation. The formation pattern of mmt-alcohol was very similar between monkey and human while the formation pattern of mmt-acid was similar between monkey and rat but different from human.

Executive summary:

The in vitro metabolic stability and metabolite profiles of mmt in rat, monkey, and human liver microsomes were independently studied at concentrations of 1 mM and 5 mM over a time-course up to 5 hours. Across all three species, there was significant loss of mmt due to metabolism as well as degradation. LC/MS and LC/MS/MS metabolite profiles indicate the formation of the two major metabolites, mmt-alcohol and acid, in the three species of liver microsomes. At an mmt concentration of 1 mM, the formation of mmt-alcohol was in the order of monkey ≥ human > rat while the formation of mmt-acid was in the order of rat = monkey > human. At a concentration of mmt of 5 mM, the formation of mmt-alcohol stayed with monkey ≥ human > rat while the formation of mmt-acid was in the order of rat > monkey > human. At 5 mM, comparable levels of mmt remaining in the active and deactivated liver microsomes as well as in the solvent control were observed (ranged from 62.3% to 76.6%), suggesting saturation of the metabolism process.

In addition, several other potential metabolites and degradation products were revealed from LC/MS profiles.