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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.
Reason / purpose for cross-reference:
reference to other study
Objective of study:
metabolism
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
Radiolabelling:
no
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.
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.





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).
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.
Type:
metabolism
Results:
See Details on Metabolites and Any Other Information on Results below.
Details on absorption:
Not applicable
Details on distribution in tissues:
Not applicable
Details on excretion:
Not applicable
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) testing results:
Not applicable

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.

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.
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.
Reason / purpose for cross-reference:
reference to other study
Objective of study:
metabolism
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 lung 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, it 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.
Radiolabelling:
no
Species:
other: Human, rat and monkey lung microsomes
Strain:
other: Human: mixed gender, non-smoker donors; Rat: Sprague-Dawley; Monkey: Cynomolgus
Sex:
male/female
Details on test animals or test system and environmental conditions:
Although female rodents appeared to be more sensitive to the toxicological effects of mmt, female lung microsomes from rat and monkey were not commercially available at the time this study was conducted. In this study, human lung microsomes pooled from mixed-gender non-smoker donors, rat lung microsomes pooled from male Sprague-Dawley rats, and lung microsomes pooled from male cynomolgus monkeys were used.

-Pooled human lung microsomes non-smoker, mixed gender: BRI ID = STM-2139; Supplier = XenoTech, LLC; Supplier Lot = 1210054
-Pooled male cynomolgus monkey lung microsomes: BRI ID = STM-2178; Supplier = XenoTech, LLC; Supplier Lot = 1210293
-Pooled male rat lung microsomes: BRI ID = STM-2138; Supplier = XenoTech, LLC; Supplier Lot = 1010153

Lung 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 10 mg/mL with 250 mM sucrose before being spiked into the incubation mixture.
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 and 30 mM phenacetine were prepared independently in acetonitrile, corresponding to 200x target incubation concentration of 75 μM and 150 μM, respectively.

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 Controls (testosterone and phenacetine in acetonitrile)
- Justification for use and choice of vehicle: No information
- Concentration in vehicle: 75 uM and 150 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.





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.

Lung 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 2.5 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 lung 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 detatils; Both concurrent, vehicle and concurrent, no treatment controls were used
Positive control reference chemical:
Phenacetin and testosterone were used independently as positive controls of the metabolic reaction with formation of corresponding metabolite acetaminophen and 6-beta-hydroxytestosterone, respectively.
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 into 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: lung 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 lung 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 was performed using a Waters Model Quattro-Micro LC/MS/MS system controlled with MassLynx software (version 4.0).
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 lung microsomes.
Type:
metabolism
Results:
See Details on Metabolites and Any Other Information on Results below.
Details on absorption:
Not applicable
Details on distribution in tissues:
Not applicable
Details on excretion:
Not applicable
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 rat lung microsomal samples, with minimal formation of m/z 233.1 in human and monkey and no formation of mmt-acid as the expected molecular ion in human or monkey microsomes.

Interestingly, two peaks of different retention times (RRT = 0.90 and 0.93) were observed in the rat lung microsome sample 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 lung microsomal samples from all three species, and both ions displayed similar characteristic fragmentation pattern as mmt-acid.

In addition, several other putative / potential metabolites and degradation products of mmt were observed with ESI LC/MS analysis. Under negative ESI LC/MS, an ion/peak of m/z 420.5 was observed in active monkey and rat lung microsomes but none or negligible in deactivated systems. In contrast, the abundance of pseudo-molecular ion of m/z 156.2 was higher in deactivated than in active lung 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 lung microsomes of all three species, and m/z 321.5 in active rat and monkey lung microsomes only. The abundance of ions/peaks of m/z 182.0 and 195.3 were higher in deactivated than in active lung microsomes of all three species. An ion/peak of m/z 331.5 was also detected at higher abundance in deactivated than that in active lung microsomes of all three species.
Bioaccessibility (or Bioavailability) testing results:
Not applicable

Phenacetin and testosterone were used independently as positive controls of the metabolic reaction with formation of corresponding

metabolite acetaminophen and 6β-hydroxytestosterone, demonstrating validity of the in vitro assay conditions.

For all three species investigated, LC/UV assay data indicated that mmt disappearance occurred in all test systems investigated, including active and deactivated lung 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 lung microsomes from all three species.

At 1 mM, the rates of mmt disappearance were lower than those at high concentrations in all corresponding test systems tested except for rat. The results indicate that mmt degradation occurred during the incubation The higher rate of mmt disappearance in solvent control versus the deactivated microsomes could be due to that the presence of active microsomal proteins which protected mmt from degradation. In active rat lung 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 lung microsomes as well as in the solvent control were observed (ranged from 51.8% to 75.4%), suggesting saturation of the metabolism process.

Apparent intrinsic clearance of mmt at 1 mM in human, monkey and rat were 1.2, 1.0 and 1.7 μL/min/mg microsomal protein, respectively. Apparent intrinsic clearance of mmt at 5 mM in human, monkey and rat were the same at 0.48 μL/h/mg microsomal protein.

Conclusions:
In an in-vitro metabolism study using rat, monkey and human lung microsomes, a LC/UV assay data indicated that mmt disappearance occurred in all test systems investigated, including active and deactivated lung microsomes as well as the solvent control during the 5-hour incubation at both the 1 uM and 5uM mmt concentrations. mmt disappearance followed first order kinetics in active lung microsomes from all three species at both concentrations. The two known metabolites, mmt-alcohol and mmt-acid, were observed in rat lung microsomal samples, with minimal formation of mmt-alcohol in human and monkey and no formation of mmt-acid. Species difference and similarity were observed in metabolite formation; in general, human and monkey microsomal samples had more similarities than rat and human microsomal samples for the major metabolites.
Executive summary:

The in vitro metabolic stability and metabolite profiles of mmt in rat, monkey, and human lung microsomes were independently studied at 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 indicated the formation of the major metabolite mmt-acid in rat lung microsomes only and mmt-alcohol in lung microsomes of all three species (highest in rat, marginal in monkey and human). Species difference and similarity was observed in metabolite formation although, in general, human and monkey microsomal samples had more similarities than rat and human microsomal samples for the major metabolites. In addition, several other potential metabolites and degradation products were revealed from LC/MS profiles.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was well documented with sufficient information on methods and results and followed accepted scientific principles focusing on the metabolism parameters of toxicokinetic studies..
Objective of study:
other: Comparative pneumotoxicity and metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were treated subcutaneously with mmt and CMT (cyclopentadienyl manganese tricarbonyl) and broncheo-alveolar lavage fluid was investigated for signs of pulmonary toxicity. The correlation between pulmonary toxicity and the presence of manganese in the lungs was studied as was the influence of metabolism by means of the application of an inhibitor and the extraction of metabolites with an apolar solvent.
GLP compliance:
not specified
Radiolabelling:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Farms (Wilmington MA)
- Age at study initiation: no data
- Weight at study initiation: 175-200 g
- Fasting period before study: no data
- Housing: Polycarbonate cages, containing wood-chips bedding, fitted with filter tops
- Individual metabolism cages: no
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25 C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs

Route of administration:
subcutaneous
Vehicle:
propylene glycol
Details on exposure:
Piperonyl butoxide (an inhibitor of CYP-dependent mono-oxygenases) was given at a dose of 400 mg/kg in corn oil by intraperitoneal injection 1 hour before the subcutaneous injection with mmt or CMT.
Duration and frequency of treatment / exposure:
Single sc injection.
Remarks:
Doses / Concentrations:
0.5, 1.0 and 2.5 mg Mn/kg as mmt or CMT.
No. of animals per sex per dose / concentration:
4 to 5 male rats.
Control animals:
yes, concurrent vehicle
Positive control reference chemical:
mmt was compared with CMT, which is identical to mmt, except that it lacks the methyl side chain. One of the objectives of the study was to study the possibility that side-chain metabolism may lead to detoxification.
Details on study design:
Pulmonary damage was assessed by measurement of bronchoalveolar lavage parameters and total Mn levels, 24 hr after administration of mmt or CMT through subcutaneous injection (sc). The parameters measured were: LDH (lactate dehydrogenase), albumin, protein, albumin/protein (%) and lung Mn.

Pulmonary, hepatic and blood NPSH (non protein sulfhydryl) and TRM (thiobarbituric acid reactive materials) content were assessed at 1.5, 6 or 24 hr following sc injection of CMT and mmt to examine oxidative stress.

Plasma was investigated for LDH, sorbitol dehydrogenase and blood urea nitrogen.

The effects of the cytochrome P450 monooxygenase inhibitor piperonyl butoxide on organomanganese-induced changes in pulmonary lavage parameters and lung content 24 hr after CMT or mmt administration were also examined.

CMT and mmt were quantitatively extracted from tissue samples with heptane.



Details on dosing and sampling:
What follows is reproduced in slightly adapted form from the original publication:

Animals were killed 24 hr after organomanganese administration unless specified otherwise (NPSH and TRM analyses were in some cases also performed 1.5 and 6 h after treatment). This time point was selected because previous studies have shown it to be the time of peak pulmonary toxicity in rats following sc injection of mmt. After the onset of pentobarbital-induced anesthesia (60 mg/kg, ip), animals were exsanguinated via the abdominal aorta with heparinized syringes. The thoracic cavity was then opened and the trachea was isolated and cannulated with polyethylene tubing. The lungs were lavaged twice with 9.6 ml of 0.9% sodium chloride.

In all but the highest dosage groups, the recovery of lavage fluid exceeded 90% (see attached Table 1). The two lavage samples were pooled. The lungs and liver were then removed and homogenized in ice-cold 1.15% KCI. In the heptane extraction studies, tissue homogenates were prepared in a dark room using photographic lights with red filters to avoid possible photochemical decomposition of CMT or mmt. All lavage fluid, whole blood, and tissue homogenates were placed on ice immediately. Plasma and lavage fluid supernatants were obtained by centrifugation (600g for 10 min) and stored on ice. All enzymatic analyses were completed on the same day the animals were killed. The remaining samples were stored at -20·C until further analyses could be completed.
Statistics:
Reproduced from original publication: "Results are expressed as mean ± SE. Multiple comparisons were made by analysis of variance followed by the Newman-Keuls test. In cases in which the variances were non-uniform, the data were subjected to log transformation prior to statistical analysis. Lavage albumin levels were compared to pulmonary Mn content by Spearman's rank correlation procedure."
Preliminary studies:
The authors refer to the study of McGinley et al. (1987), which is summarized in Section 7.1.1 Basic toxicokinetics.
Details on absorption:
Not applicable as the test compound was applied by subcutanous injection.
Details on distribution in tissues:
See attached Table 1 and Figure 1 for manganese levels in the lungs.
Details on excretion:
Not investigated.
Metabolites identified:
not specified
Details on metabolites:
The study makes clear that in the lungs, both CMT and mmt are converted to metabolites that cannot be extracted anymore with pentane, using a technique that results in quantitative extraction of mmt and CMT. Thus it can be concluded that rather polar metabolites are produced.

What follows is reproduced in adapted form from the original publication.

At high dose,(2.5 Mn/kg bw), CMT produced 56% (5/9) lethality within 24 h. At this dose, mmt was lethal to only 17% (1/6) of the treated animals. Animals receiving the high dose of either mmt or CMT exhibited labored breathing prior to death and frothy fluid was observed in the trachea at necropsy suggesting that lethality was was most likely due to pulmonary edema and/or inflammation. No deaths were observed at the low dose of either manganese compound.

Plasma LDR, SDH, and BUN levels were not increased over control levels in any treatment group, suggesting thte absence of marked hepatic or renal damage.

Both CMT and mmt induced dose-dependent pulmonary damage as assessed by bronchoalveolar lavage parameters (attached Table 1). Lavage LDH levels were increased by only the high dose of either organomanganese compound, whereas significant increases in lavage protein and albumin content were observed in either the low or high doses. Lavage protein and LDH levels in control and mmt-treated (1 mg Mn/kg) rats were similar to previous studies. CMT is the more potent pneumotoxicant as evidenced by higher lavage LDH, albumin and protein content after equimolar administration of CMT compared with mmt. The lavage albumin-to-protein ratios were significantly elevated over control levels in all treatment groups; similar elevations were observed following either CMT or mmt, regardless if the dose. The lung Mn content was significantly elevated in animals treated with CMT or mmt. These elevations were dose dependent and CMT produced a greater pulmonary Mn burden at equivalent doses to mmt.

A second series of experiments were performed to provide information on the potential mechanism(s) of action of CMT and mmt. Specifically, their effects on pulmonary NPSH and TRM content were assessed at 1.5, 6, or 24 hr following sc injection of CMT or mmt to examine the potential role of oxidative stress in CMT- or mmt-induced pulmonary injury. In addition, the effects of the cytochrome P450 monooxygenase inhibitor piperonyl butoxide on organomanganese-induced changes in pulmonary lavage parameters and lung Mn content 24 hr after CMT or doses of 1 mg Mn/kg mm and 0.5 or 1 mg Mn/kg CMT were administered. Both compounds resulted in significantly elevated pulmonary Mn burdens (attached Figure1). CMT administration produced a dose-dependent increases in this parameter. Pulmonary Mn burdens were virtually identical in 0.5 mg Mn/kg CMT- and 1.0 mg Mn/kg mmt-treated animals. The Mn levels observed in this experiment were significantly greater than observed in the initial experiment after similar doses (the reasons are not known).

Piperonyl butoxide (PB) pretreatment had no effect on control lung Mn content, but protected against the 0.5 mg Mn/kg CMT- and 1.0 mg Mn/kg mmt-induced lung Mn accumulation. A similar degree of protection was observed in these groups. PB did not result in statistically significant reductions in Mn burden after 1.0 mg Mn/kg CMT treatment.

The lung homogenates were subjected to heptane extractions to determine if parent organomanganese or metabolites were present. No more than 2% of the pulmonary Mn was extractable regardless of the treatment group, indicating that the retained Mn was not in the form of heptane soluble, parent compound.

Both CMT and mmt produced elevations in lavage albumin content (attached Fig. 2). CMT administration produced dose-dependent increases in this parameter. Identical results were obtained with lavage protein levels. The responses were virtually identical in 0.5 mg Mn/kg CMT- and 1.0 mg Mn/kg mmt-treated animals. The lavage albumin levels observed in this experiment were significantly greater than observed in the initial experiments (Table 1). Piperonyl butoxide pretreatment had no effect on control lavage albumin content, but protected against 0.5 mg Mn/kg CMT and 1.0 mg Mn/kg mmt. PB had no protective effect on the highest CMT concentration.

Table 2 (attached) shows NPSH pulmonary levels 24 hr after treatment with CMT and mmt, with or without PB pretreatment. Pulmonary NPSH levels were elevated 2- to 2.5- fold over control by CMT and mmt. PB pretreatment partially prevented the increase in NPSH after 0.5 mg Mn/kg CMT or 1.0 mg Mn/kg mmt, but had no effect on CMT higher concentration.

In Figure 3 (attached), mean lavage albumin content is plotted versus pulmonary Mn content for all groups of animals treated with 1 mg Mn/kg or less of mmt or CMT. All groups follow the same dose-response relationship regardless of the form in which Mn was administered or PB pretreatment status.

Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
It can be concluded that: 1) CMT is a stronger pulmonary toxicant than mmt; 2) The difference is possibly the result of mmt side-chain metabolism; 3) The pulmonary toxicity of both compounds is quenched by pre-treatment with piperonyl butoxide, an inhibitor of CYP; 4) Pulmonary toxicity correlates with the presence of manganese in the lungs; 5) mmt and CMT are nearly completely metabolized in the lungs to manganese-containing compounds that cannot be extracted with the apolar solvent heptane; 6) No increases of thiobarbituric acid reactive material was observed, which points to the absence of lipid peroxidation; the pulmonary non-protein sulfhydryl levels decreased first and increased later, which made interpretation difficult. 7) Taken together, the results of this study emphasize the importance of the lung as a target organ in mmt toxicity.
Executive summary:

The acute pneumotoxic effects of cyclopentadienyl manganese tricarbonyl (CMT) and methylcyclopentadienyl manganese tricarbonyl (mmt) were compared to delineate the role of the methyl side chain in the toxicity of these organomanganese compounds and to further understand the mechanisms by which these compounds act. Specifically, lung manganese (Mn) burdens and the pneumotoxic response, as measured by bronchoalveolar lavage parameters, were determined in male Sprague-Dawley rats 24 hr after sc administration of 0.5, 1.0 or 2.5 mg Mn/kg as CMT or mmt. The pneumotoxic response to either compound was characterized by large increases in lavage albumin and protein content with smaller increases in lactate dehydrogenase levels. CMT was approximately twice as potent as mmt. This difference in potency may be due to methyl side chain oxidation, a metabolic detoxification pathway unavailable to CMT. Lung Mn content was significantly elevated after treatment with either CMT or mmt. Heptane extraction studies revealed that Mn was accumulated in a nonlipid soluble form, suggesting the accumulation of metabolites rather than heptanes soluble parent mmt or CMT. A strong correlation between pulmonary Mn content and toxicity was observed, suggesting a causal relationship between the accumulation of CMT or mmt metabolites and toxicity. Piperonyl butoxide diminished both the pneumotoxicity and Mn accumulation resulting from CMT or mmt, suggesting both phenomena are due to monooxygenase metabolites. Pulmonary nonprotein sulfhydryl (NPSH) levels were increased twofold 24 hr after administration of either CMT or mmt. Depletion of NPSH was not observed 1.5 or 6 hr after administration. The mechanisms of this response are unclear but may be due to the metabolism of CMT or mmt to instable compounds which release inorganic Mn within pulmonary cells.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not mentioned
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was well documented with sufficient information on methods and results.
Reason / purpose for cross-reference:
reference to same study
Objective of study:
other: Role of metabolism in the toxicity of mmt in rats
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study evaluated the role of mmt biotransformation in mmt acute toxicity. The effect of a CyP inducer, phenobarbital, on the acute toxicity and the biliary excretion of manganese and the labelled material were investigated.
GLP compliance:
no
Radiolabelling:
yes
Remarks:
[3H]mmt
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: not mentioned
- Age at study initiation: not mentioned
- Weight at study initiation: 180-230 g
- Fasting period before study: no
- Housing: not mentioned
- Individual metabolism cages: no
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: not mentioned


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark
Route of administration:
other: Intraperitoneal and intravenous
Vehicle:
other: ip: 30 mg/kg; specific activity, 0.157 mCi/mmol dissolved in corn oil (3 ml/kg). iv: 10 mg/kg; specific activity, 0.27 mCi/mmol dissolved in a volume of Polyethylene glycol (PEG) 200 equal to 0.25 ml/kg
Details on exposure:
ip: 30 mg/kg; specific activity, 0.157 mCi/mmol dissolved in corn oil (3 ml/kg)
iv: 10 mg/kg; specific activity, 0.27 mCi/mmol dissolved in a volume of PEG 200 (0.25 ml/kg)


Duration and frequency of treatment / exposure:
Single administration.
Remarks:
Doses / Concentrations:
Biliary excretion studies:
ip: 30 mg/kg (mmt)
iv: 10 mg/kg (mmt)
Effect of phenobarbital and mmt on bile flow:
iv: 10 mg/kg (mmt)
ip: 60 mg/kg (phenobarbital)
No. of animals per sex per dose / concentration:
Biliary excretion studies:
iv: 6 rats
ip: 3 rats
Effect of phenobarbital and mmt on bile flow: See table 1
Control animals:
yes
Positive control reference chemical:
No.
Details on study design:
- Dose selection rationale: selection of doses was based on an acute toxicity study, where the test substance was applied orally or by intraperitoneal injection.
Details on dosing and sampling:
- Tissues and body fluids sampled : bile
- Time and frequency of sampling:
- ip administration: 0, 1, 2, 3, 4, 5 and 6 hours after dosage (determination of radioactivity and manganese)
- iv: 0, 10, 20, 30, 40 and 50 minutes after dosage (determination of radioactivity).
Statistics:
Student's t test was used for determination of statistical significance. The Fisher's exact probability test was used in th case of the quantal data.
Preliminary studies:
The acute toxicity of mmt was investigated in rats with or without phenobarbital pre-treatment.
Details on absorption:
Not measured.
Details on distribution in tissues:
Not measured.
Details on excretion:
Biliary excretion was measured. See attached figures from the publication.
Test no.:
#1
Toxicokinetic parameters:
other: Vmax (maximum rate of metabolism) for phenobarbital pretreated rats administered 30 mg/kg ip was determined to be 0.84 mg mmt/kg bw/hr (based on biliary tritium excretion).
Test no.:
#2
Toxicokinetic parameters:
other: Vmax (maximum rate of metabolism) for phenobarbital (PB) pretreated rats administered 10 mg/kg by iv was determined to be 5.7 mg mmt/kg bw/hr (biliary tritium excretion); Vmax for rats w/o PB pretreatment was determined to be 3.0 mg mmt/kg bw/hr
Metabolites identified:
no
Details on metabolites:
N.A.

Table 1 - Effect of Phenobarbital and mmt on bile flowa

Treatment

mmtb

(10 mg/kg, iv)

Bile flow

(ml/100 g/hr)

Control

-

0.386 ± 0.005 (5)

 

+

0.398 ± 0.009 (6)

Phenobarbitalc

-

0.490 ± 0.014d (3)

 

+

0.485 ± 0.010e (4)

a Values represent the mean ± SE for (N) rats.

b A (+) indicates treatment with mmt (10 mg/kg, iv)

c Rats received phenobarbital (60 mg/kg, ip) 72, 48 and 24 hrs prior to bile duct cannulation

d Significantly different from the control group which did not receive mmt, p<0.05

e Significantly different from the mmt-treated control group, p<0.05

Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
Phenobarbital pretreatment doubles the Vmax for mmt metabolism by liver microsomes. The oxidative bioactivation by the phenobarbital-induced cytochromes is accompanied by an increased biliary excretion of manganese and radiolabeled material, which likely stems from metabolites.
Executive summary:

In a metabolism study, 30 mg/kg of ([³H]mmt) was administered in a single dose by intraperitoneal injection, to three male Sprague-Dawley rats, pre-treated with phenobarbital. The maximum rate of metabolism (Vmax) was 0.84 mg/kg bw/h.

In a concurrent study, 10 mg/kg [³H]mmt was administered intravenously to six untreated rats (control) and to four rats pre-treated with phenobarbital. The control rats excreted tritium at a rate of 3.0 mg/kg bw/h, while phenobarbital pre-treated rats almost doubled the rate of excretion to 5.7 mg/kg bw/h. Also a study was conducted to assess the effect of phenobarbital and mmt on bile flow. Phenobarbital pre-treated rats rats showed increased bile-flow rates. Phenobarbital induction doubles the Vmax of mmt metabolism by liver microsomes, and appears to have a protective action on lung toxicity, decreasing mortality and shifting the site of tissue damage to the liver.

 

.
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Scientifically valid, well-designed and documented study similar to OECD Guideline 417
Objective of study:
absorption
excretion
metabolism
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
yes
Remarks:
Not all data on animal husbandry was given, also concentration in the vehicle was omitted in materials and methods.
GLP compliance:
not specified
Radiolabelling:
yes
Remarks:
[3H] mmt
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 180-230 g
- Fasting period before study: no data
- Individual metabolism cages: yes
- Diet: ad libitum
- Water: ad libitum
- After the single treatment with mmt the rats were placed individually in metabolism cages with free access to feed and water.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): temperature controlled room (no information on actual temperature)
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs

Route of administration:
other: oral (gavage) and intravenous
Vehicle:
other: gavage: corn oil, 2.5 ml/kg bw; intravenous: vehicle not mentioned
Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): not provided
- Concentration in vehicle: 125 mg in 2.5 mL corn oil gavage.
- Some groups of rats were given intraperitoneal injections of 60 mg phenobarbital per kg bw on three subsequent days prior to iv, followed on the fourth day by the injection with mmt.



Duration and frequency of treatment / exposure:
Single administration.
Remarks:
Doses / Concentrations:
125 mg/kg gavage or 10 mg/kg intravenous
No. of animals per sex per dose / concentration:
4 male rats per assay.
Control animals:
no
Positive control reference chemical:
A positive control was only used for the in vitro metabolism of mmt. Aminopyrine was the compound chosen, given that its biotransformation is mediated exclusively by cytochromes P-450.
Details on study design:
In view of the diversity of methods applied, parts of the 'Materials and Methods' section of the publication are reproduced below in adapted form.

Collection and Isolation of Urinary, Biliary, and Fecal Metabolites

Urine and feces were collected for 48 hr and processed as described below. To prevent chemical, photochemical, or bacterial decomposition of metabolites, the urine was collected in a foil-wrapped container held at -78 C with dry Ice. In all cases, the pH of the collected urine was at least 9-9.5. For neutralization, the urine from an individual rat was stirred magnetically in a large beaker and 85% H3PO4 was added in small portions (to prevent excessive foaming from evolution of CO2) as the pH was brought down to 4.0. The resultant solution was then filtered through a small pad of Celite-545 to remove sediment, and a small volume of water was used to rinse the pad. The combined rinse and filtrate was passed at a flow rate of 1-2 ml/min through a column of XAD-2 resin (2.5 X 15 cm) which had been packed in 0.5% acetic acid. The column was then rinsed with a small volume of 0.5% acetic acid until the effluent just turned clear. This was followed with methanol/water (3:7, v/v), and then 100% methanol. Alternatively, the raw urine was filtered and extracted with ether, acidified to pH 2-3 as described above, and extracted with ethyl acetate.

Although mmt itself is quite sensitive to light in solution, mmt metabolites did not show this sensitivity until they had been separated from most of the other colored materials in the urine. In general, however, all solutions of mmt and its metabolites were protected from light as much as possible by working in subdued light or wrapping containers and glass columns with aluminum foil.

Fecal material was dispersed and extracted by stirring with water (10 ml/g) for several hours in an Erlenmeyer flask, followed by filtration through a Celite pad.

Bile was collected after either ip or iv administration of mmt by using previously described methods.

In Vitro Biotransformation

For preparation of lung microsomes, the lungs were removed and dissected free of tracheal cartilage and blood vessels and rinsed with ice-cold buffer. After weighing, the lungs were chopped very thoroughly with a single-edged razor blade on a piece of plate glass resting on crushed ice. The mince was then homogenized with a Teflon/glass homogenizer (Glenco) in ice-cold 0. 1 M phosphate buffer (pH 7.4) containing 1 mM EDTA, 3 ml/g of lung.The homogenate was first centrifuged at 10,000 g for 30 min and the supernatant fraction was then centrifuged at 105,000 g for 1 hr. Liver microsomes were prepared by the calcium-precipitation method , and were found to retain their activity for up to 2 weeks if stored as a frozen pellet at -80oC.
For incubations, microsomes were resuspended in 0. 1 M phosphate buffer (pH 7.4) containing 1 mM EDTA, to a protein concentration of 3-4 mg/m. Incubations were carried out with 0.5 ml of this suspension as previously described.

Incubations were terminated by addition of 1 ml of ice-cold saturated NaCI solution. Unmetabolized mmt was determined by extraction with hexane containing biphenyl (50 ppm) as an internal standard, followed by gas-chromatographic analysis.

Chromatographic Methods

Analytical HPLC separations were performed on a C18 reverse-phase column (Whatman Partisil-ODS, 4.6 mmx 30 cm) eluted successively with 1% acetonitrile in 0.05 M ammonium acetate buffer, pH 4.0 (1.5 ml/min for 30 min), methanol/water, 3:7 (v/v) (1.5 ml/min for 21 min), and methanol/water, 1:1 (v/v) (1.5 mI/min for 15 min). The column effluent was monitored at 254 nm, and fractions of various sizes were collected and analyzed for 3H and/or Mn. For preparative HPLC separations a larger column (9 mm X 60 cm) was used with a flow rate of 5 ml/min but a similar elution scheme.
Thin-layer chromatographic separations were performed on silica gel plates of either 0.25-mm (analytical) or 2.0-mm (preparative) thickness, eluting with solvent system A (ethyl acetate/hexane (3:7, v/v)) or B (CHCl3 /methanol (4:1, v/v)). Compounds were visualized under UV light (254 nm) or in I2 vapor. Manganese-containing compounds were detected by spraying the plate with 1% hypochlorite solution (commercial bleach) followed by heating to decompose the organometallic compounds and dry the plate; manganese residues appeared as tan-brown deposits (MnO2) but some other constituents also produced brown spots at this stage. For confirmation of the presence of manganese, plates treated as above were sprayed with an excess of 3% H2O (which decomposes MnO2 to Mn2+ ), heated to dryness, sprayed with 1% diphenylcarbazide in 95% ethanol, and then oversprayed with 25% aqueous ammonia; spots containing inorganic manganese developed a bright pink color.
Details on dosing and sampling:
See above: "Details on study design"
Statistics:
No data.
Details on absorption:
Oral aborption is at least equal to the urinary excretion, which was on average 81%.
Details on distribution in tissues:
Not investigated.
Details on excretion:
Gavage: 74 - 89% of the radioactivity was excreted via the urine and 2-4% via the feces during the 48 hours following the treatment.
IV: 11.66% and 23.52% was excreted in bile with and without phenobarbital pre-treatment, respectively.

See 'Remarks on results including tables and figures'.
Metabolites identified:
yes
Details on metabolites:
Two metabolites were identified in the urine: (CO)3MnC5H4CO2H (I) and (CO)3MnC5H4CH2OH (II), representing about 67% and 14% of the urinary radioactivity, respectively. The urinary radioactivity accounted for 81% of the total applied radioactivity on average.

What follows is adapted from the results section of the original publication.

Excretion

During the first 48 hours after administration, the urinary and fecal excretion of mmt metabolites amounted to 81% (range 74-89%, N=4) and 2-4% respectively, of the tritium in the original dose. Because of their relative small amount, the fecal metabolites were not investigated further.

Concentration of metabolites

The metabolites were concentrated by XAD-2 column chromatography. The brown residue thus obtained was dissolved in methanol and filtered. This solution was analyzed by reverse-phase chromatography. See attached Figure 1. Two major peaks contained tritium; these were also the only two peaks to contain manganese (metabolite I and II).

The peaks for metabolites I and II correspond to approximately 67% and 14% of the total urinary tritium, respectively, and in both peaks the 3H/Mn ratio was approximately 1.3-1.4 times greater than that of the mmt administered.

Using various extraction techniques and preparative thin layer chromatography , the two metabolites were isolated. They were identified with the aid of IR and UV spectrometry, nuclear magnetic resonance and mass spectrometry.

Metabolite II was isolated by preparative TLC after extraction from raw urine at pH > 8; it had RF values of 0.23 in solvent system A and 0.57 in solvent B. In chloroform solution its IR spectrum indicated the presence of an OH group (3600 cmı) and extremely intense M(C=O) absorption bands at 2015 and 1950 cm-1. Its UV spectrum in hexane was very similar to that of mmt, with maxima at 241, 243, and 328 nm. The mass spectrum of metabolite II showed a fragmentation pattern like that of mmt but shifted 16 amu higher. The main feature of the spectrum is the stepwise loss of three CO groups from the parent ion.

Metabolite I was isolated from raw urine by extracting with ether to remove metabolite II, acidifying to pH 2, extracting with ethyl acetate, and fractionating the latter by preparative TLC. It had RF values of 0.16 in solvent system A and 0.31 in solvent B, and its partitioning behavior showed it to be an organic acid. In chloroform its IR spectrum showed intense M(C=O) absorption bands at 2020 and 1960 cm-1 and carboxyl group absorption bands at 3500 and 1730 cm-1. Its UV spectrum in hexane showed absorption maxima at 241 (sh), 243, 274 (sh), and 283 (sh) nm. Its mass spectrum showed the pattern of successive loss of three CO groups characteristic of mmt and related metal carbonyl complexes.

Metabolite I reacted with diazomethane in ether/methanol, giving a new organomanganese derivative having RF 0.48 in solvent system A. In chloroform, its IR spectrum showed intense M(C=O) absorptions at 2015 and 1945 cm-1, and an ester carbonyl band at 1720 cmı. In hexane its UV spectrum showed maxima at 205 and 335 nm.

Biliary metabolites after iv administration of mmt (10 mg/kg), 11.66 ±0.72% of the administered tritium (mean ± SD) was collected in the bile within 30 min from six normal rats, whereas in four phenobarbital-pretreated rats 23.52 ± 2.67% of the administered tritium was excreted in the first 30 min. Extractions and TLC measurements showed that metabolites I and II accounted for approximately 49% and 18% respectively, of the total biliary tritium.

In vitro biotransformation The biotransformation of mmt was studied in vitro, along with that of aminopyrine as a standard for comparison, in preparations of liver or lung microsomes supplemented with an NADPH-generating system. Preliminary studies with pooled liver microsomes from groups of 5-6 normal or phenobarbital-treated rats showed that the reaction rates were linear for at least 20 min, except in cases where mmt was completely consumed before this time. Data in Table 3 (attached) show that mmt and aminopyrine, whose biotransformation is mediated exclusively by cytochromes P-450, show parallel responses to changes in incubation conditions. In general, the mmt reactions were more sensitive to a given change in incubation conditions, and the pattern typical of cytochrome P-450 involvement may clearly be seen for both mmt and aminopyrine (i.e., dependence, inhibition by CO and N-decylimidazole, and induction by phenobarbital pretreatment). With liver microsomes from control rats, mmt had an apparent KM of 78 µM and a Vmax of 3.12 nmol per mg of protein per mm. With liver mmcrosomes from phenobarbital-treated rats, the apparent KM remained unchanged but the Vmax doubled (Fig. 4, attached). In incubations with microsomes from phenobarbital-treated rats up to 90% of the mmt, initially present at 0.5 mM, was converted during the 15-min incubation period. mmt metabolism was also linear for up to 20 min with several preparations of lung microsomes from normal and phenobarbital-treated rats. However, phenobarbital pretreatment did not alter the activity of lung microsomes toward mmt as it did for liver microsomes (Table 4). Another difference between liver and lung microsomes is the apparently decreased sensitivity of the latter to changes in incubation conditions (Tables 3 and 4).

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
1) After a single oral gavage treatment of rats with radiolabelled mmt on average 74-98% of the label was excreted via the urine and 2-4% via the feces.
2) Two metabolites were identified in the urine. In both, the methyl side chain of the pentadienyl ring was oxidized, in metabolite I to the carboxylic acid and in metabolite II to the alcohol. These metabolites represented 67% and 14% of the tritium in the urine.
2) The same metabolites were found in the bile excreted after intravenous injection.
3) The metabolism of mmt in the liver and in the lungs depends on CYP; whereas the liver metabolism is strongly stimulated by pretreatment with phenobarbital, the lung metabolism is not.

Executive summary:

What follows is the abstract of the original publication.

The biotransformation and excretion of methylcyclopentadienyl manganese tricarbınyI (mmt) has been studied in vivo in the rat, and In vitro by using rat liver and lung microsomes. Orally administered 3H-mmt is efficiently absorbed, metabolized, and excreted in the urine as two major metabolites, (CO)3MnC5H4CO2H and (CO)3MnC5H4CH2OH, which account for 67% and 14% of the urinary tritium, respectively. These metabolites are also excreted in significant quantities in bile, but undergo reabsorption and excretion by the kidney since only a small fraction of the administered tritium appears in the feces. In vitro mmt was rapidly metabolized by a cytochrome P-450-dependent process inducible in liver but not in lung microsomes. In vivo induction by phenobarbital doubles the rate of biliary excretion of mmt metabolites.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not mentioned
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was well documented with sufficient information on methods and results including distribution and excretion of manganese (Mn) from rats chronically fed diets containing a single dose of mmt.
Reason / purpose for cross-reference:
reference to same study
Objective of study:
distribution
Qualifier:
no guideline followed
Principles of method if other than guideline:
Mice were exposed via their diet to one dose of mmt for a period of 12 months. Feed consumption, bodyweight and spontaneous motor activity were followed during this period. At the end of the exposure period, manganese (Mn) concentrations were determined in various tissues, blood and urine.
GLP compliance:
no
Radiolabelling:
no
Species:
mouse
Strain:
other: ddY
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Sankyo Laboratory Service, Co. Ltd (Japan)
- Age at study initiation: 6-weeks old
- Weight at study initiation: 29.6 ± 2.1 g
- Housing: polycarbonate cages containing a wood-chip bedding, fitted with filters tops
- Individual metabolism cages: no
- Diet: ad. libitum
- Water:ad. libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hr/12hr


Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): standard laboratory mouse chow
- Storage temperature of food: at the temperature of the laboratory (22-25°C)

STABILITY OF TEST MATERIAL: experimental food containers were covered with metal lid to prevent photochemical degradation.
Duration and frequency of treatment / exposure:
12 months, 7 days per week (animals had free access to the food).
Remarks:
Doses / Concentrations:
The mmt exposed group was given a diet containing 0.5 g Mn/kg of standard laboratory mouse chow in the form of mmt.

The control group received standard laboratory mouse chow (130 mg Mn/kg, phytate 1.0%, crude fiber 3.4 %, methionine 0.3% and arginine 1.3%, type F2).
No. of animals per sex per dose / concentration:
6 animals.
Control animals:
yes
Positive control reference chemical:
No.
Details on dosing and sampling:
- Body fluids sampled: urine, blood
- Time and frequency of sampling: at the end of the exposure period of 12 months; Urine for sampling was pooled over 24 h before decapitation and blood was sampled immediately upon decapitation.
- Measurement of Mn content in the following organs: liver, kidney, pancreas, thyroid gland, sublingual gland, prostate gland, lung, spleen, bone, hair, muscle and brain cortex.
Statistics:
Data were statiscally analyzed by the Student's t-test.
Details on absorption:
Mn concentrations in blood was significantly higher in the mmt-treated rats versus the control group.

See attached tables and figures.
Details on distribution in tissues:
There was a significant increase in Mn levels found in the liver, kidney, pancreas, sublingual gland, prostate gland, lung and muscle in the group exposed to mmt compared to the control group. The Mn content was highest in the kidney, followed by the liver, thyroid gland, sublingual gland and prostate gland. The Mn content in the mmt-group was 4.4-1.5 times higher than in the control group.

In the control group, the Mn content was highest in the thyroid gland, followed by the kidney, hair and liver.

See attached tables and figures.
Details on excretion:
As measured by the levels of creatinine in the urine, Mn content was significantly higher in the mmt-treated rats versus the control group.

See attached tables and figures.
0.7g</font>

The mean daily food intake decreased during the first month but then became constant in all groups. Daily food intake per mouse was 3.6 ± 0.9 g (mean ± SD) in the control group and 3.1 ± 0.7 g in the mmt group. There was no significant difference of intake between the groups. At 20 and 80 days after the start of the experiment, the body weight of the mmt group was significantly lower than of the control group. A consistent trend of weight suppression appeared after 9 months. See attached tables and figures.

Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
Chronic oral exposure to mmt increases Mn levels in blood and urine of mice as well as in the liver, kidney, pancreas, sublingual gland, prostate gland, muscles and lung, but no significant increases were noted in the thyroid, spleen, bone and brain. In general, this study demonstrates that chronic exposure to mmt in rats, leads to higher concentrations in blood and urine and higher tissue concentrations than the control group.
Executive summary:

Abstract reproduced from the original publication.

The disposition and toxicity of methylcyclopentadienyl manganese tricarbonyl (mmt), a potential substitute for lead in gasoline, was studied to investigate the different adverse effects in ddY mice after chronic oral administration at 0.5 g Mn/kg in food for 12 months. There was no significant difference in intake between the control mice and the mice exposed to mmt (mmt group), but those given mmt suppressed weight significantly. The manganese content in the organs of the mmt group was 4.4 – 1.5 times significantly higher than that of the control group. In the mmt- group, the manganese content was highest in the kidney, followed by the liver, thyroid gland, sublingual gland and prostate gland. The blood manganese level in the mmt group was about 8 times higher than that in the control group. The urinary excretion of manganese in the mmt group was 5.4% of the daily oral intake. The organometallic form of the manganese involved is apparently absorbed more readily than inorganic forms. The stronger toxicity of mmt to the tissue than that of inorganic manganese is attributed to the significantly higher blood and tissue levels of manganese in the mmt group.  

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was well documented with sufficient information on methods and results including distribution of manganese (Mn) from rats given a single subcutaneous injection of mmt.
Objective of study:
distribution
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were exposed to mmt by means of a single subcutaneous injection at a dose level of 4 mg/kg bw. The presence of inorganic and organic manganese was determined in blood and several tissues at several time points up to 96 hours post injection. Similarly treated rats were investigated for protein and lactate dehydrogenase levels in BAL fluid and urea content in plasma, following the same time schedule above.
GLP compliance:
no
Radiolabelling:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Farms, Wilmington
- Age at study initiation: 50-70 days
- Weight at study initiation: 200 - 300 g
- Fasting period before study: no
- Housing: polycarbonate cages fitted with filter tops
- Individual metabolism cages: no
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12hr/12hr
Route of administration:
subcutaneous
Vehicle:
propylene glycol
Duration and frequency of treatment / exposure:
Single subcutaneous (sc) injection
Remarks:
Doses / Concentrations:
mmt was prepared at a concentration of 3 mg/ml in propylene glycol vehicle and administered via sc injection at a dose of 4 mg/kg bw.
No. of animals per sex per dose / concentration:
No data
Control animals:
yes, sham-exposed
Details on dosing and sampling:
Samples were collected for disposition studies and clinical chemical determinations at 1.5, 3, 6, 12, 24, 48 and 96 hours post-injection.
Statistics:
The following is stated in the publication. "Data are expressed as mean + SEM. Multiple comparisons of the tissue Mn and plasma urea and sorbitol dehydrogenase leveis were made by analysis of variance followed by the Newman-Keuls multiple range test. Because of heteroscedasticity, the lavage data were compared by the Kruskal-Wallis test, followed by the nonparametric Newman-Keuls test. Lavage protein levels were correlated to time after mmt dosing by the non-parametric Spearman rank correlation coefficient. A P value less than 0.05 was required for significance."
Preliminary studies:
Preliminary studies in rats were performed to determine the dose which produces mild lung injury but no lethality.
Details on distribution in tissues:
The following Mn levels were found in control animals in mg/kg: 0.09 ± 0.01 in blood; 1.51 ± 0.22 in lung; 2.49 ± 0.36 in liver; 1.29 ± 0.23 in kidneys; 0.45 ± 0.01 in brain. No hexane extractable Mn was found in the control animals.

In all investigated tissues of the exposed animals, Mn concentration reached peak levels at 3 - 6 h post-injection. The Mn levels were highest in the lung, followed by the kidney, liver and blood. Between 1.5 and 24 h post-injection, the mmt-derived Mn concentration in the lungs was approximately 13-fold greater than in the blood. The liver to blood ratio of mmt-derived Mn was also constant between 1.5 and 24 h and averaged about 4. The kidney to blood ratio also averaged approximately 4 during this time period. These results indicate that mmt or its metabolite(s) were accumulated and retained in these tissues. Brain Mn concentrations were not significantly higher over control levels in mmt-treated animals. Tissue mmt-derived Mn was retained for long periods. Lung. liver, kidney and blood levels were still enhanced at 96 hours post-injection. The results are graphically presented in the attached Figure 1.

In the kidneys, 50% of the mmt-derived Mn was extractable into heptane. In the liver and lung, heptane-extractable Mn levels were less than 0.1 ppm (limit of detection in the heptane extracts).

The Mn contained in control tissues was trichloroacetic-precipitable (TCA). TCA-precipitable Mn levels were not significantly increased over control levels in lung, liver or kidney, 3 hr after mmt administration.

Plasma urea and sorbitol dehydrogenase levels averaged 16 ± 2 mg/dl and 0.7 ± 0.2 units/ml respectively in control animals. These parameters were not significantly elevated over control levels at any time following mmt injection (data not shown in the publication).      

Pulmonary lavage protein concentrations were significantly different among the various groups. Test protein levels were significantly above controls levels at all sacrifice times (see attached table). Because of the variability in the response, significant differences were not detected among the mmt-treated groups. However, lavage protein levels increased from 0 hr (control) to reach a peak level at 24 -48 hr. This trend was statistically significant as evidence by a Spearman rank correlation coefficient of 0.60 between lavage protein and time after mmt dosing (from 0 to 48 hr). In mmt-treated rats, lavage lactate dehydrogenase levels were elevated over control levels but the changes were not statistically significant (no data provided).

Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
Rat tissues sensitive to mmt-induced toxicity were found to accumulate and retain mmt-derived Mn to levels higher than those in the blood, and the tissue most sensitive to mmt (lungs) accumulated the Mn to the greatest extent. The correlation of tissue sensitivity with the ability to accumulate mmt-derived Mn provides evidence that the accumulation is related to the toxicity.
Executive summary:

Abstract from original publication is reproduced below.

The disposition and toxicity of methylcyclopentadienyl manganese tricarbonyl (mmt) was studied in Sprague-Dawley rats after subcutaneous administration at a dose of 4 mg/kg. Blood, lung, liver and kidney Mn levels were increased between 1.5 and 96 hr after mmt injection, with peak organ levels occurring at 3 - 6 hr. At this time the mmt - derived Mn concentration in lung, liver and kidney averaged 13 -, 4- and 4 -fold higher, respectively, than in the blood, indicating the accumulation and retention of mmt (or metabolite) in these tissues. Maximal pulmonary toxicity, as assessed by pulmonary lavage protein levels, occurred 24 -48 hr after injection. Plasma urea and sorbitol dehydrogenase levels were not increased at any time after mmt injection. The maximal pulmonary toxicity occurred after peak Mn accumulation, and the organ-specific toxicity of mmt correlated with its accumulation and retention.

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study contains valuable information on the toxicokinetics of mmt and is well documented. It is published in a peer-reviewed scientific paper.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were treated with oral or intravenous doses of radiolabelled (54Mn) mmt and the retention, excretion and tissue distribution of the radiolabel was investigated. In addition, radiolabelled mmt metabolism was studied in vitro in tissue homogenates.
GLP compliance:
no
Remarks:
.
Radiolabelling:
yes
Species:
rat
Strain:
other: COBS
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Weight at study initiation: 200-225 g
- Fasting period before study: group I, II and III were fasted 24 hrs
Route of administration:
other: oral (gavage) or intravenous (iv) injection
Vehicle:
other: oral: Wesson Oil (brand name); cooking oil; intravenous: ethanol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
no data

VEHICLE
- Justification for use and choice of vehicle (if other than water): ethanol (iv) was used as a diluent because of the insolubility of mmt in water. Wesson oil was also used as a vehicle for gavage studies, due to the lipophilic nature of mmt.
- Concentration in vehicle: not stated
- Amount of vehicle (if gavage):
-Group I, II and III: 0.2 ml Wesson oil (radiolabelled mmt)
- Group IV: 0.1 ml ethanol (radiolabelled MnCl2)
- Group V: 0.1 ml ethanol (mmt)
- Group VI: 0.1 ml in distilled water (radiolabelled MnCl2)
Duration and frequency of treatment / exposure:
Single administration, with a maximum observation period of 80 days
Remarks:
Doses / Concentrations:
See "Any other information on materials and methods".
No. of animals per sex per dose / concentration:
6 animals per group (6 groups in total).
Control animals:
no
Details on study design:
No data.
Details on dosing and sampling:
TOXICOKINETIC STUDY (excretion)
- Tissues and body fluids sampled (delete / add / specify): urine, feces, whole body
- Time and frequency of sampling:
-whole body: 0, 10, 20, 30, 40, 50, 60, 70 and 80 days (2.5 and 0.5 mg mmt)-oral dose
-urine and feces: 0, 4, 6, 8, 10, 12, 14, 16 and 18 days (2.5 mg mmt)-oral dose
-whole body: 0, 10, 20, 30, 40, 50 and 60 days (mmt and MnCl2)- iv
-urine and feces: 0, 5, 10, 15, 20 and 25 days (mmt and MnCl2)- iv

Statistics:
No data.
Preliminary studies:
No preliminary studies mentioned.
Type:
absorption
Results:
No estimate possible, as it cannot be determined with certainty how much of the fecal radioactivity has been absorbed.
Type:
distribution
Results:
No quantitative data presented. Distribution similar to that normally found for Mn, with the exception that high concentrations were observed in lungs and abdominal fat.
Type:
excretion
Results:
Rapid clearance first week; thereafter slow until approximately 1% of dose remained after about 75 days.
Details on absorption:
Absorption after oral exposure (gavage) cannot be estimated from the excretion data, as the radioactivity was excreted via the urine and via the feces. It is not known how much of the fecal radioactivity has originally been absorbed. However, the authors state for the dose of 2.5 mg mmt: "There was an initial rapid excretion in both urine and feces. The urine contained less 54Mn than the feces and the urine/feces ratio varied from about 0.68 to 0.25. During the first 24 hours, the rats excreted 73% of the initial dose, and 36% of the 54Mn excreted was present in the urine." See attached Figure 2, 3 and 4.

Fecal excretion was also found after intravenous injection, which demonstrates that the oral absorption is larger than indicated by the urine. Actually, the higher fecal than urinary excretion after intravenous suggests that a large proportion of the fecal excretion after oral exposure represents absorbed material. This is confirmed by the virtually complete fecal excretion of Mn from MnCl2.
Details on distribution in tissues:
The following is stated with respect to distribution: "The tissue distribution of 54Mn among the organs of the rat after a single oral dose of mmt followed a pattern similar to that reported for the normal distribution of Mn, except for the high concentration of 54Mn found in the lungs and to a lesser extent in abdominal fat. One day after exposure, the highest concentrations of 54Mn were found in the liver, lungs, kidneys, urinary bladder, pancreas, and abdominal fat. Nine days after exposure, the highest concentration of 54Mn were found in the kidneys, liver, pancreas and lungs."
Details on excretion:
See under "Details on absorption". See also the attached Figure 1, which shows the total body retention of radiolabel versus time.
Metabolites identified:
yes
Details on metabolites:
The heptane-extraction experiments indicate that all radioactivity in the feces is ionic mmt and most (>90%) of the radioactivity in the urine is as well.

Metabolism thus results in the mineralization of nearly all of the mmt. The formation of nonextractable radiolabel, presumably ionic manganese, is also demonstrated by the metabolism studies with tissue homogenates.

What follows is reproduced and adapted from the original publication.

Oral Retention, Excretion, Tissue Distribution

Figure 1 shows the whole-body retention of 54Mn following a single intragastric administration of radioactive 54Mn labeled mmt to two different groups of rats (I and III). Two dose levels were used, and in both cases, a rapid clearance of 54Mn occurred from the animals in the first few days after dosing. The rapid clearance resulted from (1) excretion of 54Mn via the urine following absorption, and (2) passage of the radioactivity through the gastrointestinal tract and excretion in the feces. The amount in the feces included both unabsorbed and endogenously secreted 54Mn. It is apparent that the retention curves include an additional component that has a longer biological half-life.

The initial dose level of mmt also affected retention. A higher percentage of the lower dose (0.5 mg mmt) was retained (32% as opposed to 27%), but the total amount retained was greatest for the higher dose.

The excretion of 54Mn in the urine and feces for the high dose concentration (2.5 mg mmt) is shown in Fig. 2. There was an initial rapid excretion of 54Mn in both urine and feces. The urine contained less 54Mn than the feces, and the urine/feces ratio varied from about 0.68 to 0.25. During the first 24 hours, the rats excreted 73% of the initial dose, and 36% of the 54Mn excreted was present in the urine.

 

Intravenous retention, excretion

A comparison of the retention of 54Mn following a single intravenous dose of 0.34 mg Mn given as mmt and as MnCl, is shown in Fig. 3. Retention of 54Mn was similar following the administration of equal loads of Mn in the form of mmt or MnCl. An additional group of rats (Group IV) was given a tracer level (0.2 pg Mn) of 54Mn given as MnCI2, in order to compare 54Mn retention in rats with other reports in the literature. Although the retention of 54Mn was similar for both mmt and MnCI2, the route of excretion was different (Fig. 4). Following the iv administration of 54MnCl2, the 54Mn was excreted via the feces and only a trace was found in the urine. After iv administration of mmt, 54Mn was excreted both in the feces and urine.

 

Presence of Organic Extractable Materials

Following heptane extraction of the feces and urine, no organic extractable materials containing 54Mn were obtainable from the feces of either the mmt or MnCl2 -intravenously dosed rats. In animals dosed with mmt, organic extractable 54Mn was found in urine on Days 1 and 2 after exposure; the total amounted to about 2.3% of the dose. No organic extractable 54Mn was found in the urine from animals dosed with MnCl2.

In vitro biotransformation

Table 1 (attached) shows the results of the in vitro biotransformation of mmt in various tissues of the rat. The liver showed the highest activity for metabolism of mmt with 64.2 ± 10.3% of the dose rendered organic nonextractable (ionic) in 20 min which is equivalent to 8.02 ng/min/mg tissue. The corresponded values for lung, kidney and brain were 26.9% (1.12 ng/min/mg), 2.59% (0.11 ng/min/mg) and 1.64% (0.07 ng/min/mg) respectively, at dose of 0.25 µg mmt/mg tissue. Loss of isotopic manganese occurred in all samples ranging from 14.8% from the liver samples to 55.9% for brain. No significant loss occurred in the blanks.

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
The manganese from mmt is excreted via the feces and the urine. The importance of fecal excretion after intravenous injection shows that the oral absorption of mmt is larger than indicated by urinary excretion after oral exposure. The lack of extraction in heptane from excreta as well as from the reaction mixtures of in vitro metabolism experiments indicates nearly complete metabolization and suggests the formation of inorganic manganese species. Metabolism and elimination is rapid enough as to prevent accumulation. The manganese from mmt differs from the manganese from MnCl2 in that higher radiolabel was found in the lungs and the abdominal fat and that considerable urinary excretion occurs.
Executive summary:

Whole-body retention, excretion, and tissue distribution of 54Mn were studied in rats following oral and intravenous dosing of methylcyclopentadienyl manganese tricarbonyl (mmt). An initial rapid excretion of most of the 54Mn occurred following both routes of exposure. Extraction of the urine and feces after dosing indicated that the mmt was metabolized and that the 54Mn was excreted in the inorganic form. The high levels of 54Mn found in the urine after mmt dosing are not typical of normal Mn excretion. The liver, kidneys, and lungs contained the highest concentrations of 54Mn following administration of mmt. In vitro experiments indicated that mmt was metabolized in the liver, lung, kidney, and to a small extent in the brain. Metabolism of mmt by kidney homogenate supported the hypothesis that biotransformation occurred in the kidney and explains the high levels of urinary excretion of inorganic 54Mn. The whole body retention curves for 54Mn labeled mmt and 54MnCl2 were very similar and are consistent with the hypothesis that mmt is rapidly metabolized. Both curves for 54Mn reflect the kinetics of inorganic Mn.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not mentioned
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was well documented with sufficient information on methods and results.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were exposed to a single dose of methylcyclopentadienyl manganese tricarbonyl (mmt) as well as modulators of biotransformation activity according to the scheme presented in the attached Table 1. Animals were pretreated with biotransformation modulators prior to treatment to mmt to obtain information on the type of enzymatic process(es) that activate mmt as a potential lung toxicant. The effects of biotransformation modulators (cytochrome P450 CYP inhibitors) on toxicity in terms of lung weight changes and biochemical effects in the bronchioalveolar lavage (BAL) fluid was studied.
GLP compliance:
no
Radiolabelling:
no
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: MRC Toxicology Unit Carshalton, Surrey, UK
- Age at study initiation: both species: 8-10 weeks
- Weight at study initiation: rats: 170-200 g
Route of administration:
intraperitoneal
Vehicle:
other: Oil
Duration and frequency of treatment / exposure:
Single administration
Remarks:
Doses / Concentrations:
See attached Table 1.

4 or 6 mg/kg bw in the assays aimed at determining effects on lung weights and enzyme activities in bronchio alveolar lavage (BAL) fluid.
No. of animals per sex per dose / concentration:
Effects on lung weight and BAL biochemistry: 5 female rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Rats were exposed to mmt as well as modulators of biotransformation activity according to the scheme presented in Table 1. The biotransformation modulators were applied to obtain information on the type of enzymatic process(es) that activate the toxicants. Methodological details are provided in the table below.
Details on dosing and sampling:
See table below in section "Any other information on materials and methods incl. tables".

Furthermore it is stated in the publication (adapted): "Lungs, for wet weight determinations, were removed after the animals (from LD50 experiments) were cardiac bled under terminal ether anesthesia.

In ancillary experiments, rats were sacrificed by decapitation and mice by cervical dislocation. Similar lung weights were obtained after both procedures. Lung weights were obtained 3 and 4 days after challenge with pulmonary toxins, they were normalized to the body weight at the time of dosing in order to avoid the effects of toxicity-related losses in body weight.

Rats were lavaged 24 hr after challenge. The lungs were lavaged 3 times and the total volume recovered was approximately 8 ml.
The dealkylation of pentoxyresorufin was measured on the day of microsome preparation. A 50-µl aliquot of lung microsomes (approximately 0.8-1.0 mg of lung microsomal protein) was added to a cuvette containing 10 mM pentoxyresorufin and 250 µM NADPH in a total volume of 2.4 ml of Tris buffer, pH 7.6 at room temperature.

Lavage fluid was stored at -40C until γ-glutamyltranspeptidase and alkaline phosphatase activity were measured colorimetrically, at 540 and 410 nm.
Statistics:
Students' t test
Details on absorption:
Not investigated.
Details on distribution in tissues:
Not investigated.
Details on excretion:
Not investigated.
Metabolites identified:
no

mmt when dosed to rats at or near the LD50, caused weight loss, panting respiration and cyanosis associated with a massive increase in lung weight.

mmt caused a doubling of lung wet weight between 3 to 5 days. Lung dry weight and water content were also increased. Pulmonary damage involved loss of type I pneumocytes, proliferation of type II cells and an influx of macrophages. Fatalities normally occurred at 3 or 5 days, when lung weight was maximal.

mmt increased both γ-glutamyltranspeptidase (GGT) and alkaline phosphatase (ALT) enzyme activity in lavage fluid, the latter much more drastically than the former; GGT activity was not altered anymore after 24 hours.

Pretreatment with OOS-MeP(S), bromophos, DPEA (2,4 -dichloro(6 -phenylphenoxy)ethylamine) and p-xylene as inhibitors of cytochrome P450 CYP isozymes, all gave effective protection against pulmonary toxicity of mmt. They also decreased both overall toxicity and the 3-day increase in lung weight.

The prevention of lung damage by the pretreatment resulted in non-pulmonary effects in some cases, such as cholinergic symptoms in case of OOSMeP(O), signs of neurotoxicity above 40 mg/kg bw in case of mmt and violent seizures above 1.5 mg/kg bw and a lethal neurotoxic dose of 4 mg/kg bw. The threshols for neurotoxicity of mmt was NOT altered by the pre-treatment that resulted in the absence of lung enlargement.

Pentoxyresorufin dealkylation was strongly inhibited in vitro in lung microsomes by the pre-treatment (except beta-naphtoflavone). Pulmonary toxicity appeared to be inversely correlated with deactivation.

Detailed graphical presentations of the results are attached.

Conclusions:
Interpretation of results (migrated information): other: mmt is bioactivated in the lung by pulmonary CYP2B1
Pretreatment with OOS-MeP(S), bromophos, DPEA or p-xylene reduces/prevents the pneumotoxicity of mmt in rats. These treatments also inhibit the pulmonary microsomal dealkylation of pentoxyresorufin, an action attributed to CYP2B1 the most likely candidate for the bioactivation of these toxins. The result clearly indicate that CYP2B1 is involved in the local conversion of mmt into a pulmonary toxicant.
Executive summary:

The pulmonary toxicity of mmt depends on its bioactivation by the cytochrome P-450 (CYP) system. A range of compounds, that modifies the activity of specific CYP isoenzymes, has been used to establish those particular isoenzymes involved in bioactivation. Pulmonary toxicity was assessed by measurement of lung weight and changes in the γ-glutamyltranspeptidase (GGT) and alkaline phosphatase (ALT) enzymes in bronchoalveolar lavage fluid. Pretreatment with O, O, S – Trimethylphosphorodithioate, bromophos, ρ-xylene and 2, 4 – dichloro-(6-phenylphenoxy)ethylamine all inhibited the dealkylation of pentoxyresorufin, an indicator of CYP2B1 activity, also prevented pulmonary toxicity when treated with mmt. Modification of the activity of CYP1A1, CYP2E1 and CYP4B1 did not alter the pulmonary toxicity of mmt. These results indicate that pulmonary CYP2B1 is responsible for the bioactivation and pulmonary toxicity of mmt in rats.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was well documented with sufficient information on methods and results providing limited information on absorption, distribution, and excretion.
Objective of study:
toxicokinetics
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were exposed to manganese chloride (MnCl2) or methylcyclopentadienyl tricarbonyl (mmt) and the concentrations of manganese (Mn) in the plasma were determined at several different time points by means of atomic absorption spectrometry.
GLP compliance:
no
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan, Inc.
- Age at study initiation: 2 months old
- Weight at study initiation: 210-230 grams
- Fasting period before study: yes, animals were fasted 12 hr prior to compound administration
- Housing: no data
- Individual metabolism cages: no data
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Animals housed in a temperature-controlled environment with a 12:12 hr light/dark room


Route of administration:
other: MnCl2: intravenous (iv) and oral gavage; mmt: oral gavage
Vehicle:
other: MnCl2: sterile saline and mmt: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: No data


VEHICLE
- Justification for use and choice of vehicle (if other than water): poor solubility of mmt in water
- Concentration in vehicle:
-MnCl2: 6.0 mg Mn/kg (1.0 ml/kg)
-mmt: 20 mg/kg (3.3 ml/kg); equivalent to 5.6 mg Mn/kg
Duration and frequency of treatment / exposure:
Single dose
Remarks:
Doses / Concentrations:
mmt: 20 mg/kg (3.3 ml/kg); equivalent to 5.6 mg Mn/kg
MnCl2: 6.0 mg Mn/kg (1.0 ml/kg)
No. of animals per sex per dose / concentration:
mmt (oral): 4 male rats and 4 female rats
manganese chloride (iv): 5 male rats
manganese chloride (oral): 4 male rats
Control animals:
no
Positive control reference chemical:
No.
Details on study design:
- Dose selection rationale: the dose regimen for the MnCl2 administration was chosen because it was known to be associated with a significant reduction of succinic dehydrogenase and aconitase in rat brain. Rationale for mmt dose regimen was not provided.
Details on dosing and sampling:
TOXICOKINETIC STUDY (Absorption, distribution, excretion): measurement of Mn
- Tissues and body fluids sampled: plasma
- Time and frequency of sampling:
-MnCl2: 0, 0.05, 0.17, 0.33, 0.5, 1, 2, 4, 8 and 12 hr.
-mmt: 0, 0.17, 0.5, 1, 2, 4, 8, 12, 24, 48, 120, 168, 288, 384 and 456 hr

Mn concentrations in plasma were determined by atomic absorption spectrophotometry (AAS).

The detection limit for this method was 0.2 ng Mn/ml of assay solution.
Statistics:
All data are presented as mean ± SD. Statistical analysis for comparison of two means was performed using one-way ANOVA. In all cases, a probability level of p<0.05 was considered as a criterion of significance.
Preliminary studies:
N.A.
Details on absorption:
Oral dose of MnCl2: A single dose of manganese chloride by oral gavage to rats resulted in a rapid appearance of Mn in the plasma. The Cmax (0.3 µg/ml) was achieved within 0.5 hr of the oral dose. The absolute oral bioavailability (F) of Mn following oral MnCl2 was 13.2% at a dose of 6 mg Mn/kg. See Table 1.

Oral dose of mmt: Following a single dose of mmt by oral gavage to rats, Mn appeared in the plasma and attained a Cmax between 2 and 12 hr after dosing. Although the absolute dose of Mn in the mmt dose (5.6 mg Mn/kg) was comparable to that of manganese chloride (6 mg Mn/kg), the Cmax
(0.931 µg/ml) following oral administration of mmt was significantly higher (about 3 fold higher) than that following oral administration of MnCl2 (0.3µg/ml). The absolute oral bioavailability (F) for mmt was not calculated since an iv study was not performed. See Table 2.
Details on distribution in tissues:
Only concentrations in plasma were determined.
Details on excretion:
Oral dose of MnCl2 : Plasma Mn returned to normal level 12 hrs after dosing. Oral dosing of MnCl2 resulted in a significant increase in terminal t1/2 compared to rats receiving iv injection. By adjusting the apparent clearance (CL) and apparent volume of distribution (Vd) with the oral bioavailability (F), the clearance in the oral dose group remained unchanged; however, the Vd in the oral dosing group was significantly increased (2.5 fold) compared to the iv dosing group. See Table 1.

Oral dose of mmt: The elimination of Mn after Tmax was monophasic, with an average elimination t1/2 of 55hr. mmt-derived Mn was eliminated very slowly from the rats. The rats receiving mmt had an apparent oral clearance (CL) of 0.089 l/hr kg. Accordingly, the AUC in the rats dosed with mmt was about 37-fold larger than that in rats dosed with MnCl2. See Table 2.
Metabolites identified:
no
Details on metabolites:
N.A.

Table 1 - Toxicokinetic parameters of Mn in Sprague-Dawley rats following intravenous or oral administration of MnCl2

 

Intravenous dose

Oral dose

t1/2α(h)

0.17 ± 0.03

 

t1/2β(h)

1.83 ± 0.63

4.56 ±1.30**

AUC (mM . h)

14.8 ± 3.60

1.95 ± 0.51**

CLs(L/h . kg)

0.43 ± 0.13

 

Vβ(L/kg)

1.16 ± 0.51

 

Vc(L/kg)

0.14 ± 0.03

 

Cmax(µg/ml)

 

0.30 ± 0.11

Tmax(h)

 

0.25 ± 0.21

F (%)

 

13.19

Rats (220 ± 10 g) were administered either an iv-bolus or oral dose of MnCl2 (6.0 mg Mn/kg). Parameters were computed from the plasma concentration – time curves of each animal. Data represent the mean ± SD, n=5 for iv dose, and n=4 for oral dose studies.

*p>0.05; **p<00.01 compared to values in the male group

Table 2 - Toxicokinetics parameters of Mn in Sprague-Dawley rats following oral administration of mmt

 

Male

Female

Combineda

t1/2(e)(h)

42.0 ± 3.35

68.4 ± 13.9**

55.2 ± 17.0

Ke(β) (h-1)

0.02 ± 0.00

0.010 ± 0.002**

0.01 ± 0.00

AUC (mM . h)

51.8 ± 10.9

93.1 ± 30.7*

72.5 ± 30.7

Cmax(µg/ml)

0.79 ± 0.36

1.07 ± 0.46

0.93 ± 0.41

Tmax(h)

5.50 ± 4.43

10.0 ± 2.31

7.75 ± 4.06

CL/F (L/h.Kg)

0.11 ± 0.03

0.07 ± 0.02*

0.09 ± 0.03

Vβ/F(L/kg)

6.81 ± 1.73

6.25 ± 1.49

6.53 ± 1.53

Rats (220 ± 10g) were administered with oral dose of mmt (20 mg/kg, i.e. 5.6 mg Mn/kg). Parameters were computed from the plasma concentration-time curves of each animal. Data represent the mean ± SD, n=4 for both sexes

aThe data from both male and female rats were combined to estimate mean ± SD, n=8

*p>0.05; **p<00.01 compared to values in the male group

Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
The study allows for the comparison of the toxicokinetics in rats of inorganic manganese (MnCl2) and mmt based on plasma Mn concentrations after a single oral dose. Much higher and more prolonged concentration-time profiles were found for the mmt-derived Mn. After oral dosing of mmt, the mmt-derived Mn entered the blood circulation at a slower rate, but to a much greater extent as compared to the oral dosing of inorganic MnCl2. The relatively complete absorption and slow elimination of mmt-derived Mn in mmt-treated rats contribute to its higher plasma concentration-time profiles. The elimination of mmt-derived Mn also appears to be gender dependent.
Executive summary:

What follows is the abstract of the original publication. The toxicokinetics of manganese (Mn) was investigated in male and female rats either following a single intravenous (iv) or oral dose of MnCl2 (6.0 mg Mn/kg), or following a single oral dose of mmt (20 mg mmt/kg or 5.6 mg Mn/kg). The plasma concentrations of Mn were quantified by atomic absorption spectrofotometry (AAS). Upon iv administration of MnCl2, Mn rapidly disappeared from blood with a terminal elimination t1/2of 1.83h and CLsof 0.43 L/h/kg. The plasma concentration-time profiles of Mn could be described by C = 41.9e-4.24t+2.1e-0.44t. Following oral administration of MnCl2, manganese rapidly entered the systemic circulation (Tmax= 0.25 h). The absolute oral bioavailability was about 13%. Oral dose of mmt resulted in a delayed Tmax(7.6 h), elevated Cmax(0.93 µg/ml), and prolonged terminal t1/2(55.1 h). The rats receiving mmt had an apparent clearance (CL/F = 0.09 L/h kg) about 37-fold less than did those who were dosed with MnCl2. Accordingly, the area under the plasma concentration-time curves (AUC) of Mn in mmt-treated rats was about 37-fold greater than that in MnCl2-treated rats. A gender-dependent difference in toxicokinetic profiles of plasma Mn was also observed. Female rats dosed with mmt displayed a greater AUC than that of male rats. Although the apparent volume of distribution of Mn was similar in both sexes, the apparent clearance in males was about twice that observed in females. The results indicated that after oral administration, the mmt-derived Mn displayed higher and more prolonged plasma concentration-time profiles than MnCl2-derived Mn.

Description of key information

Eight publically available studies on the toxicokinetics of mmt were summarized in this section. In addition, three acute toxicity studies from the public domain were summarized in Section 7.2.4, which also cover aspects of mmt toxicokinetics. The studies give insight in the oral absorption, excretion, distribution and metabolism of the substance. Much attention is paid to the role of lung metabolism in lung toxicity and the protective role of liver metabolism, whereby inhibitors and inducers of CYP-dependent mono-oxygenases play an important role. Two internal in vitro metabolism studies demonstrate differences in mmt metabolite formation between rats and humans in lung microsomes. Another aspect studied was the difference between the toxicokinetics of mmt and inorganic manganese in relation to their toxicity.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
50
Absorption rate - inhalation (%):
100

Additional information

Conclusions

1. Based on excretion, kinetics and metabolism data obtained in rats, it can be concluded that mmt is largely and possibly completely absorbed by this species after oral exposure and inhalation exposure.

2. In rats, more than 90% of an oral dose is eliminated in a few days. Thereafter, elimination is slower. The possibility cannot be excluded that, depending on dose, regular exposures result in increased concentrations in certain organs, in particular the liver and the kidneys, as is shown by an repeated-dose study with mice and a kinetic study with rats. However, it is expected that termination of exposure will lead to a reduction of the concentrations.

3. In rats, less than half of oral doses has been found to be excreted via the urine. The remaining is excreted via the feces. Urinary excretion in mice appears to be much lower than in rats.. Biliary excretion is substantial.

4. Pretreatment with phenobarbital, an inducer of CYP, caused a shift from fecal to urinary excretion and increased biliary excretion.

5. Pulmonary toxicity of mmt is strongly reduced by pretreatment with phenobarbital. However, the pretreatment does not result in a reduction of neurotoxicity.

6. mmt is metabolized by homogenates of rat liver, lungs, kidneys and brain to compounds that cannot be extracted from the reaction mixture with heptane (ratio of rates: liver=1; lungs: 0.14; kidneys: 0.014 and brain: 0.009).

7. Pretreatment with phenobarbital stimulates liver mmt metabolism but not lung mmt metabolism.

8. Material excreted via the urine and the bile by the rat consists largely of CMT-COOH and CMT-CH2OH (mmt oxidized at the methyl side chain of the cyclopentadienyl ring). These metabolites are much less acutely toxic than mmt.

9. Pulmonary toxicity depends on the CYP2B1 which is present in the lungs of the rat and thus is not directly caused by mmt.

10. Comparison with CMT (mmt without the methyl side chain at the pentadienyl ring) shows that the pulmonary toxicity is not caused by the metabolic oxidation of the methyl side chain.

11. The protection against pulmonary toxicity in the rat depends on the methyl-side-chain oxidation in the liver. Liver metabolism of mmt can thus be regarded as a detoxifying process.

12. The toxic metabolites that cause the pulmonary toxicity have as yet not been identified. However, the fact that methyl-side-chain oxidation is not involved leaves the involvement of the manganese atom.

13. In an 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. At 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 5mM mmt, 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.

14. In an in vitro metabolism study using rat, monkey and human lung microsomes, a LC/UV assay data indicated that mmt disappearance occurred in all test systems investigated, including active and deactivated lung microsomes as well as the solvent control during the 5-hour incubation at both the 1 uM and 5uM mmt concentrations. mmt disappearance followed first order kinetics in active lung microsomes from all three species at both concentrations. The two known metabolites, mmt-alcohol and mmt-acid, were observed in rat lung microsomal samples, with minimal formation of mmt-alcohol in human and monkey and no formation of mmt-acid. Species difference and similarity were observed in metabolite formation; in general, human and monkey microsomal samples had more similarities than rat and human microsomal samples for the major metabolites.