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Endpoint:
dermal absorption in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
Principles of method if other than guideline:
The disposition of 14C-MEKO was determined in the male F344 rat following dermal administration.
GLP compliance:
not specified
Radiolabelling:
yes
Remarks:
14C-methyl ethyl ketoxime
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: 11-13 weeks old
- Weight at study initiation: 210-265 g
- Fasting period before study: no data
- Housing: During experiments, rats were housed individually in glass metabolism cages which provided for the separate collection of urine, feces, CO2 and other volatiles.
- Diet (e.g. ad libitum): Purina Rodent Chow #5002, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 +/- 1.6
- Humidity (%): 50 +/- 20
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
open
Vehicle:
acetone
Duration of exposure:
Single dermal exposure
Doses:
Dose levels evaluated: 2.7 and 270 mg C14-MEKO/kg body weight
No. of animals per group:
3 male rats per dose
Control animals:
no
Details on study design:
Dermal dose formulations were prepared in acetone to deliver ~20 uCi in a single dose volume of 200 uL and were applied to a 12 square cm area from which the hair had been clipped the previous day. Dose site areas were inspected for damage prior to dosing and any animals with damage in the clipped area were excluded from the study. Dose sites were protected from grooming by a nonocclusive foam appliance with a cloth cover and metal shield.
Dose:
2.7 mg/kg
Parameter:
percentage
Absorption:
ca. 26 %
Remarks on result:
other: 72 hours
Remarks:
Volatilisation from the dose site prior to placement in the metabolism cage may account for the low absorption.
Dose:
270 mg/kg
Parameter:
percentage
Absorption:
ca. 13 %
Remarks on result:
other: 72 hours
Remarks:
Volatilisation from the dose site prior to placement in the metabolism cage may account for the low absorption.
Conclusions:
Following dermal application of C14-methyl ethyl ketoxime at 2.7 and 270 mg/kg body weight, approximately 26 and 13% of the 2.7 and 270 mg C14 MEKO/kg doses, respectively, were absorbed. Volatilization from the dose site prior to placement in the metabolism cage may account for the low absorption.
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Objective of study:
other: Disposition
Qualifier:
no guideline followed
Principles of method if other than guideline:
The disposition of 14C-MEKO was determined in the male F344 rat following intravenous (i.v.) administration.
GLP compliance:
not specified
Radiolabelling:
yes
Remarks:
14C-methyl ethyl ketoxime
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: 11-13 weeks old
- Weight at study initiation: 210-265 g
- Fasting period before study: no data
- Housing: During experiments, rats were housed individually in glass metabolism cages which provided for the separate collection of urine, feces, CO2 and other volatiles.
- Diet (e.g. ad libitum): Purina Rodent Chow #5002, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 +/- 1.6
- Humidity (%): 50 +/- 20
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intravenous
Vehicle:
other: isotonic saline
Details on exposure:
Isotonic was used for iv doses, which were administered via the lateral tail vein at a volume of 0.8 mL/kg body weight.
Duration and frequency of treatment / exposure:
Single intravenous (iv) exposure
Dose / conc.:
2.7 mg/kg bw/day (nominal)
Remarks:
A single dose level of 2.7 mg C14-MEKO/kg bw
No. of animals per sex per dose / concentration:
3 male rats per dose
Control animals:
no
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Adipose, blood, kidney, liver, muscle, skin, testis, urine, faeces, and volatiles.
- Time and frequency of sampling: 2, 8, 24 and 72 hours after iv administration

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: 0 and 8 hours after iv administration
- From how many animals: (samples pooled): 3
- Method type(s) for identification: HPLC
Details on distribution in tissues:
About 7% of the administered radioactivity remained in the tissues after 72 hours.
Details on excretion:
The i.v. dose of 2.7 mg/kg was principally excreted as CO2 (48.8%) with excretion in urine, and as expired volatiles, accounting for 21.4 and 11.4%, respectively.
Metabolites identified:
yes
Details on metabolites:
MEKO was biotransformed to at least five polar metabolites that could only be partially resolved by anion exchange chromatography. Incubation with glucuronidase, but not sulphatase, changed the urinary metabolic profile. Methyl ethyl ketone was a major component in the volatiles.
Conclusions:
An intravenous dose of C-14 methyl ethyl ketoxime at 2.7 mg/kg body weight was primarily excreted as CO2 (48.8%) with excretion in urine, and as expired volatiles, accounting for 21.4 and 11.4%, respectively. About 7% of the administered radioactivity remained in the tissues after 72 hours. MEKO was biotransformed to at least five polar metabolites that could only be partially resolved by anion exchange chromatography. Incubation with glucuronidase, but not sulphatase, changed the urinary metabolic profile. Methyl ethyl ketone was a major component in the volatiles.
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Objective of study:
other: Disposition
Qualifier:
no guideline followed
Principles of method if other than guideline:
The disposition of 14C-MEKO was determined in the male F344 rat following oral administration.
GLP compliance:
not specified
Radiolabelling:
yes
Remarks:
14C-methyl ethyl ketoxime
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: 11-13 weeks old
- Weight at study initiation: 210-265 g
- Fasting period before study: no data
- Housing: During experiments, rats were housed individually in glass metabolism cages which provided for the separate collection of urine, feces, CO2 and other volatiles.
- Diet (e.g. ad libitum): Purina Rodent Chow #5002, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 +/- 1.6
- Humidity (%): 50 +/- 20
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- Dose formulations contained ~20 uCi radiolabel and an appropriate amount of non-radiolabeled MEKO dissolved in sufficient water to allow oral delivery by gavage of the target dose in a total of 5 mL/kg body weight.


Duration and frequency of treatment / exposure:
Single oral exposure
Dose / conc.:
2.7 mg/kg bw/day (nominal)
Remarks:
mg C14-MEKO/kg bw
Dose / conc.:
27 mg/kg bw/day (nominal)
Remarks:
mg C14-MEKO/kg bw
Dose / conc.:
270 mg/kg bw/day (nominal)
Remarks:
mg C14-MEKO/kg bw
No. of animals per sex per dose / concentration:
3 male rats per dose
Control animals:
no
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Adipose, blood, kidney, liver, muscle, skin, testis, urine, and faeces,
- Time and frequency of sampling: 2, 8, 24 and 72 hours after oral administration

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: 0 and 8 hours after oral administration
- From how many animals: (samples pooled): 3
- Method type(s) for identification: HPLC



Details on distribution in tissues:
Five to six percent of the oral dose of MEKO remained in major tissues after 72 hours.
Details on excretion:
Oral doses of methyl ethyl ketoxime were primarily excreted as CO2 (71-49%) in decreasing percentage as the dose increased. Excretion in urine (13-26%) and as volatiles (5-18%) increased as the dose increased.
Metabolites identified:
yes
Details on metabolites:
MEKO was biotransformed to at least five polar metabolites that could only be partially resolved by anion exchange chromatography. Incubation with glucuronidase, but not sulphatase, changed the urinary metabolic profile. Methyl ethyl ketone was a major component in the volatiles.
Conclusions:
Oral doses of C14-methyl ethyl ketoxime at 2.7, 27 and 270 mg/kg body weight were primarily excreted as CO2 (71-49%) in decreasing percentage as the dose increased. Excretion in urine (13-26%) and as volatiles (5-18%) increased as the dose increased. Five to 6% of the oral dose of MEKO remained in major tissues after 72 hours. MEKO was biotransformed to at least five polar metabolites that could only be partially resolved by anion exchange chromatography. Incubation with glucuronidase, but not sulphatase, changed the urinary metabolic profile. Methyl ethyl ketone was a major component in the volatiles.
Endpoint:
basic toxicokinetics, other
Remarks:
in vivo and in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose for cross-reference:
reference to same study
Objective of study:
toxicokinetics
Qualifier:
no guideline followed
Principles of method if other than guideline:
Evaluation of biotransformation of MEKO and acetoxime in vivo and in vitro; chemical reactivity of the postulated MEKO-metabolites characterised; formation of DNA and RNA-modifications by MEKO in liver DNA and RNA from male and female rats exposed to MEKO by inhalation.
GLP compliance:
yes
Species:
other: rat, mouse and human
Strain:
other: Wistar rats and B6C3F1 mice
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS and TISSUES:
- Source of Rats and MIce: Harlan-Winkelmann, Borchen, Germany
- Source of Human Liver Specimens: Keystone Skin Bank, Exton, PA USA
Metabolites identified:
yes
Details on metabolites:
The biotransformation of MEKO was studied in liver microsomes and cytosols from male and female rats, mice and humans. MEKO was found to be oxidised to butane 2-nitronate by microsomal monooxygenases but at very low rates. No sex differences were observed. The hypothesized biosynthesis of methyl ethyl ketoxime O-sulfate and acetoxime O-sulfate in liver subcellular fractions from the corresponding nitronates or oximes was found not to occur or to occur at very low rates since formation of the stable O-sulfates could not be demonstrated using actoxime and MEKO or propane 2-nitronate and butane 2-nitronate as substrates in the presence of appropriate cofactors.

The toxicokinetic studies demonstrated the existence of two metabolic pathways and suggests the possibility of a third pathway for MEKO, the major pathway being hydrolysis to 2-butanone (MEK). This pathway was studied quantitatively. The sum of the two other pathways could be described by a Vmax and an apparent Km value. One of these two pathways was inhibited by the CYP450 inhibitor diethyldithiocarbamate. For the CYP450 dependent pathway, a maximum transformation rate of 0.008 mmol/hour/250 g rat for males and 0.010 mmol/hour/250 g rat for females were estimated. These values are the lower end of the published CYP450 dependent maximum rates in xenobiotic metabolism in vivo. With respect to the three pathways, there were no quantitative differences in the extent of MEKO metabolism between male and female rats.

Conclusions:
MEKO-biotransformation appears to be more complex than previously proposed, with interacting steps and several enzyme systems involved. The first activating step (likely P450 dependent oxidation) may have only a minor and dose-dependend contribution to the overall biotransformation of MEKO. The postulated reductive bioactivation of 2-nitrobutane formed from MEKO likely represents a very minor pathway in overall MEKO metabolism or may not occur at all. Since the extent of oxidation of MEKO is dose-dependent it may be relevant only after exposure to high MEKO-doses. The results of these studies shed considerable doubt that MEKO liver tumours in male rats result from the formation of a DNA-reactive metabolite by the biotransformation pathways.

Description of key information

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

The toxicokinetic studies demonstrated the existence of two and suggest the possibility of a third metabolic pathway for MEKO. The major pathway is hydrolysis to 2-butanone (MEK). This pathway was studied quantitatively. A Vmax and an apparent Km value could describe the sum of the two other pathways. One of these two pathways was inhibited by the CYP450 inhibitor diethyldithiocarbamate. For the CYP450 dependent pathway, a maximum transformation rate of 0.008 mmol/h/250 g rate for males and 0.010 mmol/h/250 g rat for females were estimated. These values are the lower end of the published CYP450 dependent maximum rates in xenobiotic metabolism in vivo. With respect to the three pathways, there were no quantitative differences in the extent of MEKO metabolism between male and female rats.

 

14C-MEKO disposition studies in rats and mice clearly show MEKO to be rapidly absorbed from gastro-intestinal tract and skin, undergo widespread uptake, distribute over the entire body, be extensively metabolised and not accumulate in tissues (Burka et al., 1998; Volker et al., 1999; University of Wurzburg, 2000; Pharmacon Research Foundation, Inc., 1981). Excretion as volatiles in expired air, excretion in urine and bile occurs. Low activity was found in the intestine content. MEKO and or its metabolites are primarily excreted via urine.

 

Following dermal application of14C-MEKO ketoxime at 2.7 and 270 mg/kg bw, approximately 26 and 13% of the 2.7 and 270 mg14C-MEKO/kg bw, respectively, were absorbed (Burka et al., 1998). Volatilization from the dose site prior to placement in the metabolism cage may account for the low absorption.

MEKO biotransformation appears to be more complex than previously proposed, with interacting steps and several enzyme systems involved. The first activating step (likely P450 dependent oxidation) may have only a minor and dose-dependent contribution to the overall biotransformation of MEKO. The postulated reductive bioactivation of 2-nitrobutane formed from MEKO likely represents a very minor pathway in overall MEKO metabolism or may not occur at all. Since the extent of oxidation of MEKO is dose-dependent it may be relevant only after exposure to high MEKO-doses.