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Registration Dossier

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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:
toxicokinetics
Qualifier:
no guideline followed
Version / remarks:
This study is a pilot pharmacokinetic investigation and as such is not designed to follow specific test guidelines.
Principles of method if other than guideline:
This study was performed to obtain blood samples from rats at selected time points following a single 6-hour exposure of the test atmosphere of Methyisobutylketone at two different concentration levels of Low (500 ppm) and High (1000 ppm) in order to determine the concentrations and the pharmacokinetics parameters of Diacetone alcohol (DAA), Methyl-isobutyl ketone (MIBK) and Methylisobutylcarbinol (MIBC) from the plasma.
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
SPF colony
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: 387g-468g
- Housing: three animals per cage
- Diet (ad libitum): ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice
- Water (ad libitum): tap water from municipal supply
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 25.6
- Humidity (%): 40 - 62
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose only, past flow, dynamic flow exposure unit, consisted of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports.
- Method of holding animals in test chamber: tube
- Source and rate of air: no data
- Method of conditioning air: oil-free compressor passed through a suitable filter system prior to introduction to the nebuliser
- System of generating particulates/aerosols: The test item was administered as vapours. The test item was aerosolised using a stainless steel concentric jet nebulisers (TSE Systems GmbH, Bad Homburg, Germany) located at the top of the exposure chambers and a separator glass was installed to remove any liquid particles from the atmosphere prior entering into the inhalation tower. The rate of test item use was controlled by a syringe pump.
- Temperature, humidity in air chamber: 21.0-25.5°C, 3.0-4.5%
- Air Flow In (Inner Plenum) (L/min): 29.6-30.8
- Air Flow Out (Outer Cylinder) (L/min): 23.6-25.7
- Air change rate: no data
- Method of particle size determination: not determined since the test item was evaporated and non-condensing.
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: the concentration of the test item during the exposure was measured by gas chromatography (GC/FID). Samples for analytical determination were taken once an hour during the exposure into an impinger containing ethanol as a solvent.
- Samples taken from breathing zone: yes
Duration and frequency of treatment / exposure:
single 6-hour exposure
Dose / conc.:
500 other: ppm (target)
Dose / conc.:
531 ppm (analytical)
Remarks:
2.18 mg/L
Dose / conc.:
1 000 other: ppm (target)
Dose / conc.:
1 022 ppm (analytical)
Remarks:
4.19 mg/L
No. of animals per sex per dose / concentration:
9
Control animals:
no
Details on dosing and sampling:
TOXICOKINETIC STUDY
- Tissues and body fluids sampled: plasma
- Time and frequency of sampling: 0 (on day -1 or -3), 0.5, 1, 1.5, 3, 4.5, 6, 8, 18 and 24 hours post exposure

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: plasma
- Time and frequency of sampling: 0 (on day -1 or -3), 0.5, 1, 1.5, 3, 4.5, 6, 8, 18 and 24 hours post exposure
- From how many animals: 3
- Method type(s) for identification: Gas Chromatography coupled with MS detection after electron impact ionization (GC-MS)
- Limits of quantification: DAA < 0.5 µg/mL; MIBC < 0.1 µg/mL; MIBK < 0.2 µg/mL

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): no
Key result
Toxicokinetic parameters:
other: Cmax (µg/mL)/500 ppm: DAA 45.1, MIBC 0.66, MIBK 23.5
Key result
Toxicokinetic parameters:
other: Tmax (h)/500 ppm: DAA 1.5, MIBC 0.5, MIBK 0.5
Key result
Toxicokinetic parameters:
other: t1/2 (h)/500 ppm: DAA 2.4, MIBC nc, MIBK 0.581
Key result
Toxicokinetic parameters:
other: AUC0.5-24h (h*µg/mL)/500 ppm: DAA 266, MIBC 0.499, MIBK 20.1
Key result
Toxicokinetic parameters:
other: Cmax (µg/mL)/1000 ppm: DAA 82.7, MIBC 1.36, MIBK 56.8
Key result
Toxicokinetic parameters:
other: Tmax (h)/1000 ppm: DAA 1.5, MIBC 0.5, MIBK 0.5
Key result
Toxicokinetic parameters:
other: t1/2 (h)/1000 ppm: DAA 4.91, MIBC nc, MIBK nc
Key result
Toxicokinetic parameters:
other: AUC0.5-24h (h*µg/mL)/1000 ppm: DAA 522, MIBC 1.82, MIBK 70.2
Metabolites identified:
yes
Details on metabolites:
Diacetone alcohol (DAA), Methyl-isobutyl ketone (MIBK) and Methylisobutyl carbinol (MIBC)

Mean plasma concentrations of DAA, MIBK and MIBC after an inhalation exposure to MIBK at 500 and 1000 ppm

Exposure

Analyte (µg/mL)

Sampling time

Pre-dose

0.5 h

1 h

1.5 h

3 h

4.5 h

6 h

8 h

18 h

24 h


MIBK

500 ppm

 

DAA

BLQ

41.7

44.3

45.1

43.1

29.0

15.8

12.9

0.559

BLQ

MIBK

BLQ

23.5

13.7

5.18

2.32

0.15

BLQ

BLQ

BLQ

BLQ

MIBC

BLQ

0.660

0.356

0.170

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

MIBK
1000 ppm

 

DAA

BLQ

62.6

58.7

82.7

70.8

49.9

52.8

32.4

0.84

0.73

MIBK

BLQ

56.8

33.1

23.6

12.5

0.633

0.267

0.193

BLQ

BLQ

MIBC

BLQ

1.36

0.852

0.698

0.280

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ: Below Limit of Quantification.

Toxicokinetic parameters of DAA, MIBC and MIBK after a single 6-hour inhalation
exposure of male Sprague-Dawley rats to 500 ppm and 1000 ppm of DAA

 

Concentration

Analytes

Number of point used to calculatelz

lz

t1/2

Tmax

Cmax

Cmax/
concentration#

AUC0.5-24h

AUC0.5-24h/
concentration#

Metabolite/
Administered compound

1/h

h

h

µg/mL

h*µg/mL

Cmax

AUC0.5-24h

500 ppm
(2.38 mg/L)

DAA

0.994

5

0.289

2.40

1.5

45.1

22.0

266

130

1.92

13.20

MIBC

na

na

nc

nc

0.5

0.66

0.322

0.499

0.243

0.0281

0.0248

MIBK

0.944

4

1.19

0.581

0.5

23.5

11.5

20.1

9.82

na

na

1000 ppm
(4.75 mg/L)

DAA

0.948

6

0.248

2.79

1.5

82.7

20.2

522

127

1.45

7.44

MIBC

0.995

3

0.569

1.22

0.5

1.36

0.331

1.82

0.445

0.0239

0.0260

MIBK

0.912

6

0.832

0.833

0.5

56.8

13.9

70.2

17.1

na

na

na: not applicable.

nc: not calculated as not enough quantifiable points were available.

#:Cmaxand AUC0.5-24hwere divided by the exposure concentrations of DAA (in mg/L).

 

Executive summary:

A study was performed to obtain blood samples from rats at selected time points following a single 6-hour exposure of the test atmosphere of Methylisobutylketone at target concentration levels of 500 and 1000 ppm in order to determine the concentrations and the pharmacokinetics parameters of Diacetone alcohol (DAA), Methyl-isobutyl ketone (MIBK) and Methyl-isobutyl carbinol (MIBC) from the plasma. The animals were exposed to the test atmosphere (in the form of a vapour) using a nose-only exposure system. Analytical concentrations of 531 ppm, 1022 ppm were achieved in the respective groups. No control animals were used in the study. The test atmosphere concentration was monitored based on a validated GC method (by trapping the vapors in an impinger containing ethanol). The results of the test atmosphere characterization were considered suitable for the study purposes. Heighteen male Sprague-Dawley rats were involved in the study, 9 animals in each treatment groups. Plasma samples were prepared from male rats following the end of the exposure at nine time-points (three animals at each sampling occasion) and the plasma level of DAA, MIBK and MIBC was determined. The analytical method consisted of a liquid/liquid extraction of the three analytes from plasma with Methyl tert-Butyl Ether (MtBE) followed by analysis of supernatant by Gas Chromatography coupled with MS detection after electron impact ionization (GC-MS). The toxicokinetic evaluation was performed using non-compartmental analysis on Phoenix WinNonlin software (Pharsight Corporation, Mountain View, California 94040/USA). DAA, MIBC and MIBK toxicokinetic parameters were determined from the mean plasma concentration collected from each animal/group, at each post-exposure time-point. A separate analysis was performed for each dose-level. The following toxicokinetic parameters were calculated t½, ¿z, AUC0.5-24h, Tmaxand Cmax.

The mean actual achieved concentration of the test item were:

Target Concentration

(ppm)* [mg/L]

Achieved Concentration

(ppm)* [mg/L]

Nominal Concentration

 mg/L

500 [2.05]

531 [2.18]

3.69

1000 [4.10]

1022 [4.19]

6.17

For the three analytes (DAA, MIBK and MIBC), the standard acceptance criteria for the calibration lines and Quality Control samples were met for a successful analysis of the study samples.

Study samples with concentrations above the calibration ranges were repeated diluted (for DAA principally and MIBK). DAA, MIBC and MIBK were not quantifiable in pre-dose samples in any group.

The mean plasma concentrations of DAA, MIBK and MIBC after an inhalation exposure to MIBK at 500 and 1000 ppm were:

Exposure

Analyte (µg/mL)

Sampling time

Pre-dose

0.5 h

1 h

1.5 h

3 h

4.5 h

6 h

8 h

18 h

24 h


MIBK

500 ppm

 

DAA

BLQ

41.7

44.3

45.1

43.1

29.0

15.8

12.9

0.559

BLQ

MIBK

BLQ

23.5

13.7

5.18

2.32

0.15

BLQ

BLQ

BLQ

BLQ

MIBC

BLQ

0.660

0.356

0.170

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

MIBK
1000 ppm

 

DAA

BLQ

62.6

58.7

82.7

70.8

49.9

52.8

32.4

0.84

0.73

MIBK

BLQ

56.8

33.1

23.6

12.5

0.633

0.267

0.193

BLQ

BLQ

MIBC

BLQ

1.36

0.852

0.698

0.280

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ: Below Limit of Quantification.

The toxicokinetic parameters of DAA, MIBC and MIBK after a single 6-hour inhalation exposure of male Sprague-Dawley rats to 500 ppm and 1000 ppm of MIBK were:

 

Concentration

Analytes

Number of point used to calculatelz

lz

t1/2

Tmax

Cmax

Cmax/
concentration#

AUC0.5-24h

AUC0.5-24h/
concentration#

Metabolite/
Administered compound

1/h

h

h

µg/mL

h*µg/mL

Cmax

AUC0.5-24h

500 ppm
(2.38 mg/L)

DAA

0.994

5

0.289

2.40

1.5

45.1

22.0

266

130

1.92

13.20

MIBC

na

na

nc

nc

0.5

0.66

0.322

0.499

0.243

0.0281

0.0248

MIBK

0.944

4

1.19

0.581

0.5

23.5

11.5

20.1

9.82

na

na

1000 ppm
(4.75 mg/L)

DAA

0.948

6

0.248

2.79

1.5

82.7

20.2

522

127

1.45

7.44

MIBC

0.995

3

0.569

1.22

0.5

1.36

0.331

1.82

0.445

0.0239

0.0260

MIBK

0.912

6

0.832

0.833

0.5

56.8

13.9

70.2

17.1

na

na

na: not applicable.

nc: not calculated as not enough quantifiable points were available.

#:Cmaxand AUC0.5-24hwere divided by the exposure concentrations of DAA (in mg/L).

After a 6-hour inhalation exposure to MIBK:

·        MIBK was on average quantifiable from 0.5 to 4.5 h after the end of exposure to 500 ppm and for up to 8 h at 1000 ppm. Maximum concentrations of MIBK were at 0.5 h after the end of exposure for both MIBK concentrations,

·        DAA was on average quantifiable from 0.5 to 18 h after the end of exposure to MIBK at 500 ppm and for up to 24 h at 1000 ppm. Maximum concentrations of DAA were at 1.5 h after the end of exposure for both MIBK concentrations,

·        MIBC plasma concentrations were very low but nevertheless quantifiable from 0.5 to 1.5 h after the end of exposure to 500 ppm of MIBK and for up to 3 h at 1000 ppm. Maximal plasma concentrations of MIBC were at 0.5 h after the end of exposure for both MIBK concentrations.

·        the elimination rates and half-live of DAA and MIBK were not significantly changed between both exposure concentrations, indicating that the excretion pathway was not saturated.

The plasma concentration values of DAA were higher than those of MIBC and MIBK after administration of MIBK, this indicates that DAA may represent a major metabolite of MIBK in rats. MIBC was considered to be a minor metabolite as it represented approximately 2.5% of the plasma MIBK exposure.

Based on AUC0-24hand Cmax(both normalized by exposure concentration), plasma DAA, MIBC and MIBK exposure increased dose proportionally, between 500 and 1000 ppm of MIBK.
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
This study describes the principal metabolites detected in guinea pig serum after ip injection of MIBK; their half-lives and clearance times; elucidates the initial steps in the rate of MiBK
GLP compliance:
not specified
Radiolabelling:
no
Species:
guinea pig
Strain:
not specified
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation:
- Weight at study initiation: 250-450 g
- Housing: no data
- Diet : Purina Lab Chow ad libitum
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data
Route of administration:
intraperitoneal
Vehicle:
corn oil
Duration and frequency of treatment / exposure:
single dosing
Remarks:
Doses / Concentrations:
450 mg/kg
No. of animals per sex per dose / concentration:
no data
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
Blood was collected by heart puncture from four animals at each of the following times after the dose was administered: 1, 2, 4, 6, 8, 12, and 16 hr. Only one blood sample was obtained from each guinea pig. Whole blood was centrifuged and serum collected. Serum was then placed in sealed culture tubes, refrigerated, and assayed within 48 hr.
- Method type(s) for identification: The concentrations of aliphatic ketones and their metabolites were measured in duplicate by direct on-column injection of undiluted serum. All analyses were performed on a Varian 2100 Gas Chromatograph equipped with a flame ionization detector. Columns were all-glass, U-shaped, 6 ft x 4 mm id in size, packed with either Chromosorb 101,2 80-100 mesh, 0.4% Carbowax 1500 coated on Carbopack A3, or 15% diethylene glycol succinate2 coated on Gas Chrom P, 80-100 mesh. Nitrogen was used as the carrier gas for ail columns.
The recoveries of MiBK added to serum were greater than 99%. The coefficient of variation was 1.1%. The limit of detection was at least 0.1 pg/ml of serum. The ketones were stable in serum for several weeks if kept under refrigeration. Red blood cell-plasma distributions were determined and the results indicated that over 90% of the ketone was distributed in the plasma fraction. Half-lives in serum were estimated by extrapolating the linear portion of the decay curve to zero time.
Toxicokinetic parameters:
half-life 1st: 66 min (MIBK)
Toxicokinetic parameters:
other: clearance time: 6 h (MIBK)
Toxicokinetic parameters:
other: clearance time: 16 h (HMP)
Toxicokinetic parameters:
other: MIBC concentration was to low for quantification
Metabolites identified:
yes
Details on metabolites:
4-hydroxy-4-methyl-2-pentanone (HMP)
4-methyl-2-pentanol (MIBC)

Two metabolites, 4-hydroxy-4-methyl-2-pentanone (HMP) and 4-methyl-2-pentanol (MIBC), were detected in serum after ip administration of MiBK to guinea pigs. The half-life and clearance times of MIBK were 66 min and 6 hr, respectively. 4-hydroxy-4-methyl-2-pentanone was the principal metabolite and was cleared in 16 hr. The concentration of 4-methyl-2-pentanol was too low for quantification.

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
Executive summary:

4-hydroxymethyl-4-methyl-2-pentanone (HMP, DAA) was identified in serum as the major metabolite following a single intraperitoneal administration of 450 mg/kg of methyl isobutyl ketone (MIBK) to guinea pigs. MIBK and HMP had serum half-lives of 78 minutes and 66 minutes, and clearance times of 6 hours and 16 hours, respectively.

The authors stated that a hydroxylation product like HMP would commonly be eliminated in urine as O-sulfate or O-glucuronide or may enter the intermediary metabolism to be eliminated as CO2 or to be incorporated into tissues.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
distribution
metabolism
Principles of method if other than guideline:
MIBK was administered by gavage and by inhalation to verify if the route of administration could influence the MIBK and metabolites concentration in plasma, liver and lung
GLP compliance:
not specified
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Canada Inc. (St-Constant, Québec)
- Age at study initiation: No data
- Weight at study initiation: 200-250 g
- Fasting period before study: No data
- Housing: stainless steel cages
- Individual metabolism cages: yes/no
- Diet : Charles River rat chow , ad libitum
- Water : ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
other: Oral and inhalation
Vehicle:
other: Mazola corn-oil for the oral route
Details on exposure:
For inhalation exposures, MiBK vapors were generated with a syringe pump by injecting the solvents in a negative pressure air conduit connected to the inhalation 500-l gas chamber. Air flow rate was 15 cu. ft/min. Animals were housed individually in small stainless-steel wire cages inside the chamber. During the 4-h, total body exposure (12:00 to 16:00 h) MiBK concentrations inside the chamber were monitored via an infrared spectrophotometer. Animals never exceeded 5% of the chamber volume (12 maximum).
Duration and frequency of treatment / exposure:
Oral: daily for 3 days
Inhalation: 4h/day for 3 days
Remarks:
Doses / Concentrations:
Oral route : 1.5, 3, or 6 mmol/kg
Inhalation: 200, 400, or 600 ppm
No. of animals per sex per dose / concentration:
6
Control animals:
yes
Details on dosing and sampling:
One hour after the last gavage (16:00 h) or immediately after the last vapor exposition session, animals were killed by decapitation. Heparinized blood was collected and centrifuged at 4°C for 15 min at 2000 x g. A portion of about 2 g of the median hepatic lobe was excised, weighed and homogenized with 5 ml of ice-cold 6% (w/v) perchloric acid solution. The homogenate was centrifuged at 4°C for 15 min at 2000 x g and the remaining supernatant prepared for further extraction. The lungs were rapidly removed, weighed, homogenized and centrifuged as previously described for liver samples. The supernatant of plasma, liver or lung (1 ml) was then extracted with 3 ml methylene chloride for 15 min. After centrifugation (15 min at 4°C and 2000 x g), the aqueous phase was removed. Internat standards (2-hexanol, 80 µg/ml) were added to the organic phase and the organic phase reduced to 500 µl under nitrogen flow. MiBK concentrations were determined on this extract by gas chromatographic analysis. Methylene chloride was the solvent used for the extraction of the ketone and their metabolites from biological fluid. MiBK and its respective metabolite contents were analysed from the resulting extraction. The extraction efficiencies for all the analysed substances were 90% or better.
MiBK and its respective metabolites were analyzed on a 5890A Hewlett-Packard gas chromatograph equipped with a flame ionisation detector.
Statistics:
The data were submitted to an ANOVA followed by Fisher's PLSD test, using a significance level of 0.05. Linear regression analyses were also performed where applicable
Details on distribution in tissues:
MiBK was administered daily, by gavage or by inhalation, for 3 days. With MiBK gavage solutions of 1.5, 3.0, or 6.0 mmol/kg, plasma MiBK concentrations were 5.3, 8.4, and 16.1 µg/ml, respectively (Table 1 attached). With a 3-day MiBK 4-h/day inhalation session, plasma MiBK concentrations were similar to those obtained by gavage. Concentrations of 5.0, 8.1 and 14.3 µg/ml were detected after exposure to 200, 400 or 600 ppm MiBK, respectively. MiBK and 4-OHMiBK concentrations in plasma were found to increase in a dose-related manner with the administered dose of MiBK, irrespective of the route of administration. After treatment by gavage, 4-MPOL concentration was too low for quantification (<0.25 µg/ml) and could not be detected with any MiBK orally administered doses used in this study. Interestingly, however, 4-MPOL was detectable at concentrations of 4.0 and 4.8 µg/ml after 400 and 600 ppm MiBK inhalation.
Metabolites identified:
yes
Details on metabolites:
4-methyl-2-pentanol (4-MPOL) = methylisobutyl carbinol (MIBC)
4-hydroxymethyl iso-butyl ketone (4-OHMiBK) = 4-hydroxy-4 methyl-2-pentanone (HMP) = diacetone alcohol (DAA)

Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
Executive summary:

Plasma MIBK concentrations were 5.3, 8.4, and 16.1 µg/mL in rats at 1 hour after the last of 3 daily gavage exposures to 1.5, 3.0, and 6.0 mmol/kg MIBK (150, 300, or 601 mg/kg-day), indicating rapid and exposure level-related oral absorption into the bloodstream. In the rat, inhalation exposures to atmospheric concentrations of 200, 400, or 600 ppm MIBK for 4 hours resulted in absorption of the same amount of MIBK as from the oral administration of 1.5, 3.0, or 6.0 mmol/kg, respectively. Concentrations of MIBK and its principal metabolite, 4-hydroxy-4-methyl-2-pentanone, in rat plasma, liver, and lung tissue were positively related to exposure level shortly after the last of 3 daily oral or inhalation exposures.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
The metabolic fate of MIBK, MIBC (4 -MPOL) and DAA (HMP) was investigated in Charles River CD-1 mice by measuring blood and brain concentrations
GLP compliance:
not specified
Radiolabelling:
no
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: ca. 4 week old
- Weight at study initiation: 21-27 g
- Fasting period before study: no data
- Housing: no data
- Individual metabolism cages: no
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 +/-1
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 0.25 mmol/ml
- Amount of vehicle (if gavage): 10 ml/kg
Duration and frequency of treatment / exposure:
single administration
Remarks:
Doses / Concentrations:
2.5 mmol HMP/kg
5 mmnol MIBK/kg
5 mmnol 4-MPOL/kg
No. of animals per sex per dose / concentration:
6
Control animals:
no
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled : blood, brain
- Time and frequency of sampling: 15, 30, 60 and 90 min after treatment
- From how many animals: (samples pooled or not)
- Method type(s) for identification : The concentrations of MiBK and their principal metabolites were analyzed by gas chromatography (GC). The apparatus used was a Hewlett-Packard Model 5830 A, equipped with FID and glass column (6 ft x 2 mm i.d. packed with Carbopack 0.1% SP-1000).
The column oven temperature was initially 100°C and increased to 200°C at a rate of 8°C/min. A final temperature of 200°C was maintained for 0.5 min to give a total cycle time of 13 min. The temperatures of the injection port and the detection port were 200°C and 250°C, respectively. The flow of carrier nitrogen gas was 20 ml/min, while that of hydrogen and air to the FID were 30 and 240 ml/min, respectively.
- Extraction procedures: A blood sample (0.2 ml) was added to 2 ml of methylene chloride containing 40,ug of 4-heptanol as an internal standard. The samples were mixed (Vortex) for 45 s and centrifuged at 645 x g for 15 min. The organic phase was retained for analysis and was reduced to 0.5 ml under a flow of nitrogen. The brains were weighed and homogenized in a glass homogenizer with 2 ml of methylene chloride containing 40 pg of 4-heptanol as internai standard. The homogenates were centrifuged at 645 x g for 15 min and the organic phase was retained for analysis. The recoveries for the MiBK and their principal metabolites added to the blood were greater than 90%.
- Limits of detection and quantification: no data
Metabolites identified:
yes
Details on metabolites:
MIBK is reduced to 4-MPOL (MIBC) as well as oxidized to HMP. The concentration of the reduced metabolite in the brain was twice that seen in the blood at the 15- and 30-min time intervals.
The administration of 4-MPOL (MIBC) results in the appearance of both MIBK and HMP in both blood and brain.
The administration of HMP did not result in the appearance of any biotransformation products.
Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
Executive summary:

The metabolic fate of 5 mmol/kg methyl isobutyl ketone (MIBK), 5 mmol/kg 4-methyl-2-pentanol (4 -MPOL, MIBC), 2.5 mmol/kg 4-hydroxymethyl-4-methyl-2-pentanone (HMP, DAA) was investigated in Charles River CD-1 mice by measuring blood and brain concentrations at 15, 30, 60 and 90 minutes following intraperitoneal injection (Granvil et al., 1994). MIBK is reduced to 4-MPOL as well as oxidized to HMP. The concentration of the reduced metabolite in the brain was twice that seen in the blood at the 15- and 30-min time intervals. The administration of 4-MPOL results in the appearance of both MiBK and HMP in both blood and brain. Administration of HMP resulted in levels from approximately 430, 300, 160 and 50 mg/ml blood and 430, 300, 150 and 60 mg/g brain at 15, 30, 60 and 90 minutes after dosing. No HMP metabolites were detected.

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Objective of study:
absorption
metabolism
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
yes
Remarks:
only 1 dose level tested
GLP compliance:
yes
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Source: Charles River Laboratories, L'Arbresle, France
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: mean body weight of 237 g (range: 226 to 250 g; first arrival) and mean body weight of 205 g (range: 199 to 214 g; second arrival).
- Fasting period before study: fasted for an overnight period of at least 14 hours
- Housing: 3 rats of the same group were housed in a suspended wire-mesh cage
- Individual metabolism cages: no
- Diet (e.g. ad libitum): A04 C pelleted maintenance diet, batch no. 00622 (UAR, Villemoisson, Epinay-sur-Orge, France), ad libitum
- Water (e.g. ad libitum): tap water (filtered with a 0.22 µm filter), ad libitum
- Acclimation period: 7 to 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hr of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Each test item was administered as a solution in the vehicle. Each test item was mixed with the required quantity of vehicle in order to achieve a concentration of 2.5 mmol/mL and then homogenized using a magnetic stirrer. The quantities of test items used for the dosage forms were calculated taking into consideration their respective molecular weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not provided
- Concentration in vehicle: 2.5 mmol/mL
- Amount of vehicle (if gavage): Not reported
- Lot/batch no. (if required): batch no. 89H0149 and 70K0127
- Purity: Not reported

HOMOGENEITY AND STABILITY OF TEST MATERIAL: Not reported
Duration and frequency of treatment / exposure:
Single dose exposure
Remarks:
Doses / Concentrations:
501 mg/kg body weight (5 mmol/kg body weight; 2.5 mmol/mL)
No. of animals per sex per dose / concentration:
Test 1: 9 males
Test 2: 3 males
Control animals:
no
Positive control reference chemical:
None used.
Details on study design:
- Dose selection rationale: The 5 mmol/kg (500 mg/kg) dose was selected for this study because it is in the range of doses (1.5/6 mmol/kg) used in
previous rat oral MIBK metabolism studies (Duguay and Plaa, 1995), and approaches the limit dose (1000 mg/kg) that would be used in any OECD guideline toxicity studies for MIBC.

- Rationale for animal assignment (if not random): The required number of animals was selected according to body weight and clinical condition and allocated to the groups, according to a computerized stratification procedure, so that the average body weight of each group was similar.
Details on dosing and sampling:
Test 1 (Study No. 20991 PAR):
PHARMACOKINETIC STUDY (Absorption and metabolism)
- Tissues and body fluids sampled: blood, plasma
- Time and frequency of sampling:
Blood samples from the first 3 animals/group were sampled at 0.125, 0.75, and 3 hours post-dosing
Blood samples from the second 3 animals/group were sampled at 0.25, 1, and 4.5 hours post-dosing.
Blood samples from the third 3 animals/group were sampled at 0.5, 1.5, and 6 hours post-dosing.

Test 2 (Study No. 22776 PAR):
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: blood, plasma
- Time and frequency of sampling: Sampled at 9 and 12 hours post-dosing
- From how many animals: 3 animals
- Method type(s) for identification: GC-MS
- Limits of detection and quantification: >0.005 mmol/L
- Other: Blood samples (1 mL per sampling) were taken into tubes containing lithium heparinate, from the orbital sinus of the animals. For the blood sampling, the animals were lightly anesthetized by isoflurane. The blood samples were kept on ice (+4 °C) until centrifugation to obtain plasma (4000 rpm for 10 min at +4°C). The plasma was stored frozen in individual tubes at -20 °C until analyzed for MIBK, methyl isobutyl carbinol (MIBC), and the common metabolite, 4-hydroxy-4-methyl-2-pentanone (HMP, which is also known as DAA).
Statistics:
Statistical analysis was not performed (not required).
Details on absorption:
Following oral administration of MIBK at a nominal dose-level of 5 mmol/kg to male rats, the mean (±SD) plasma parent material levels increased quickly from the first quantifiable time-point at 0.125 hours post-gavage to reach a Cmax (0.644 ± 0.221 mmol/L) at 0.25 hours, post-gavage. Thereafter, the plasma levels fell slowly to time-point 9 hours (0.0584 ± 0.0396 mmol/L) post-gavage, after passing through a second peak at time-point 3.0 hours post-gavage (0.550 ± 0.167 mmol/L).
Toxicokinetic parameters:
AUC: 3.558 mmol*h/L (MIBK)
Toxicokinetic parameters:
AUC: 13.756 mmol*h/L (HMP)
Toxicokinetic parameters:
AUC: 0.089 mmol*h/L (MIBC)
Toxicokinetic parameters:
Cmax: 0.644 mmol/L at 0.25 h (MIBK)
Toxicokinetic parameters:
Cmax: 2.030 mmol/L at 9.0 h (HPM)
Toxicokinetic parameters:
Cmax: 0.014 mmol/L (MIBC)
Toxicokinetic parameters:
half-life 1st: 2.529 h (MIBK)
Toxicokinetic parameters:
half-life 1st: 4.831 h (HMP)
Toxicokinetic parameters:
half-life 1st: 4.657 h (MIBC)
Metabolites identified:
yes
Details on metabolites:
methyl isobutyl carbinol (MIBC)
4-hydroxy-4-methyl-2-pentanone (HMP)

No mortality or morbidity was noted. No clinical signs were observed.

After administration of MIBK, the plasma primary metabolite (MIBC) levels rose slowly after gavage to a Cmax at time-point 1.0 hours (0.0118 ± 0.0046 mmol/L) and then declined slowly to time-point 9 hours (0.00610 ± 0.00043 mmol/L) post-dosing; again a secondary peak was noted at time-point 3.0 hours post-gavage (0.0143 ± 0.0001 mmol/L). The Cmax of parent MIBK (0.64 mmol/L) was reached at 0.25 hours, and levels were maintained until 3 hours then decreased with a half life of 2.5 hour. MIBK and its metabolite were no longer detected at the time-point 12 hours after dosing. The plasma secondary metabolite (HMP) levels, increased slowly after gavage to reach a Cmax at 9 hours (2.03 ± 0.07 mmol/L) post-dosing and then decline at the last time-point 12 hours after dosing (1.32 ± 0.45 mmol/L) with a half life of 3.8 hours. MIBK, as well as MIBC and HMP plasma levels showed a slight to considerable inter-animal variability. The area under the curve (AUC)(0 to12 h) for MIBK was 3.56, for MIBC was 0.09 and for HMP was 13.76 mmol*h/L. No other metabolites were present in the blood. 

Table 1: Selected Pharmacokinetic Parameters after Oral Administration of MIBK

Test Substance

Analyte

Cmax

[mmole/L]

Time of Cmax[hr]

Half Life [hr]

AUC0-12hr 

[mmole*hr/L]

% Total AUC

MIBK

   Total AUC

MIBC

0.014

NA

4.657

0.089

 0.05

MIBK

0.644

0.25

2.529

3.558

20

HMP

2.030

9

4.831

13.756

79

17.436

Conclusions:
MIBK is rapidly absorbed into the blood, and is metabolized to HMP, which is the major material in blood after 3 hours; metabolism of MIBK to MIBC is negligible.
Executive summary:

The absorption and metabolism of MIBK was studied in male Sprague-Dawley rats orally administered a single dose of 5 mmol/kg body weight of MIBK in corn oil, equivalent to 501 mg/kg body weight, by gavage. MIBK was rapidly absorbed into the systemic circulation following oral exposure, with a mean maximum plasma concentration (Cmax) of 0.644 mmol/L occurring at 0.25 hours [(time to maximum plasma concentration (tmax)] post-administration. MIBK was detected at very low levels (0.006 mmol/L) at 9 hours post-administration. The plasma levels of methyl isobutyl carbinol (MIBC) were very low (<0.012 mmol/L) all over the study. The major material in the blood was 4-hydroxymethyl-4-methyl-2-pentanone (referred to as HMP based on the chemical name) [i.e., diacetone alcohol (DAA], with a Cmax of 2.03 mmol/L at 9 hours and remained detectable at 12 hours post-dosing. Neither MIBK nor MIBC were detectable in 12-hour samples. No compounds other than HMP and MIBK were detected in the blood. The 12-hour area under the plasma concentration time curve (AUC0-12 h) for MIBK, MIBC and HMP were 0.089, 3.558 and 17, 436mmol·hour/L, respectively. HMP and MIBK represented 79% and 20% of the total AUC, respectively. The plasma elimination half-life (t1/2) of MIBK and HMP were 2.529 and 4.831hours, respectively. Based on the results of this study, MIBK is rapidly absorbed into the blood in rats following oral exposure and is rapidly and extensively metabolized to HMP (the major metabolite in blood after 3 hours).

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment (paper in Japanese)
Objective of study:
excretion
metabolism
Principles of method if other than guideline:
Rats were injected intraperitoneally with methyl isobutyl ketone (MIBK) in a single dose, and the amount of MIBK in the expired air and in the urine were studied
GLP compliance:
not specified
Radiolabelling:
no
Species:
rat
Strain:
Wistar
Sex:
not specified
Details on test animals or test system and environmental conditions:
Age about 7-10 weeks
Body weight 230 ± 20g
Route of administration:
intraperitoneal
Vehicle:
olive oil
Remarks:
Doses / Concentrations:
100, 200 and 300 mg/kg BW
No. of animals per sex per dose / concentration:
25
Control animals:
yes
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled:
blood: 1hour post exposure (200 mg/kg)
urine, exhalated air: 0-0.5, 0.5-1, 1-1.5, 1.5-2, 2-3, 3-4, 4-6, 6-9, 9-12, 12-18, 18-24 hours

METABOLITE CHARACTERISATION STUDIES
- Method type(s) for identification: head space GC-MS method. Breath was collected on charcoal tubes and then extracted with CS2. Urine was analysed by a combination of forced vaporization method with anhydrous sodium sulfate. 0.2ml bllod was taken from the external jugular vein 1 hour after the administration, filled with 2ml distilled water, sealed with teflon liner with a rubber stopper and an aluminum seal, after warming (30min) at 60 ¿, the 0.5 ml head space gas were quantified by GC.

TREATMENT FOR CLEAVAGE OF CONJUGATES: yes (ß- glucuronidase)
Type:
other: MIBK excretion in air
Results:
Cmax = 18.7 +/-5.8 mg/h at 0.-05 h after 200 mg/kg ip
Type:
other: MIBK excretion in air
Results:
t1/2 = 0.6 hour
Type:
other: MIBK total excretion in air
Results:
41.4 +/- 8.7% of the dose (200 mg/kg)
Type:
other: MIBK excretion rate in urine
Results:
maximum: 16.9 ± 3.3µg/hr 0-3 hours after administration of 200 mg/kg
Type:
other: MIBK total excretion in urine
Results:
0.19±0.11% of the dose of 200 mg/kg
Type:
other: 4M2P total excretion in urine
Results:
0.31 ± 0.18 % of the dose (200 mg/kg)
Type:
other: 4M2P t1/2 excretion in urine
Results:
3.2 hours
Details on excretion:
Excretion in air: see figures 2 and 4
Excretion in urine: see figures 3 and 5
Toxicokinetic parameters:
other: Blood MIBK concentration = 35 ± 2 µg/ml at 1 hour after dosing
Metabolites identified:
yes
Details on metabolites:
The gas chromatogram of urine ß- glucuronidase treated for three hours was seen is a match to 4-methyl-2-pentanol (4M2P) and 4-hydroxy-4-methyl-2-pentanone (4H4M2P, diacetone alcohol). 4H4M2P was detected in urine but not quantified.
Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
Executive summary:

Rats were injected intraperitoneally with 100 mg/kg, 200 mg/kg and 300 mg/kg of methyl isobutyl ketone (MIBK) in a single dose, and the amount of MIBK in the expired air and in the urine were studied. One of the metabolites was identified as 4-methyl-2-pentanol (4M2P) in the urine by gas chromatography-mass spectrometry. The concentration of MIBK in the exhaled air attained its maximum within 0.5 hour. Thereafter it decreased with a half life of 0.6 hour, and 41.1 ± 8.7 (m ± SD) % of the total amount injected was exhaled within 24 hours. The concentration of MIBK in the urine attained its maximum within 3 hours after injection. Then it decreased with a half life of 1.8 hours and 0.19 ± 0.11 % of the total amount administered was excreted in 18 hours. The concentration of 4-methyl-2-pentanol (4M2P) in the urine attained its maximum in 3-6 hour and decreased gradually thereafter. Its half life was 3.2 hours and 0.31 ± 0.18 % of the total amount was excreted in 12 hours.

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
distribution
Principles of method if other than guideline:
Determination of the distribution of MIBK between in rat and human blood in vitro
GLP compliance:
not specified
Radiolabelling:
no
Details on dosing and sampling:
Determination of MIBK partitioning between RBCs and plasma of rat blood in vitro:
Four rats (350-450 g) were each anesthetized with 1 ml of Nembutal sodium solution. Blood was drawn by cardiac puncture. After the blood samples were pooled and chilled. One-half milliliter of an ice-cold isotonic buffer solution saturated with MIBK was added to each blood sample. After gentle mixing, RBCs and plasma were separated. The contents of MIBK present in the RBCs and plasma were determined determined using a GC equipped with a flame ionization detector.

Determination of MIBK partitioning between RBCs and plasma of hunan blood in vitro:
Blood samples were collected from human volunteers. The samples were pooled and chilled and a solution of MIBK. RBCs and plasma were separated and extracted before the content of MIBK was determined.

Determination of MIBK distribution in plasma components in vitro:
Ice-cold aqueous solutions of MIBK were added to chilled human plasma. After mixing, samples were assayed for the initial concentration of the added solvent in the plasma and the other plasma samples were treated for proteins extraction. Both the supernatant and the precipitate were analysed of MIBK concentrations.

Determination of MIBK distribution in RBCs in vitro.
After removing plasma from chilled fresh human blood samples an ice-cold solution MIBK was added to RBCs. MIBK concentrations were determined in RBC membranes and hemoglobin.
Details on distribution in tissues:
In Vitro Distribution of MIBK between Plasma and RBCs in Rat Blood
Partitioning of MIBK between plasma (3.68+/-0.08 mg/10ml) and RBCs(3.82+/-0.06 mg/10ml) in vitro was roughly equal. In this experiment, aliquots of MIBK in aqueous solution were also added to the buffer solutions in the same volumes as those added to the 10-ml blood samples. The amount of MIBK in the aliquots was 7.29 ± 0.25 mg. If the recovery of each solvent from the buffer solution is taken as 100%, then recovery of the added solvents from RBC and plasma samples was in the range of 96.8-110%.

In Vitro Distribution of MIBK between Plasma and RBCs in Human Blood
Partitioning of MIBK between human plasma (2.34+/-0.08 mg/5ml) and RBCs(1.98+/-0.06 mg/5ml) in vitro was roughly equal.

Distribution of Volatile Organic Solvents in Human Plasma
A large fraction of MIBK was found in the precipitates (79.7%). The recovery of MIBK from plasma precipitates plus supernatants was 99% with the amount found in the plasma sample taken as 100%.
Metabolites identified:
no
Executive summary:

Lam et al. (1990) found that 49.4% of MIBK added to human blood in vitro at 0.8 mg/mL resided with RBCs. For human RBCs, 68% of MIBK was associated with hemoglobin. In human plasma, 80% of MIBK was associated with plasma proteins. Therefore, the majority of MIBK in human blood was associated with protein

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
distribution
Principles of method if other than guideline:
Determination of the distribution of MIBK between the plasma and RBCs of inhalation-exposed rats
GLP compliance:
not specified
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Spraguc-Dawley Farm (Houston, TX).
- Age at study initiation: no data
- Weight at study initiation: 300g
- Fasting period before study: no data
- Housing: in pairs in the vivarium at the Johnson Space Center
- Individual metabolism cages: no data
- Diet (ad libitum): Purina Formulab Chow No. 5008
- Water (ad libitum): tap water
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Vehicle:
other: air
Details on exposure:
Groups of four to five rats were exposed to MIBK in a 30-liter inhalation chamber. The vapor of MIBK was generated by bubbling nitrogen through the liquid solvent in an impinger, which was placed in a water bath (ambient temperature). The concentrated vapor was mixed with house air (10 liters/min) in a 2-liter erlenmyer flask; the diluted vapor was then fed into the chamber. After the chamber atmosphere reached a steady-state concentration of approximately 500 ppm for at least 0.5 hr, rats were placed through a small door into the chamber one at a time, 10 min apart. Chamber vapor concentrations were monitored by gas chromatography.
Duration and frequency of treatment / exposure:
Single 2-hour exposure
Remarks:
Doses / Concentrations:
512 +/- 37 ppm (analytical conc.)
No. of animals per sex per dose / concentration:
5
Control animals:
no
Details on dosing and sampling:
Determination of MIBK partitioning between RBCs and plasma in blood from exposed rats.
After a 2-hr exposure to MIBK each rat was decapitated and blood was collected immediately. RBC or plasma sample from rats exposed MIBK were extracted with methylene chloride. The concentrations of MIBK in the methylene chloride extracts were determined using a GC equipped with a flame ionization detector.
Details on distribution in tissues:
The blood solvent concentrations, which are equal to the sum of the concentrations in the RBCs and plasma, are shown in Table 1. The average percentage of MIBK in blood found in the RBCs was 51.2. The ratio of the amount of MIBK in RBCs to that in plasma was 1.0. The results of this inhalation study show that MIBK was carried in RBCs and plasma in approximately equal portions. It should be pointed out that the momentary drop in chamber concentration each time an animal was removed for blood collection might have contributed to the varying of blood concentrations in the exposed animals. However, the variation did not affect the partitioning of the solvent between plasma and RBCs.
Metabolites identified:
no

TABLE 1: DISTRIBUTION OF MIBK BETWEEN THE PLASMA AND RBCs OF INHALATION-EXPOSED RATS

Solvent

(ppm)

Rat

(code)

Plasma

(µg/ml)"

RBCs

(µg/ml)"

Plasma + RBCs

(µg/ml)

RBCs x100/RBCs + Plasma

MIBK

512 ± 37 ppm

MI

13.76

13.97

27.73

50.4

M2

13.09

14.40

27.49

52.4

M3

11.16

11.34

22.50

50.0

M4

11.10

12.50

23.60

52.9

M5

12.51

12.67

25.18

50.3

Average

25.30

51.2

Executive summary:

The distribution of MIBK in blood was studied by Lam et al. (1990). In rats exposed to MIBK at 512 ppm for 2 h, MIBK reached a concentration of 25.3µg/mL in blood with 51.2% distributed to red blood cells (RBCs) and the balance in plasma immediately after the exposure.

Description of key information

The low molecular weight (100.16 g/mol), log Pow (1.9), and water solubility along with the physical state (liquid) of methyl iso-butyl ketone (MIBK) favour its absorptionviavarious routes of exposure (oral, dermal, and inhalation). Consistent with this prediction, pharmacokinetic analysis of MIBK demonstrated that the compound was rapidly absorbed into the systemic circulation of rats following oral administration with maximum plasma concentrations occurring at 0.25 hours post-dosing. Signs of toxicity and systemic effects observed in experimental animals following acute oral and acute and repeated inhalation exposure to MIBK are also indicative of systemic absorptionviathese routes of exposure. Following absorption, MIBK was slowly eliminated from the plasma and remained detectable up to 9 hours post-dosing in rats, but not beyond 12 hours. Distribution of MIBK to tissues was demonstrated by the systemic effects in liver and kidneys in mice and rats after repeated inhalation exposure. In developmental toxicity studies, fetal effects were only observed at doses that caused maternal toxicity. Toxicological evidence of central nervous system effects suggests that MIBK may cross the blood-brain barrier in mice and rats. Metabolic data indicate that MIBK is rapidly and extensively metabolized to its major metabolite 4-hydroxymethyl-4-methyl-2-pentanone [i.e., diacetone alcohol (DAA)] and to a minor extent (0.05%) to methyl i-butyl carbinol (MIBC). Metabolism to DAA occursviaoxidation by the mixed function oxidase and metabolism to MIBC occursviareduction by alcohol dehydrogenase. Hydroxylation products of MIBK, such as MIBC and DAA, are expected either to be conjugated with sulfate or glucuronic acid and excreted in urine or to enter intermediary metabolism to be converted to carbon dioxide. Based on the available metabolic data and taking into consideration its low molecular weight and log Pow value, MIBK is not expected to bioaccumulate. The available data and the physicochemical properties of MIBK suggest that dermal absorption of liquid MIBK could be significant.

ABSORPTION

Gastrointestinal Absorption

The relative uptake of MIBK from the gastrointestinal tract has not been quantified in humans or in animals. Effects observed in laboratory animals following oral exposure to MIBK provide qualitative evidence that it is absorbed from the gastrointestinal tract in toxicologically relevant quantities.

The absorption and metabolism of MIBK was studied in male Sprague-Dawley rats orally administered a single dose of 5 mmol/kg body weight of MIBK in corn oil, equivalent to 501 mg/kg body weight, by gavage (Guillaumat, 2004). MIBK was rapidly absorbed into the systemic circulation following oral exposure, with a mean maximum plasma concentration (Cmax) of 0.644 mmol/L occurring at 0.25 hours [(time to maximum plasma concentration (tmax)] post-administration. MIBK was detected at very low levels (0.006 mmol/L) at 9 hours post-administration. The plasma levels of methyl isobutyl carbinol (MIBC) were very low (<0.012 mmol/L) all over the study. The major material in the blood was DAA with a Cmax of 2.03 mmol/L at 9 hours and remained detectable at 12 hours post-dosing. Neither MIBK nor MIBC were detectable in 12-hour samples. No compounds other than DAA and MIBK were detected in the blood. The 12-hour area under the plasma concentration time curve (AUC0-12 h) for MIBK, MIBC and DAA were 0.089, 3.558 and 17, 436 mmol·hour/L, respectively. DAAand MIBK represented 79% and 20% of the total AUC, respectively.The plasma elimination half-life (t1/2) of MIBK and DAA were 2.529 and4.831hours, respectively. Based on the results of this study, MIBK is rapidly absorbed into the blood in rats following oral exposure and is rapidly and extensively metabolized to DAA (the major metabolite in blood after 3 hours).

Plasma MIBK concentrations were 5.3, 8.4, and 16.1 µg/mL in rats at 1 hour after the last of 3 daily gavage exposures to 1.5, 3.0, and 6.0 mmol/kg MIBK (150, 300, or 601 mg/kg-day), indicating rapid and exposure level-related oral absorption into the bloodstream (Duguay and Plaa, 1993, 1995).

Respiratory Tract Absorption

MIBK vapor is absorbed relatively well in humans.Hjelm et al.(1990) measured the pulmonary retention of inhaled MIBK in eight men exposed to MIBK at 10, 100, or 200 mg/m3(2.4, 24, or 49 ppm) for 2 h by comparing the exhaled concentration with the inhaled concentration. Regardless of the exposure concentration, about 60% of the inhaled MIBK was retained by the body. The respiratory retention rate was fairly constant during the 2-h exposure. The blood concentration of MIBK rose quite rapidly, and no plateau was reached during the 2-h exposure.

Another study, however, shows that MIBK blood concentration can reach a plateau in 2 h. Dick et al. (1992) conducted a neurobehavioral study in which MIBK concentrations in the blood and the breath were measured in human volunteers exposed to MIBK at 88 ppm for 4 h. The mean concentrations in 13 men and 12 women combined are presented in the following Table. MIBK reached plateau concentrations in the blood and breath as early as 2 h into the exposure.

TABLE: Blood and Breath Concentrations in Volunteers Exposed to MIBK at 88 ppm (Dick et al. 1992)

2-h Exposure      4-h Exposure      90-min Post-Exp.       20-h Post-Exp.

MIBK in blood (µg/mL)

10.6                    10.5                     0.2                           NDa

MIBK in breath (ppm)

0.6                       0.6                      0.1                           ND

aND, below detection limit.

MIBK absorption is not as well studied in rodents as in humans. Duguay and Plaa (1993, 1995) measured the plasma concentration of MIBK given by inhalation to ratsin a study on MIBK's potentiation of the cholestatic effects of taurocholate or manganese bilirubin in rats. MIBK reached about 5, 8, or 14µg/mL in the plasma 1 h after a 4-h exposure of rats at 200, 400, or 600 ppm.

In rats, plasma MIBK concentrations were 5.0, 8.1, and 14.3 µg/mL immediately following the last of 3 daily 4-hour inhalation exposures to 200, 400, or 600 ppm MIBK (819, 1639, and 2458 mg/m3)1, indicating rapid and exposure level-related respiratory absorption into the bloodstream (Duguay and Plaa, 1995). In the rat, inhalation exposures to atmospheric concentrations of 200, 400, or 600 ppm MIBK for 4 hours resulted in absorption of the same amount of MIBK as from the oral administration of 1.5, 3.0, or 6.0 mmol/kg, respectively.

A study was performed to obtain blood samples from rats at selected time points following a single 6-hour exposure of the test atmosphere of MIBK at target concentration levels of 500 and 1000 ppm in order to determine the concentrations and the pharmacokinetics parameters of DAA, MIBK and MIBC from the plasma (Balazs, 2016). The animals were exposed to the test atmosphere (in the form of a vapour) using a nose-only exposure system. Analytical concentrations of 531 ppm, 1022 ppm were achieved in the respective groups. No control animals were used in the study. The test atmosphere concentration was monitored based on a validated GC method (by trapping the vapors in an impinger containing ethanol). The results of the test atmosphere characterization were considered suitable for the study purposes. Heighteen male Sprague-Dawley rats were involved in the study, 9 animals in each treatment groups. Plasma samples were prepared from male rats following the end of the exposure at nine time-points (three animals at each sampling occasion) and the plasma level of DAA, MIBK and MIBC was determined. The analytical method consisted of a liquid/liquid extraction of the three analytes from plasma with Methyl tert-Butyl Ether (MtBE) followed by analysis of supernatant by Gas Chromatography coupled with MS detection after electron impact ionization (GC-MS). The toxicokinetic evaluation was performed using non-compartmental analysis on Phoenix WinNonlin software (Pharsight Corporation, Mountain View, California 94040/USA). DAA, MIBC and MIBK toxicokinetic parameters were determined from the mean plasma concentration collected from each animal/group, at each post-exposure time-point. A separate analysis was performed for each dose-level. The following toxicokinetic parameters were calculated t½, lambdaz, AUC0.5-24h, Tmaxand Cmax.

The mean actual achieved concentration of the test item were:

Target Concentration

(ppm)* [mg/L]

Achieved Concentration

(ppm)* [mg/L]

Nominal Concentration

 mg/L

500 [2.05]

531 [2.18]

3.69

1000 [4.10]

1022 [4.19]

6.17

For the three analytes (DAA, MIBK and MIBC), the standard acceptance criteria for the calibration lines and Quality Control samples were met for a successful analysis of the study samples.

Study samples with concentrations above the calibration ranges were repeated diluted (for DAA principally and MIBK). DAA, MIBC and MIBK were not quantifiable in pre-dose samples in any group.

The mean plasma concentrations of DAA, MIBK and MIBC after an inhalation exposure to MIBK at 500 and 1000 ppm were:

Exposure

Analyte (µg/mL)

Sampling time

Pre-dose

0.5 h

1 h

1.5 h

3 h

4.5 h

6 h

8 h

18 h

24 h


MIBK

500 ppm

 

DAA

BLQ

41.7

44.3

45.1

43.1

29.0

15.8

12.9

0.559

BLQ

MIBK

BLQ

23.5

13.7

5.18

2.32

0.15

BLQ

BLQ

BLQ

BLQ

MIBC

BLQ

0.660

0.356

0.170

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ

MIBK
1000 ppm

 

DAA

BLQ

62.6

58.7

82.7

70.8

49.9

52.8

32.4

0.84

0.73

MIBK

BLQ

56.8

33.1

23.6

12.5

0.633

0.267

0.193

BLQ

BLQ

MIBC

BLQ

1.36

0.852

0.698

0.280

BLQ

BLQ

BLQ

BLQ

BLQ

BLQ: Below Limit of Quantification.

The toxicokinetic parameters of DAA, MIBC and MIBK after a single 6-hour inhalation exposure of male Sprague-Dawley rats to 500 ppm and 1000 ppm of MIBK were:

 

Concentration

Analytes

Number of point used to calculatelz

lz

t1/2

Tmax

Cmax

Cmax/
concentration#

AUC0.5-24h

AUC0.5-24h/
concentration#

Metabolite/
Administered compound

1/h

h

h

µg/mL

h*µg/mL

Cmax

AUC0.5-24h

500 ppm
(2.38 mg/L)

DAA

0.994

5

0.289

2.40

1.5

45.1

22.0

266

130

1.92

13.20

MIBC

na

na

nc

nc

0.5

0.66

0.322

0.499

0.243

0.0281

0.0248

MIBK

0.944

4

1.19

0.581

0.5

23.5

11.5

20.1

9.82

na

na

1000 ppm
(4.75 mg/L)

DAA

0.948

6

0.248

2.79

1.5

82.7

20.2

522

127

1.45

7.44

MIBC

0.995

3

0.569

1.22

0.5

1.36

0.331

1.82

0.445

0.0239

0.0260

MIBK

0.912

6

0.832

0.833

0.5

56.8

13.9

70.2

17.1

na

na

na: not applicable.

nc: not calculated as not enough quantifiable points were available.

#:Cmaxand AUC0.5-24hwere divided by the exposure concentrations of DAA (in mg/L).

After a 6-hour inhalation exposure to MIBK:

·        MIBK was on average quantifiable from 0.5 to 4.5 h after the end of exposure to 500 ppm and for up to 8 h at 1000 ppm. Maximum concentrations of MIBK were at 0.5 h after the end of exposure for both MIBK concentrations,

·        DAA was on average quantifiable from 0.5 to 18 h after the end of exposure to MIBK at 500 ppm and for up to 24 h at 1000 ppm. Maximum concentrations of DAA were at 1.5 h after the end of exposure for both MIBK concentrations,

·        MIBC plasma concentrations were very low but nevertheless quantifiable from 0.5 to 1.5 h after the end of exposure to 500 ppm of MIBK and for up to 3 h at 1000 ppm. Maximal plasma concentrations of MIBC were at 0.5 h after the end of exposure for both MIBK concentrations.

·        the elimination rates and half-live of DAA and MIBK were not significantly changed between both exposure concentrations, indicating that the excretion pathway was not saturated.

The plasma concentration values of DAA were higher than those of MIBC and MIBK after administration of MIBK, this indicates that DAA may represent a major metabolite of MIBK in rats. MIBC was considered to be a minor metabolite as it represented approximately 2.5% of the plasma MIBK exposure.

Based on AUC0-24hand Cmax(both normalized by exposure concentration), plasma DAA, MIBC and MIBK exposure increased dose proportionally, between 500 and 1000 ppm of MIBK.

Dermal absorption

Hjelm et al. (1991) evaluated the percutaneous absorption of MIBK using eight outbred female guinea pigs. lnitially, to determine blood clearance values, MIBK was infused into each animal at a rate of 0.478 µmol MIBK per minute, corresponding to 0.680 to 0.928 µmol/minute/kg body weight, for 30 min. The average blood clearance of MIBK was 201 ml/min/kg body weight. After a 2.5-h nontreatment period, the percutaneous absorption part of the study was begun. Hair on the back of each animal was clipped and epicutaneous exposure (150 min) was carried out by filling a glass cylinder, secured to the application site, with 1ml of MIBK. Arterial blood was analyzed for MIBK using gas chromatography. A maximum percutaneous uptake rate of 1.1 µmol/min/cm² was reached at 10 to 45 min after the initiation of exposure. A decrease in the uptake rate to 0.56 µmol/min/cm² was noted during the latter part of exposure (75 to 135 min after the initiation of exposure).

The penetration rate predicted from the solubility and the octanol-water partition coefficient (log P = 1.38) is 0.95 mg/cm2/hr ( Fiserova-Bergerova et al., 1990).

DISTRIBUTION

MIBK is likely to be widely distributed in the body because it is absorbed readily into the bloodstream after inhalation exposure (Hjelm et al., 1990). MIBK partitions approximately equally between red blood cells and plasma in rat and human blood; in plasma MIBK is associated primarily with proteins rather than being dissolved in plasma water (Lam et al., 1990). High lipid solubility indicates that MIBK may partition rapidly to lipid-rich tissues, such as nervous tissue.

Concentrations of MIBK and its principal metabolite, 4-hydroxy-4-methyl-2-pentanone, in rat plasma, liver, and lung tissue were positively related to exposure level shortly after the last of 3 daily oral or inhalation exposures (Duguay and Plaa, 1995) or after a single oral administration (Guillaumat, 2004; Gingell et al., 2003).

MIBK accumulated rapidly in brain tissue of mice that received a single intraperitoneal dose of 5 mmol MIBK/kg, peaking at 30 minutes post-exposure, but it was completely eliminated from the brain by 90 minutes post-exposure (Granvil et al., 1994). The brain concentration of 4-hydroxy-4-methyl-2-pentanone continued to increase throughout the 90-minute post-exposure period.

The distribution of MIBK in blood was studied by Lam et al. (1990). In rats exposed to MIBK at 512 ppm for 2 h, MIBK reached a concentration of 25.3µg/mL in blood with 51.2% distributed to red blood cells (RBCs) and the balance in plasma immediately after the exposure. Apparently, MIBK distributed similarly in human blood because Lam et al. (1990) found that 49.4% of MIBK added to human blood in vitro at 0.8 mg/mL resided with RBCs. For human RBCs, 68% of MIBK was associated with hemoglobin. In human plasma, 80% of MIBK was associated with plasma proteins. Therefore, the majority of MIBK in human blood was associated with proteins.

METABOLISM

Based on studies in rodents, MIBK is metabolized by either oxidation at the omega-1 carbon to form a hydroxylated ketone or reduction of the carbonyl group to form an alcohol.

Following a single dose of 5 mmol/kg bw of MIBK in corn oil, equivalent to 501 mg/kg body weight, by gavage to male Sprague-Dawley rats (Guillaumat, 2004; Gingell et al., 2003), the main metabolite, DAA, was detected in plasma shortly after oral exposure to MIBK. Plasma levels of DAA slowly increased to a Cmaxof 2.03 mmol/L 9 hours after dosing and remained detectable at 12 hours post-dosing; negligible levels of MIBC were also detectable in plasma after oral dosing of MIBK. Neither MIBK nor MIBC were detectable in 12-hour samples. No metabolite other than DAA and MIBC was detected in the blood. The plasma levels of MIBC were very low (<0.012 mmol/L) all over the study. The major material in the blood was DAA, with a Cmax of 2.03 mmol/L at 9 hours and remained detectable at 12 hours post-dosing. Neither MIBK nor MIBC were detectable in 12-hour samples. No compounds other than DAA and MIBK were detected in the blood. The 12-hour area under the plasma concentration time curve (AUC0-12 h) for MIBK, MIBC and DAA were 0.089, 3.558 and 17.436 mmol·hour/L, respectively. DAA and MIBK represented 79% and 20% of the total AUC, respectively. Based on the results of this study, MIBK is rapidly absorbed into the blood in rats following oral exposure and is rapidly and extensively metabolized to DAA (the major metabolite in blood after 3 hours).

DiVincenzo et al.(1976) identified 4-methyl-2-pentanol and 4-hydroxy-4-methyl-2-pentanone as MIBK metabolites in blood of guinea pigs.DiVincenzo et al. (1976) identified 4-hydroxy-4-methyl-2-pentanone and 4-methyl-2-pentanol as the MIBK metabolites in the serum of guinea pigs administered MIBK at 450 mg/kg intraperitoneally.

Duguay and Plaa (1993) could not detect 4-methyl-2-pentanol in the plasma of rats 1 h after a 4-h inhalation exposure to MIBK at 200 ppm, but 4-hydroxy-4-methyl-2-pentanone was found at 5µg/mL. However, both 4-hydroxy-4-methyl-2-pentanone (at about 6-7µg/mL) and 4-methyl-2-pentanol (at about 4-5µg/mL) were detected in the plasma of rats 1 h after a 4-h exposure to MIBK at 400 or 600 ppm (Duguay and Plaa 1993).

Hjelm et al.(1990) evaluated for these metabolites in the urine of human volunteers and found them both to be at concentrations below the detection limit of 5 nmol/L within a 3-hour post-exposure period. Blood levels of potential MIBK metabolites were not quantified in the Hjelm et al. (1990) study. Hjelm et al. (1990) suggested the source of the apparent discrepancy for why MIBK metabolites were detected in the blood of guinea pig but not in the urine of humans could be the lower dose of MIBK used in the Hjelm et al. (1990) study, or perhaps the qualitative and/or quantitative differences in metabolism of MIBK between man and guinea pig. In addition, the urinary excretion of the metabolites may be delayed and therefore the 3-hour post-exposure period may have been too short to permit detection of the metabolites in the urine.Hjelm et al.(1990) suggested that, in humans, 4-methyl-2-pentanol and 4-hydroxy-4-methyl-2-pentanone may either undergo further metabolism to be eliminated as CO2via the lungs or intermediate metabolites may be stored in tissues.

Vézina et al.(1990) found that either single or repeated oral doses of MIBK induced significant increases in hepatocellular cytochrome P-450 content and the hepatic activities of aniline hydroxylase and 7-ethoxycoumarin O-deethylase in rats, suggesting that the liver is involved in the metabolism of MIBK. Similarly, Brondeau et al. (1989) reported increased hepatic cytochrome P-450 content and glutathione-S-transferase activity in rats (but not mice) exposed once by inhalation to MIBK. Hepatic total cytochrome P-450 concentrations were significantly increased in New Zealand male rabbits treated orally with 5 mmol MIBK/kg daily for 3 days (Kobusch et al., 1987). Furthermore, the hepatic mixed-function oxidase activities for aminopyrine N-demethylation, 7-ethoxycoumarin dealkylation, and aniline hydroxylation were increased significantly.

ELIMINATION AND EXCRETION

After a single dose of 5 mmol/kg bw of MIBK in corn oil, equivalent to 501 mg/kg body weight, by gavage to male Sprague-Dawley rats (Guillaumat, 2004; Gingell et al., 2003), the half live of elimination of MIBK and 4-Hydroxy-4-methyl-2-pentanone from the blood were 2.5 and 4.8 hours respectively. Only a trace amount of a second metabolite, 4-methyl-2-pentanol, was detected at any time.

The half-life of MIBK in the serum of guinea pigs administered a single 450 mg/kg intraperitoneal dose was estimated to be 66 minutes, based on single blood samples collected from different guinea pigs at intervals up to 16 hours post-dosing (DiVincenzo et al., 1976). 4-Hydroxy-4-methyl-2-pentanone was cleared from the blood within 16 hours, and only a trace amount of a second metabolite, 4-methyl-2-pentanol, was detected at any time.

MIBK was completely eliminated from the blood of mice within 90 minutes of injection of a single intraperitoneal dose of 5 mmol/kg (Granvil et al., 1994); the blood concentration of 4-hydroxy-4-methyl-2-pentanone peaked at 60 minutes post-dosing and was decreasing at the termination of the study at 90 minutes post-dosing.

In humans, elimination of MIBK from blood following cessation of a 2-hour inhalation exposure with light exercise was biphasic, with a half-life of 11 to 13 minutes during the first 30 minutes post-exposure in subjects exposed to 100 or 200 mg/m3(Hjelm et al., 1990). The half-life in blood during the second elimination phase (60 and 180 minutes post-exposure) was 59 and 74 minutes in subjects exposed to 100 and 200 mg/m3, respectively. Blood MIBK levels were too low to permit the calculation of blood elimination half-times in subjects exposed to 10 mg/m3.In the eight men studied by Hjelm et al. (1990), about 0.04% of the MIBK dose was excreted in the urine as MIBK within 3 h after a 2-h inhalation exposure to MIBK at 10, 100, or 200 mg/m3(2.4, 24, or 49 ppm). The urinary concentrations of MIBK's metabolites, 4-methyl-2-pentanol and DAA, were below the detection limit of 5 nmol/L at 0.5 or 3 h post-exposure. The total body clearance of MIBK was 1.6 L of blood per hour per kilogram of body weight in these men (Hjelm et al. 1990). However, in another study, Hjelm et al. (1991) found that the total body clearance was 12 L of blood per hour per kilogram of body weight in guinea pigs infused with MIBK intravenously. The reason for the large difference in MIBK's total body clearance between men and guinea pigs is unknown.

Hirota (1991) studied the elimination of MIBK in rats exposed intraperitoneally at 100-300 mg/kg. The major route of MIBK elimination was exhalation via the lungs, which accounted for 41% of the dose. The concentration of MIBK in the exhaled air declined with a half-life of 0.6 h after reaching a maximum at 0.5 h after the injection. Two minor routes of MIBK elimination were urinary excretion of MIBK and 4-methyl-2-pentanol. MIBK in the urine attained a maximum concentration within 3 h of the injection and then it declined with a half-life of 1.8 h. The concentration of 4-methyl-2-pentanol reached its peak within 3-6 h of the injection and then decreased with a half-life of 3.2 h.

Additional references:

Brondeau, M.T., M. Ban, P. Bonnet, J.P. Guenier, and J. deCeaurriz. (1989) Acetone compared to other ketones in modifying the hepatotoxicity of inhaled 1,2-dichlorobenzene in rats and mice. Toxicol Letters, 49, 69–78.

Kobusch, A.B., B. Bailey, and P. du Souich. (1987) Enzyme induction by environmental agents: effect on drug kinetics. In: Plaa, G.L., P. du Souich, and S. Erill, eds. Interactions Between Drugs and Chemicals in Industrial Societies. Amsterdam: Elsevier Science Publishers, pp. 29–42.

Sato, A. and T. Nakajima. (1979) Partition coefficients of some aromatic hydrocarbons and ketones in water, blood and oil. Brit J Ind Med, 36, 231–234.

Vézina, M., and G.L. Plaa. (1987) Potentiation by methyl isobutyl ketone of the cholestasis induced in rats by a manganese-bilirubin combination or manganese alone. Toxicol Appl Pharmacol, 91, 477–483.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

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