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Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The purpose of the study was to investigate the potentiating properties of MIBK, 4MPOL, and 4-0HMIBK on the hepatotoxicity induced in rats by CHCl3. The inducing properties of MIBK were also assessed by measuring the total amount of cytochrome P-450; the rate of oxidation of aminopyrine, aniline, and 7-ethoxycoumarin; and the electrophoretic pattern of microsomal proteins probably related to cytochrome P-450.
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
other: metabolic interaction
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
Male Sprague-Dawley rats (Charles River Canada, lnc., St-Constant, Qué.) weighing 225-300 g were maintained on Charles River Rat Chow and water ad libitum. They were housed in the animal quarters for a period of acclimation of 3 days before the initiation of the experiment. Each experimental unit (six rats each) was kept in a stainless-steel wire-mesh suspended cage and a 12-h light-dark cycle was enforced.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Amount of vehicle (if gavage): 10 ml/kg
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Single
No. of animals per sex per dose:
6-10
Control animals:
yes, concurrent vehicle
Details on study design:
In a first series of experiments, the effect of either MIBK, 4MPOL, or 4-OHMIBK at a dosage of 3.75, 5.60, or 7.5 mmol/kg was assessed on CHCl3-induced liver injury. Twenty-four hours following the pretreatment, the animals were challenged ip with either corn oil or CHCl3 (0.5 mL/kg) dissolved in corn oil to give 10 mL/kg. Twenty-four hours after the challenge, the animals were weighed and anesthetized with ether; blood was removed via the abdominal aorta with heparin as anticoagulant. The liver was rapidly excised and weighed, and one section of the lateral median lobe was processed for histological examination.
In a second series, the inducing properties of MIBK on different cytochrome P-450 isozymes were studied. Groups of six to eight rats received by gavage single dosages of 1.5, 5.0, 7.5, 15.0, or 30.0 mmol/kg MIBK dissolved in corn oil. Two other groups of six to eight rats received 5.0 or 7.5 mmol/kg of MIBK every 24 h for 5 days. Control rats (n = 10) received 10 mL/kg of corn oil. Twenty-four hours following the last administration of MIBK, the animals were killed by decapitation. The liver was removed and microsomes prepared.
In a third series, the inducing properties of MIBK were assessed by conducting a gel electrophoresis on liver proteins. MIBK was administered by gavage at a dosage of 30 mmol/kg; control animals received corn oil only (10 mL/kg). The animals were killed 24 h following the single treatment with either MIBK or corn oil. The liver was removed and microsomes prepared.
Examinations:
Biochemical analyses
Liver injury was assessed by alanine aminotransferase (ALT) and ornithine carbamoyl transferase (OCT) plasma activities. Total plasma bilirubin and relative liver weight were also considered as indices of hepatotoxicity.

Morphological analyses .
Immediately after the animal was killed, the right lateral median lobe of the liver was removed and immersed in a 10% buffered Formalin fixative, dehydrated, and embedded in paraffin. Corona! sections (5 µm) from paraffin-embedded tissue were cut and stained with hematoxylin-eosin. The pattern of the lesion was assessed by a semi quantitative procedure.

Liver microsomes assay
Protein content, total cytochrome P-450, aminopyrine N-demethylation, 0-dealkylation of 7-ethoxycoumarin and hydroxylation of aniline were determined in the microsomal preparation.

Separation of microsomal proteins
Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS- PAGE) of solubilized microsomal preparations was carried out.
Details on results:
MIBK and both metabolites significantly increased the liver damage induced by CHCl3, as demonstrated by the elevation of the plasma activity of alanine aminotransferase and ornithine carbamoyl transferase and by the severity of the morphological changes. The minimally effective dosage needed to potentiate CHCl3-induced hepatotoxicity was approximately 5 mmol/kg for the three compounds.
Cytochrome P-450 content and the oxidation of aniline and 7-ethoxycoumarin were significantly increased with either a single (7.5 mmol/kg or greater) or a multiple (5.0 and 7.5 mmol.kg-1.day-1 for 5 days) administration of MIBK. An increase in the activity of the aminopyrine demethylase was also elicited by the repetitive administration of MIBK. MIBK significantly increased the 52.1- and 54.1-kDa proteins, corresponding most probably to P-450 isozymes.

The effect of corn oil, MIBK, 4MPOL, or 4-OHMIBK at dosages of 3.75, 5.6, or 7.5 mmol/kg prior to the administration of a CHCl3 challenge on various hepatic parameters appears in Table l . CHCl3 -induced liver injury was enhanced by a pretreatment of MIBK, 4MPOL, or 4-OHMIBK.

Graphical representation of the morphological patterns of the liver injury is presented in Fig. 2.

The ability of MIBK to increase the total amount of cytochrome P-450 and to induce the activity of aniline hydroxylase, ethoxycoumarin 0-deethylase, and aminopyrine N-demethylase was assessed in rats 24 h after a single treatment or after the last of a 5-day treatment regimen as shown in Table 2.

The effect of MIBK on microsomal proteins as determined by gel electrophoresis is summarized in Table 3.

Conclusions:
Not only the MIBK, but also its two metabolites, are potentiators of the liver toxicity induced by CHCl3. The ability of MIBK to potentiate haloalkane hepatotoxicity appears to be related to its capacity to induce cytochrome P-450. More than one form of cytochrome P-450 appears to be implicated in the phenomenon.
Executive summary:

The hepatonecrogenic properties of chloroform (CHCl3) can be modified by the administration of various chemicals. The ability of methyl isobutyl ketone (MIBK) and its two major metabolites, 4-methyl-2-pentanol (4MPOL) and 4-hydroxymethyl isobutyl ketone (4-OHMIBK) to potentiate the liver injury induced by CHCl3 was assessed in rats. The parent compound and both metabolites significantly increased the liver damage induced by CHCl3, as demonstrated by the elevation of the plasma activity of two transferases alanine aminotransferase and ornithine carbamoyl transferase and by the severity of the morphological changes. Moreover, the minimally effective dosage needed to potentiate CHCl3-induced hepatotoxicity was approximately 5 mmol/kg for the three compounds. We also studied the inducing properties of MIBK (cytochrome P-450 liver content and the activity of aniline hydroxylase, 7-ethoxycoumarin O-deethylase, and aminopyrine N-demethylase). Cytochrome P-450 content and the oxidation of aniline and 7-ethoxycoumarin were significantly increased with either a single (7.5 mmol/kg or greater) or a multiple (5.0 and 7.5 mmol.kg-1.day-1 for 5 days) administration of MIBK. An increase in the activity of the aminopyrine demethylase was also elicited by the repetitive administration of MIBK. With gel electrophoresis, we found that MIBK significantly increased the 52.1- and 54.1-kDa proteins, corresponding most probably to P-450 isozymes.

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented.
Qualifier:
no guideline available
Principles of method if other than guideline:
Male B6C3F1 mice, six control and six treated, were exposed to target concentrations of 0 or 1800 ppm MIBK vapors via inhalation (whole-body) for six hours/day for 7 consecutive days. Liver-specific clinical chemistry, histopathology, targeted gene expression, cytochrome P450 enzymatic activity, and hepatocellular proliferation were measured. Animals were sacrificed on the morning following their final exposure.
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
carcinogenicity
Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc. (Portage, Michigan)
- Age at study initiation: 8 weeks
- Fasting period before study: None
- Housing: One per cage in stainless steel cages with wire mesh floors and were suspended above absorben paper.
- Diet (e.g. ad libitum): LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form provided ad libitum
- Water (e.g. ad libitum): Obtained from the municipal water source provided ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1
- Humidity (%): 40-70
- Air changes (per hr): 12 - 15
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass Rochester-type whole-body exposure chambers
- Method of holding animals in test chamber: animals were housed singly
- Source and rate of air: Chamber airflow maintained at 12-15 air changes per hour
- Method of conditioning air: Not reported
- System of generating particulates/aerosols: J-tube method
- Temperature, humidity, pressure in air chamber: 22+/- 2 °C, 40-60%, and pressure not reported
- Air flow rate: Not reported
- Air change rate: 12-15 per hour
- Method of particle size determination: Miran 1A infrared spectrophotometer
- Treatment of exhaust air: Not reported


TEST ATMOSPHERE
- Brief description of analytical method used: The various vapor concentrations of MIBK were generated using a glass J-tube method (Miller et al., 1980). Liquid test material was pumped into the glass J-tube assembly and vaporized by nitrogen gas passing through the bead bed of the glass J-tube (20 liters per minute). The nitrogen was heated with a flameless heat torch (FHT-4, Master Appliance Corporation, Racine, Wisconsin) to the minimum extent necessary to vaporize the test material. The generation system was electrically grounded and the J-tubes were changed as needed. The nitrogen carrier gas with vaporized test material was mixed and diluted with filtered supply air to achieve the desired test chamber concentration. Animals in the control group were exposed to the same volume percent of nitrogen as the MIBK exposed animals.

- Samples taken from breathing zone: yes


VEHICLE (if applicable)
-Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chamber concentrations of MIBK, measured approximately in the center of the breathing zone of the animals, was determined at least once per hour with a Miran 1A infrared (IR) spectrophotometer (Foxboro/Wilks, South Norwalk, Connecticut). The IR spectrophotometer was calibrated and a standard curve compiled prior to the start of the study, using air standards prepared by vaporizing measured volumes of MIBK into Tedlar® sample bags (Series 233, SKC, Eighty Four, Pennsylvania) along with metered volumes of filtered air. The analytical concentration during the exposure was interpolated by the CAMILE TG Data Acquisition and Control System using the standard curve. The analytical system was checked prior to and after each exposure with a MIBK standard gas-bag of known concentration. Output from the infrared spectrophotometer was monitored by the CAMILE Data Acquisition and Control System, displayed, logged to memory, and printed for inclusion in the study file. Prior to exposure of animals, the distribution of vaporized test material was determined from five sample points in the breathing zone and the normal sampling point (reference point) in the chamber in order to ensure that a uniform distribution of vapor is present throughout the breathing zone of the animals. The daily nominal concentration of the test material was calculated based on the amount of test material used divided by the total airflow through the chamber each day.
Duration of treatment / exposure:
7 days
Frequency of treatment:
6 hours/day
Post exposure period:
None
Remarks:
Doses / Concentrations:
1800 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
6
Control animals:
yes, sham-exposed
Examinations:
Body weight/body weight gains; Cage-side clinical observations; Clinical chemistry (at necropsy); Liver weights; Targeted gene expression analysis of the upper third of the left liver lobe; Histopathological examination and BrdU proliferation analysis of middle third of the left liver lobe; Sample of duodenum as a positive control for proliferation analysis; Enzyme activity analysis (lower portion of the left lobe and the medial lobe of the liver).
Positive control:
None
Details on results:
Treatment-related findings included very slight hepatocytes hypertrophy with increased cytoplasic eosinophilia in the centrilobular/midzonal regions of the hepatic lobule which were consistent with increased smooth endoplasmic reticulum and induction of cytochrome P450 enzymes. CYP2B10transcript levels increased 4-fold andCYP4A10decreased 5.56-fold. This was verified by increased CYP2B10 enzyme activity (PROD) and hepatocyte proliferation. These responses are commonly observed following activation of constitutive androstane receptor (CAR) and indicate that MIBK may be an agonist ligand for CAR in mice and share a similar mode of action to that of Phenobarbital in mice. The study authors noted that this mechanism of action is not relevant to humans.

No treatment related effects were observed for clinical signs, body weights, liver weights, or clinical chemistry measurements in MIBK exposed animals. Treatment-related histopathologic changes in the liver consisted of very slight hepatocyte hypertrophy (enlargement) with altered tinctorial properties (increased cytoplasmic eosinophilia) in the centrilobular/midzonal regions of the hepatic lobule. These changes were consistent with possible increased smooth endoplasmic reticulum and induction of cytochrome P450 enzymes. Significant transcript level alterations were observed for CYP2B10 (increased 4-fold) and CYP4A10 gene expression (decreased 5.56-fold). No changes were observed in CYP1A1 or CYP3A11 gene expression levels. Significant inductions of CYP2B10 enzyme activity (PROD) and hepatocellular proliferation were also observed. These responses are commonly observed following activation of constitutive androstane receptor (CAR) and are typical for phenobarbital (PB)-like compounds. Together with literature observations, these findings indicate that MIBK may be an agonist ligand for CAR and share a similar mode of action to that of phenobarbital.

Executive summary:

In a non-guideline and non-GLP study, the effects of methyl isobutyl ketone (MIBK) on the mouse liver were evaluated (Geter, 2009). Male B6C3F1 mice were implanted with 5-bromo-2’deoxyuridine (BrdU) pumps and then exposed to 0 or 1800 ppm (n=6/group) of MIBK via whole-body inhalation for 6 hours/day for 7 days. In-life assessments included clinical signs and body weights. Mice were euthanized and assessed for clinical chemistry, gene expression analysis of the upper third of the left liver lobe, liver histopathological examination and BrdU proliferation analysis, and liver enzyme activity. There were no treatment-related effects noted for clinical signs, body weights, liver weights, or clinical chemistry assessments. Treatment-related findings included very slight hepatocytes hypertrophy with increased cytoplasic eosinophilia in the centrilobular/midzonal regions of the hepatic lobule which were consistent with increased smooth endoplasmic reticulum and induction of cytochrome P450 enzymes. CYP2B10transcript levels increased 4-fold andCYP4A10decreased 5.56-fold. This was verified by increased CYP2B10 enzyme activity (PROD) and hepatocyte proliferation. These responses are commonly observed following activation of constitutive androstane receptor (CAR) and indicate that MIBK may be an agonist ligand for CAR in mice and share a similar mode of action to that of Phenobarbital in mice. The study authors noted that this mechanism of action is not relevant to humans.

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Abstract
Type of method:
in vivo
Endpoint addressed:
carcinogenicity
Species:
mouse
Strain:
other: B6C3F1, C57BL/6, and Car/Pxr Knockout (KO)
Sex:
male/female
Duration of treatment / exposure:
10 days
Frequency of treatment:
6 h/d, 5 d/w
Post exposure period:
1-3 hours
Remarks:
Doses / Concentrations:
1800 ppm
Basis:

Control animals:
yes, sham-exposed
Examinations:
On day 1, mice were implanted with osmotic mini-pumps containing 5-Bromo-2-deoxyuridine (BrdU) 1 h following exposure and humanely euthanized 1 to 3 h following the final exposure.
Details on results:
B6C3F1 and FC57BL/6 mice had statistically significant increases in liver weights compared to controls that corresponded with hepatocellular hypertrophy and increased mitotic figures. Hepatocellular proliferation data indicated induction of S-phase DNA synthesis in B6C3F1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to control, no increase was observed in MIBK exposed CAR/PXR KO mice. Liver gene expression changes indicated a maximally-induced Cyp2b10 (CAR-associated) transcript and a slight increase in Cyp3a11(PXR-associated) transcript in B6C3F1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to controls, but not in Cyp1a1 (AhR associated) or Cyp4a10 (PPAR-a-associated) transcripts. CAR/PXR KO mice showed no evidence of activation of AhR, CAR, PXR or PPAR-a nuclear receptors via their associated transcripts.
Conclusions:
MIBK induced hepatic effects are consistent with a phenobarbital-like MOA where the initiating events are activation of the CAR and PXR nuclear receptors leading to rodent liver tumours.
Executive summary:

Methyl isobutyl ketone (MIBK) is widely used in the coatings industry. A 2007 NTP study identified that MIBK induces liver tumors in B6C3F1 male (M) and female (F) mice; however, MIBK lacks genotoxicity. Previous studies suggested that the mode of action (MOA) by which MIBK induces hepatocellular tumors in mice is through phenobarbital-like nuclear receptor associated activation. To further investigate the MOA for MIBK-induced murine liver tumors, M and F B6C3F1, C57BL/6, and CAR/PXR Knockout (KO) mice were exposed to either 0 or 1800 ppm MIBK for 6 h/d, 5 d/w for a total of 10 days. On day 1, mice were implanted with osmotic mini-pumps containing 5-Bromo-2-deoxyuridine (BrdU) 1 h following exposure and humanely euthanized 1 to 3 h following the final exposure. B6C3F1 and FC57BL/6 mice had statistically significant increases in liver weights compared to controls that corresponded with hepatocellular hypertrophy and increased mitotic figures. Hepatocellular proliferation data indicated induction of S-phase DNA synthesis in B6C3F1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to control, no increase was observed in MIBK exposed CAR/PXR KO mice. Liver gene expression changes indicated a maximally-induced Cyp2b10 (CAR-associated) transcript and a slight increase in Cyp3a11(PXR-associated) transcript in B6C3F1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to controls, but not in Cyp1a1 (AhR associated) or Cyp4a10 (PPAR-a-associated) transcripts. CAR/PXR KO mice showed no evidence of activation of AhR, CAR, PXR or PPAR-a nuclear receptors via their associated transcripts. MIBK induced hepatic effects are consistent with a phenobarbital-like MOA where the initiating events are activation of the CAR and PXR nuclear receptors leading to rodent liver tumors.

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Not stated (published study)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
The authors report the results of a number of a comparative investigation in wild-type and CAR/PXR-knockout mice following exposure by inhalation to 1800 ppm MIBK for 10 days. Parameters investigated included liver weights, liver histopathology, gene expression, and the induction of DNA synthesis.
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
carcinogenicity
Specific details on test material used for the study:
MIBK was provided by Dow and was shown by GC-MS/NMR to be 99.5% purity
Species:
mouse
Strain:
other: The study was performed in B6C3F1 mice (the strain used in the NTP carcinogenicity study); 'wild-type' C57BL/6 mice (the strain used to derive the KO mice; and C57BL/6 CAR/PXR knockout mice.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic (USA)
- Age at study initiation: 7-9 weeks
- Weight at study initiation: Not reported
- Fasting period before study: None
- Housing: Not reported
- Diet: ad libitum (NTP-2000)
- Water: ad libitum
Acclimation: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 30-70
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12/12
- Housing: group housed
Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
Mice were exposed via whole-body inhalation to MIBK vapour (0 or 1800 ppm) for 6 hours/day, five days/week for two weeks (total 10 exposures). Mice were exposed (whole body) in 1 m3 stainless steel and glass chambers. Airflow through the chamber was maintained at a flow rate of approximately 250 L/min, providing 15 air changes per hour. Chamber concentrations were generated by metering MIBK via a liquid pump into a stainless steel J-tube generator
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of MIBK were analysed using GC-FID. Samples were taken from ten positions inthe exposure chamber during the exposure period.
Duration of treatment / exposure:
6 hours
Frequency of treatment:
5 days/week for two weeks
Post exposure period:
None
Dose / conc.:
0 ppm
Remarks:
Control: measured MIBK concentration 0 +/ 0 ppm (males and females)
Dose / conc.:
1 800 ppm
Remarks:
Nominal MIBK vapour concentration. Measured concentration 1800 +/- 16 ppm (males); 1800 +/- 17 ppm (females)
No. of animals per sex per dose:
B6C3F1 mice: 8/sex/group
C57BL/6 mice: 8/sex/group
CAR/PXR KO mice: 8 males, 5-6 females/group
Control animals:
yes, sham-exposed
Details on study design:
Mice were subcutaneously implanted with BrdU-osmotic mini-pumps at least 1 hour after day 1 of the initial exposure. Mice were terminated 1-1.5 hours following the final exposure.
Examinations:
Terminal blood samples were taken for the assessment of clinical chemistry parameters (AST, ALT, ALP, GGT, total bilirubin, total protein, albumin, globulin, A:G ratio, triglycerides and cholesterol). Liver weights were recorded. The liver (left lobe) and duodenum (control tissue for BrdU incorporation) were assessed histopathologically. Liver S-phase DNA synthesis was assessed by BrdU incorporation, measured using monoclonal anti-BrdU antibodies. The number of BrdU-positive nuclei was reported for 1000 hepatocytes in each lobular zone. RNA was extracted from liver samples and screened for expression of Cyp1A1 (AhR response), Cyb2b10 (CAR response), Cyp3a11 (PXR response) and Cyp4a10 (PPAR-alpha response). Beta-actin was used as a housekeeping gene.
Positive control:
None
Details on results:
Body weight, liver weight:
Mean terminal body weights of all groups mice were unaffected by exposure to 1800 ppm MIBK. Mean relative liver weights were increased in exposed groups of mice, relative to the respective control group. Values attained statistical significance, with the exception of male wild-type (WT) C57BL/6 mice.

Liver histopathology:
Histopathology of the liver in WT mice of both strains showed findings characterised by centrilobular/midzonal hepatocyte hypertrophy and altered staining; increased mitotic figures; and vacuolisation consistent with fatty change. CAR/PXR KO mice showed a decreased response, limited to very slight centrilobular/midzonal hepatocyte hypertrophy, and vacuolisation, with no evidence of mitosis.

Clinical chemistry:
Changes in clinical chemistry parameters related to MIBK exposure were limited to increased serum ALT activity, reported for WT B6C3F1 mice (M, F); WT C57BL/6 mice (F); and C57BL/6 KO mice (M). Other statistically significant changes observed in female WT mice were not considered to be related to MIBK exposure.

Gene expression:
Both sexes of both WT mouse strains exposed to MIBK showed changes in gene expression characteristic of CAR/PXR activation. Notably, in male WT mice, transcription levels of Cyp2b10 (a marker of CAR activation) were 982 and 599 times higher than the respective controls in B6C3F1 and C57BL/9, respectively. There was no response in WT mice of either strain indicative of AhR or PPAR-alpha activation. In WT mice of both sexes, mRNA transcription levels associated with CAR, PXR, AhR and PPAR-alpha receptor activation were essentially unchanged relative to the control group.

BrdU incorporation:
A significant increase in hepatocyte staining intensity was noted in the centrilobular, midzonal and periportal zones of male WT mice of both strains exposed to MIBK. A similar statistically significant increase in staining intensity was seen for female B6C3F1 mice; the response in female C57BL/6 mice was of less intensity and did not attain statistical significance. No increase in staining intensity was seen in any hepatolobular zone in CAR/PXR KO mice of either sex.




Summary of histopathological findings

















































































































 



WT



KO



B6C3F1



C57BL/6



C57BL/6



M



F



M



F



M



F



0



1800



0



1800



0



1800



0



1800



0



1800



0



1800



N (#)



8



8



8



8



8



8



8



8



8



8



5



6



Hypertrophy (very slight)



0



0



0



0



0



0



0



0



0



8*



0



5*



Hypertrophy (slight)



0



8*



0



8*



0



8*



0



8*



0



0



0



0



Increased mitosis



2



5*



1



5*



0



5*



1



5*



0



0



0



1



Vacuolisation



0



2



0



0



0



1



0



1



0



4*



0



5*



*significantly different to controls (p<0.05)


 


Summary of clinical chemistry findings










































































































































































































































Parameter



Exposure



WT



KO



B6C3F1



C57BL/6



C57BL/6



M



F



M



F



M



F



AST (U/L)



0 ppm



61



104



125



132



90



145



1800 ppm



80



81



102



124



116



180



ALT (U/L)



0 ppm



46



52



53



53



47



65



1800 ppm



79*



80*



67



89*



62*



76



ALP (U/L)



0 ppm



122



130



101



150



83



79



1800 ppm



106*



122



107



143



96*



133*



GGT (U/L)



0 ppm



3



8



6



6



3



5



1800 ppm



6



5



4



4



4



4



TBil (mg/dL)



0 ppm



0.1



0.2



0.1



0.2



0.1



0.2



1800 ppm



0.1



0.1



0.2



0.1*



0.2



0.3



TProt (g/dL)



0 ppm



5.2



5.4



5.1



5.0



5.2



5.0



1800 ppm



5.3



5.3



5.3*



5.3*



5.4



5.4*



Alb (g/dL)



0 ppm



4.1



4.7



4.0



4.5



4.5



4.9*



1800 ppm



4.2



4.5



4.3*



4.5



4.5



4.9*



Glob (g/dL)



0 ppm



1.1



0.7



1.1



0.5



1.0



0.6



1800 ppm



1.2



0.9*



1.0



0.7*



1.0



0.6



A:G ratio



0 ppm



3.6



6.9



3.8



9.3



4.3



4.8



1800 ppm



3.5



5.5*



4.3



6.4*



5.2



11.8



Trig (mg/dL)



0 ppm



66



48



58



51



62



57



1800 ppm



74



62*



71



62*



51



43



Chol (mg/dL)



0 ppm



118



96



97



83



88



65



1800 ppm



128



123*



116*



99*



85



70



*significantly different to controls (p<0.05)


 


Summary of effects on gene expression



















































































Strain



Exposure



Relative gene expression



Cyp 1a1 (AhR)



Cyp2b10 (CAR)



Cyp3a11 (PXR)



Cyp4a10 (PPAR-alpha)



WT B6C3F1



Control



1



1



1



1



1800 ppm (M)



1.3



981.9*



2.3*



1.2



1800 ppm (F)



1.7



234.8*



4.3*



0.6



WT C57BL/6



Control



1



1



1



1



1800 ppm (M)



1.8



599.2*



2.2*



0.8



1800 ppm (F)



2.7



269.8*



4.8*



0.6



C57BL/6 CAR/PXR KO



Control



1



1



1



1



1800 ppm (M)



0.9



0.9



1.3



2.2



1800 ppm (F)



0.8



2.2



1.3



3.6



*significantly different to controls (p<0.05)


 

Conclusions:
The results of this study indicate activation of the CAR/PXR nuclear receptors in mice exposed to 1800 ppm MIBK by inhalation for 10 days.
Executive summary:

In this published paper, the authors report the results of a number of a comparative investigation in wild-type (B6C3F1, C57BL/6) and C57BL/6 CAR/PXR-knockout mice following exposure by inhalation to 0 (control) or 1800 ppm MIBK for 10 days.  Terminal blood samples were taken for the assessment of clinical chemistry parameters (AST, ALT, ALP, GGT, total bilirubin, total protein, albumin, globulin, A:G ratio, triglycerides and cholesterol). Liver weights were recorded. The liver (left lobe) and duodenum (control tissue for BrdU incorporation) were assessed histopathologically. Liver S-phase DNA synthesis was assessed by BrdU incorporation, measured using monoclonal anti-BrdU antibodies. The number of BrdU-positive nuclei was reported for 1000 hepatocytes in each lobular zone. RNA was extracted from liver samples and screened for expression of Cyp1A1 (AhR response), Cyb2b10 (CAR response), Cyp3a11 (PXR response) and Cyp4a10 (PPAR-alpha response). Beta-actin was used as a housekeeping gene.


Mean terminal body weights of all groups mice were unaffected by exposure to 1800 ppm MIBK. Mean relative liver weights were increased in exposed groups of mice, relative to the respective control group. Values attained statistical significance, with the exception of male wild-type (WT) C57BL/6 mice.  Histopathology of the liver in WT mice of both strains showed findings characterised by centrilobular/midzonal hepatocyte hypertrophy and altered staining; increased mitotic figures; and vacuolisation consistent with fatty change. CAR/PXR KO mice showed a decreased response, limited to very slight centrilobular/midzonal hepatocyte hypertrophy, and vacuolisation, with no evidence of mitosis.  Changes in clinical chemistry parameters related to MIBK exposure were limited to increased serum ALT activity, reported for WT B6C3F1 mice (M, F); WT C57BL/6 mice (F); and C57BL/6 KO mice (M). Other statistically significant changes observed in female WT mice were not considered to be related to MIBK exposure.  Both sexes of both WT mouse strains exposed to MIBK showed changes in gene expression characteristic of CAR/PXR activation. Notably, in male WT mice, transcription levels of Cyp2b10 (a marker of CAR activation) were 982 and 599 times higher than the respective controls in B6C3F1 and C57BL/9, respectively. There was no response in WT mice of either strain indicative of AhR or PPAR-alpha activation. In WT mice of both sexes, mRNA transcription levels associated with CAR, PXR, AhR and PPAR-alpha receptor activation were essentially unchanged relative to the control group.  A significant increase in hepatocyte BrdU staining intensity was noted in the centrilobular, midzonal and periportal zones of male WT mice of both strains exposed to MIBK. A similar statistically significant increase in staining intensity was seen for female B6C3F1 mice; the response in female C57BL/6 mice was of less intensity and did not attain statistical significance. No increase in staining intensity was seen in any hepatolobular zone in CAR/PXR KO mice of either sex.


The results of this study therefore indicate activation of the CAR/PXR nuclear receptors in mice exposed to 1800 ppm MIBK by inhalation for 10 days.

Endpoint:
mechanistic studies
Remarks:
CAR/PXR activation in mouse hepatocytes
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
22 July 2020 - 27 May 2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
This mechanistic study has no recognised test guideline, but was performed to accepted scientific principles and to GLP.
Qualifier:
no guideline available
Principles of method if other than guideline:
- Principle of test: Cultured primary mouse hepatocytes were exposed to concentrations of MIBK
- Short description of test conditions: Cutures were exposed to non-cytotoxic concentrations of MIBK for 96 hours
- Parameters analysed / observed: Activation of the CAR, PXR, PPAR-alpha and AhR were assessed by measurement of target genes, associated enzyme activities and cell proliferation
GLP compliance:
yes
Type of method:
in vitro
Endpoint addressed:
carcinogenicity
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Sasol
- Purity: 99.8%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: stable
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: stable
- Reactivity of the test material with the incubation material used (e.g. plastic ware): non-reactive

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none
- Preliminary purification step (if any): none
- Final concentration of a dissolved solid, stock liquid or gel: 300 mM
Species:
mouse
Strain:
C57BL
Sex:
male
Details on test animals or test system and environmental conditions:
Commercially sourced cryopreserved primary male C57BL/6 mouse hepatocytes
Route of administration:
other: in vitro
Vehicle:
DMSO
Remarks:
0.1% final concentration
Details on exposure:
Cells were exposed to MIBK (dissolved in DMSO) at final concentrations of 10, 30, 100 and 300 uM for 96 hours. The highest concentration was selected on the basis of preliminary solubility and cytotoxicity assessments.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
96 hours
Frequency of treatment:
Single exposure
Post exposure period:
None
Dose / conc.:
0 other: uM
Remarks:
Solvent control
Dose / conc.:
10 other: uM
Dose / conc.:
30 other: uM
Dose / conc.:
100 other: uM
Dose / conc.:
300 other: uM
Control animals:
yes, concurrent vehicle
Details on study design:
Cultured male C57BL/6 mouse primary hepatocytes were exposed for 96 hours to concentrations of MIBK previously shown to be non-toxic in this assay system. Cytotoxicity was measured using ATP depletion. Cells were assessed for mRNA transcription using primers specific for Cyp1a1, Cyp1a2, Cyp2b10, Cyp3a11, Cyp410; murine beta-actin was used as an internal standard. Cells were assessed for replicative DNA synthesis (RDS) immunohistochemically by BrdU incorporation. Cell protein content was determined using the Lowry method. Cell enzyme activities (EROD, PROD, BROD, BQ) were also determined. Positive control compounds phenobarbital and EGF were also assessed. The negative control was the vehicle DMSO (0.1% final concentraion).
Examinations:
Cytotoxicity (ATP depletion)
mRNA analysis using primers specific for Cyp1a1, Cyp1a2, Cyp2b10, Cyp3a11, Cyp410; and murine beta-actin (internal standard)
Replicative DNA synthesis (RDS): immunohistochemically by BrdU incorporation
Protein determination: Lowry method
Enzyme activities: EROD, PROD, BROD, BQ
Positive control:
Phenobarbital (CAR activation control)
EGF (cell proliferation control)
Details on results:
Preliminary miscibility test
The results of this test showed a limit of 300 mM MIBK in the assay solvent DMSO

Preliminary cytotoxicity assay
Treatment with MIBK at concentrations of up to and including 300 uM did not show any cytotoxicity

MAIN ASSAY
Cytotoxicity
Cell viability was reduced at 10 uM (78% of controls) and at 300 uM (61% of controls). The finding at 10 uM was considered to be spurious in the absence of a concentration-response relationship.

mRNA analysis
Cyp2b10 mRNA expression was induced at all concentrations of MIBK, with a maximum of ~1.5 times at 300 uM. The positive control (PB) induced expression by ~1.7 times at 250 uM and ~1.8 times at 500 uM.
Cyp3a11 mRNA expression was unaffected by treatment with MIBK. The positive control (PB) induced expression by ~1.6 times at 250 uM and ~1.6 times at 500 uM.
Cyp1a1 mRNA expression was unaffected by treatment with MIBK. The positive control (PB) induced expression by ~2.4 times at 250 uM and ~3.3 times at 500 uM.
Cyp1a2 mRNA expression was marginally (~1.3 times) induced at MIBK concentrations of 100 and 300 uM, with no concentration-response relationship. The positive control (PB) induced expression by ~1.7 times at 250 uM and ~2.1 times at 500 uM.
Cyp4a10 mRNA expression was unaffected by treatment with MIBK or PB.

Enzyme activity
PROD activity was increased (148% of controls) by exposure to 10 uM MIBK; this result was considered to be spurious in the absence of effects at higher concentrations. The positive control PB increased PROD activity at 500 um (155%).
BROD activity was not increased by exposure to MIBK. The positive control PB increased BROD activity at 500 um (250%).
EROD activity was not increased by exposure to MIBK. The positive control PB increased EROD activity at 500 um (411%).
BQ activity was not increased by exposure to MIBK. The positive control PB increased BQ activity at 500 um (380%).

RDS
Exposure to MIBK or PB at any concentration did not induce RDS
The positive control EGF induced RDS by ~96-fold relative to controls.

Summary of mRNA induction in mouse hepatocytes

















































































Treatment



Cytotoxicity (% control)



Relative mRNA induction



Cyp1a1



Cyp1a2



Cyp2b10



Cyp3a11



Cypo4a10



DMSO



-



1.000



1.000



1.000



1.000



1.000



PB 250 µM



102



2.360**



1.694***



1.730***



1.645**



1.079



PB 500 µM



89



3.328***



2.118***



1.791***



1.605**



1.045



MIBK 10 µM



78**



0.965



1.263



1.367**



1.240



1.345



MIBK 30 µM



98



1.240



1.263



1.327**



1.096



1.229



MIBK 100 µM



101



1.066



1.331*



1.384***



1.138



1.147



MIBK 300 µM



61***



0.985



1.337*



1.536***



1.317



1.060



**significantly different to controls p<0.01; ***p<0.001


 


Summary of enzyme induction in mouse hepatocytes









































































Treatment



Cytotoxicity (% control)



Enzyme activity (% control)



EROD



PROD



BROD



BQ



DMSO



-



100.0



100.0



100.0



100.0



PB 250 µM



102



259.66**



132.40



135.01



170.57



PB 500 µM



89



410.99***



155.03*



248.86**



378.82**



MIBK 10 µM



78**



150.82



117.72*



119.64



118.31



MIBK 30 µM



98



145.14



124.41



125.97



105.63



MIBK 100 µM



101



105.22



94.34



86.59



73.67



MIBK 300 µM



61***



126.20



113.17



95.86



78.59



*significantly different to controls p<0.05; **p<0.01; ***p<0.001


 


Summary of replicative DNA synthesis in mouse hepatocytes



















































Treatment



Cytotoxicity


(% control)



Replicative DNA synthesis


(fold change)



DMSO



-



1.0



EGF 25 ng/mL



-



95.5***



PB 250 µM



102



1.5



PB 500 µM



89



3.4***



MIBK 10 µM



78**



0.9



MIBK 30 µM



98



1.8



MIBK 100 µM



101



1.1



MIBK 300 µM



61***



1.3



*significantly different to controls p<0.05; **p<0.01; ***p<0.001


 


 


 

Executive summary:

In this non-standard mechanistic study performed to investigate the hepatic carcinogenicity of MIBK in mice, cultured primary male B57CL/6 mouse hepatocytes were exposed to concentrations of MIBK (10, 30, 100 and 300 uM) previously shown to be non-toxic, for 96 hours.  Negative (solvent) controls were also used positive controls were phenobarbital and EGF.  Activation of the CAR, PXR, PPAR-alpha and AhR were assessed by measurement of target genes, associated enzyme activities and cell proliferation.  Cell viability was reduced at 10 um (78% of controls) and at 300 um (61% of controls). The finding at 10 uM was considered to be spurious in the absence of a concentration-response relationship.


Cyp2b10 mRNA expression was induced at all concentrations of MIBK, with a maximum of ~1.5 times at 300 uM. The positive control (PB) induced expression by ~1.7 times at 250 uM and ~1.8 times at 500 uM.  Cyp3a11 mRNA expression was unaffected by treatment with MIBK. The positive control (PB) induced expression by ~1.6 times at 250 uM and ~1.6 times at 500 uM.  Cyp1a1 mRNA expression was unaffected by treatment with MIBK. The positive control (PB) induced expression by ~2.4 times at 250 uM and ~3.3 times at 500 uM.  Cyp1a2 mRNA expression was marginally (~1.3 times) induced at MIBK concentrations of 100 and 300 uM, with no concentration-response relationship. The positive control (PB) induced expression by ~1.7 times at 250 uM and ~2.1 times at 500 uM.  Cyp4a10 mRNA expression was unaffected by treatment with MIBK or PB.  PROD activity was increased (148% of controls) by exposure to 10 uM MIBK; this result was considered to be spurious in the absence of effects at higher concentrations. The positive control PB increased PROD activity at 500 um (155%).  BROD activity was not increased by exposure to MIBK. The positive control PB increased BROD activity at 500 um (250%).  EROD activity was not increased by exposure to MIBK. The positive control PB increased EROD activity at 500 um (411%).  BQ activity was not increased by exposure to MIBK. The positive control PB increased BQ activity at 500 um (380%).  Exposure to MIBK or PB at any concentration did not induce replicative DNA synthesis.  The positive control EGF induced RDS by ~96-fold relative to controls.


The results of this study indicate that MIBK is a weak CAR activator in male C57BL/6 mouse hepatocytes in vitro.  There was no evidence of PXR, AhR or PPAR-alpha activation in this study.  The weak activation of CAR in this study was not associated with a proliferative response.

Endpoint:
mechanistic studies
Remarks:
CAR/PXR activation in human hepatocytes
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
22 July 2020 - 2022
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
This mechanistic study has no recognised test guideline, but was performed to accepted scientific pr inciples and to GLP.
Qualifier:
no guideline available
Principles of method if other than guideline:
- Principle of test: Cultured primary human hepatocytes were exposed to concentrations of MIBK
- Short description of test conditions: Cutures were exposed to non-cytotoxic concentrations of MIBK
for 96 hours
- Parameters analysed / observed: Activation of the CAR, PXR, PPAR-alpha and AhR were assessed
by measurement of target genes, associated enzyme activities and cell proliferation
GLP compliance:
yes
Type of method:
in vitro
Endpoint addressed:
carcinogenicity
Species:
human
Strain:
other: Not applicable (human)
Sex:
male
Details on test animals or test system and environmental conditions:
Commercially sourced cryopreserved primary male human hepatocytes from three donors
Route of administration:
other: in vitro
Vehicle:
DMSO
Remarks:
Final concentration 0.1%
Details on exposure:
Cells were exposed to MIBK (dissolved in DMSO) at final concentrations of 10, 30, 100 and 300 uM for 96 hours. The highest concentration was selected on the basis of preliminary solubility and cytotoxicity assessments.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
96 hours
Frequency of treatment:
Single exposure
Post exposure period:
None
Dose / conc.:
0 other: uM
Remarks:
Solvent control (DMSO 0.1%)
Dose / conc.:
10 other: uM
Dose / conc.:
30 other: uM
Dose / conc.:
100 other: uM
Dose / conc.:
300 other: uM
Control animals:
yes, concurrent vehicle
Details on study design:
Cultured male human primary hepatocytes were exposed for 96 hours to concentrations of MIBK previously shown to be non-toxic in this assay system. Cytotoxicity was measured using ATP depletion. Cells were assessed for mRNA transcription using primers specific for CYP1A1, CYP2B6, CYP3A4 and CYP4A11; human beta-actin was used as an internal standard. Cells were assessed for replicative DNA synthesis (RDS) immunohistochemically by BrdU incorporation. Cell protein content was determined using the Lowry method. Cell enzyme activities (EROD, PROD, BROD, BQ) were also determined. Positive control compounds phenobarbital and EGF were also assessed. The negative control was the vehicle DMSO (0.1% final concentraion).
Examinations:
Cytotoxicity (ATP depletion)
mRNA analysis using primers specific for Cyp1a1, Cyp2b6, Cyp3a4, Cyp4a10; and human beta-actin (internal standard)
Replicative DNA synthesis (RDS): immunohistochemically by BrdU incorporation
Protein determination: Lowry method
Enzyme activities: EROD, PROD, BROD, BQ
Positive control:
Phenobarbital (CAR activation control)
EGF (cell proliferation control)
Details on results:
Preliminary miscibility test
The results of this test showed a limit of 300 mM MIBK in the assay solvent DMSO

Preliminary cytotoxicity assay
Treatment with MIBK at concentrations of up to and including 300 uM did not show any cytotoxicity in hepatocytes from any of the three donors.

MAIN ASSAY
Cytotoxicity
Cell viability was not reduced at any concentration of MIBK
.
mRNA analysis
CYP1A1 mRNA expression was marginally (~1.3 times controls) induced at 300 uM MIBK in hepatocytes from one donor. The positive control (PB) induced expression of CYP1A1 by ~1.3 to 1.4 times in hepatocytes from all donors at 1000 uM.
CYP2B6 mRNA expression was marginally (~1.6 times controls) induced at 300 uM MIBK in hepatocytes from one donor. The positive control (PB) induced expression of CYP2B6 by 6.7-14 times at 1000 uM in hepatocytes from all donors.
CYP3A4 mRNA expression was unaffected by treatment with MIBK in hepatocytes from any of the three donors. The positive control (PB) induced expression of CYP3A4 by ~27 to 44 times at 1000 uM in hepatocytes from all donors.
CYP4A11 mRNA expression was unaffected by treatment with MIBK in hepatocytes from any of the three donors. The positive control (PB) reduced expression of CYP4A11 in hepatocytes from all donors.

.
Enzyme activity
PROD activity was not increased by exposure to MIBK in hepatocytes from any of the three donors. The positive control PB marginally increased PROD activity at 1000 um.
BROD activity was not increased by exposure to MIBK in hepatocytes from any of the three donors. The positive control PB increased BROD activity at 1000 um (up to 175%).
EROD activity was not increased by exposure to MIBK in hepatocytes from any of the three donors. The positive control PB increased BROD activity at 1000 um (up to 215%).
BQ activity was not increased by exposure to MIBK in hepatocytes from any of the three donors. The positive control PB increased BROD activity at 1000 um (up to 1567%).

RDS
Exposure to MIBK at 300 uM increased RDS slightly (~1.7 times) in hepatocytes from one donor. Treatment with PB concentration did not induce RDS. Treatment with EGF increased RDS in hepatocytes from all donors by ~6 to 35-fold.

 


 


 


Summary of cytotoxicity in human hepatocytes






















































































Treatment



Cytotoxicity (% control)



Donor IXL



Donor HU8340



Donor HUM181551A



Plate 1



Plate 2



Plate 1



Plate 2



Plate 1



Plate 2



Control



100 ± 11



100 ± 11



100 ± 9



100 ± 7



100 ± 9



100 ± 6



PB 500 µM



102 ± 14



100 ± 4



101 ± 4



94 ± 7



91 ± 7



91 ± 7



PB 1000 µM



98 ± 12



98 ± 10



100 ± 4



89 ± 3**



90 ± 6*



86 ± 9*



MIBK 10 µM



111 ± 15



97 ± 11



98 ± 6



104 ± 5



102 ± 5



104 ± 11



MIBK 30 µM



99 ± 6



96 ± 9



99 ± 10



102 ± 4



101 ± 5



105 ± 8



MIBK 100 µM



103 ± 8



97 ± 8



100 ± 4



100 ± 7



99 ± 4



103 ± 8



MIBK 300 µM



101 ± 5



96 ± 5



98 ± 3



102 ± 6



102 ± 6



101 ± 11



*significantly different to controls p<0.05; **p<0.01


Summary of mRNA transcription in human hepatocytes



































































































































Treatment



Donor IXL



Donor HU8340



Donor HUM181551A



CYP1A1



CYP2B6



CYP3A4



CYP4A11



CYP1A1



CYP2B6



CYP3A4



CYP4A11



CYP1A1



CYP2B6



CYP3A4



CYP4A11



Control



1.000



1.000



1.000



1.000



1.000



1.000



1.000



1.000



1.000



1.000



1.000



1.000



PB 500 µM



1.245



7.203



15.414



2.104



1.212



6.759



40.486



 0.649



1.278**



8.006***



23.231***



0.584*



PB 1000 µM



1.261



14.032***



30.128***



 0.609



1.313**



8.524***



43.834***



0.523*



1.398***



11.160***



27.096***



0.515*



MIBK 10 µM



1.085



1.264



1.656



1.128



0.905



1.207



1.347



1.481



0.918



0.946



0.845



1.126



MIBK 30 µM



1.217



1.314



1.597



1.933



1.039



1.015



1.115



1.265



0.986



1.276



1.064



0.990



MIBK 100 µM



1.178



1.146



1.280



0.869



0.970



1.015



1.070



1.433



0.983



1.146



1.084



1.435



MIBK 300 µM



1.315



 1.655



2.276



1.986



1.010



1.183



1.136



1.218



1.001



1.363



1.169



1.315



*significantly different to controls p<0.05; **p<0.01; ***p<0.001


 


Summary of enzyme activities in human hepatocytes



































































































































Treatment



Donor IXL



Donor HU8340



Donor HUM181551A



EROD



PROD



BROD



BQ



EROD



PROD



BROD



BQ



EROD



PROD



BROD



BQ



Control



100



100



 100



100



100



100



100



100



100



100



100



100



PB 500 µM



164*



115



147



293***



172



118



156***



1567***



174



140



158



572***



PB 1000 µM



215*



129



146



418***



225



135



164***



1448***



209



145



175



447***



MIBK 10 µM



76.2



73.5



84.0



106.7



131



90.6



95.2



108



114



155



92.3



101



MIBK 30 µM



112



78.7



89.6



83.6



142



94.4



93.6



89.1



86.4



106



96.1



95.6



MIBK 100 µM



80.8



61.5



73.8



100



135



113



90.7



99.5



96.6



154



89.4



 83.2



MIBK 300 µM



63.1*



63.9



59.0*



81.3



138.3



91.0



94.5



98.8



91.2



 129



87.1



76.2



*significantly different to controls p<0.05; **p<0.01; ***p<0.001


 


 

Conclusions:
The results of this study do not indicate the potential of MIBK to activate the CAR, PXR, AhR or PPAR-alpha receptors in primary human hepatocytes.
Executive summary:

Cell viability was not reduced at any tested concentrations of MIBK.  mRNA analysis showed that CYP1A1 mRNA expression was marginally (~1.3 times controls) induced at 300 uM MIBK in hepatocytes from one donor. The positive control (PB) induced expression of CYP 1A1 by ~1.3 to 1.4 times in hepatocytes from all donors at 1000 uM. CYP2B6 mRNA expression was marginally (~1.6 times controls) induced at 300 uM MIBK in hepatocytes from one donor. The positive control (PB) induced expression of CYP2B6 by 6.7-14 times at 1000 uM in hepatocytes from all donors.  CYP3A4 mRNA expression was unaffected by treatment with MIBK in hepatocytes from any of the three donors. The positive control (PB) induced expression of CYP3A4 by ~27 to 44 times at 1000 uM in hepatocytes from all donors.  CYP4A11 mRNA expression was unaffected by treatment with MIBK in hepatocytes from any of the three donors. The positive control (PB) reduced expression in hepatocytes from all donors.  PROD activity was not increased by exposure to MIBK in hepatocytes from any of the three donors. The positive control PB marginally increased PROD activity at 1000 um.  BROD activity was not increased by exposure to MIBK in hepatocytes from any of the three donors. The positive control PB increased BROD activity at 1000 um (up to 175%).  EROD activity was not increased by exposure to MIBK in hepatocytes from any of the three donors. The positive control PB increased BROD activity at 1000 um (up to 215%).  BQ activity was not increased by exposure to MIBK in hepatocytes from any of the three donors. The positive control PB increased BROD activity at 1000 um (up to 1567%).  Exposure to MIBK at 300 uM increased replicative DNA synthesis (RDS) slightly (~1.7 times) in hepatocytes from one donor, but this is considered. Treatment with PB concentration did not induce RDS. Treatment with EGF increased RDS in hepatocytes from all donors by ~6 to 35-fold.  The results of this study do not indicate the potential of MIBK to activate the CAR, PXR, AhR or PPAR-alpha receptors in primary human hepatocytes.

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guidelines.
Qualifier:
no guideline available
Principles of method if other than guideline:
Male and female rats (4/sex/treatment group) were administered corn oil (vehicle control) or MIBK (1000 mg/kg bw/day) and another group of male rats (n=4) were administered d-limonene (positive control; 300 mg/kg bw/day) for 10 consecutive days by oral gavage. Approximately 24 h after the final dose the kidneys were excised and the left kidney prepared and evaluated for histological changes including protein (hyaline) droplet accumulation, immunohisto-chemical staining for a2µ-globulin (a2µ) nephropathy (a2µ-N), and proliferating cell nuclear antigen (PCNA) to quantitate renal cell proliferation. The right kidney was prepared for quantitation of total protein and a2µ using an enzyme-linked immunosorbent assay (ELISA).
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
carcinogenicity
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
10 consecutive days
Frequency of treatment:
Daily
Post exposure period:
Rats were euthanized 24 hours following the final dose.
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
nominal conc.
(5 mL/kg body weight/day)
No. of animals per sex per dose:
4/sex/dose
Control animals:
yes, concurrent vehicle
Details on results:
MIBK elicited an increase in protein droplets, accumulation of alpha2µ, and renal cell proliferation in male, but not female rats, responses characteristic of alpha2µ-mediated nephropathy. MIBKproduced identical histopathological changes in the male rat kidney when compared to d-limonene, an acknowledged inducer of alpha2µ-nephropathy except that the grade of severity tended to be slightly lower with MIBK. MIBK did not induce any effects in female rats.

Exposure to MIBK or d-limonene did not affect body weight gain, terminal body weights, and total kidney weight over the length of this study; however, there was a slight increase in absolute kidney weights in female rats administered MIBK. The kidney:body weight ratio was increased slightly in male and female rats as compared to their respective controls. MIBK-treated male rats showed an identical range of changes in the kidney as compared to the changes noted in the kidneys from d-limonene treated male rats. The severity of droplet accumulation was graded as mild in 3/4 male rats, and moderate in 1/4 rats. Thus, the various effects appeared at a slightly lower grade than with d-limonene. All of the female rat kidneys were judged to be within normal limits with no renal alterations produced by MIBK administration. No histopathological evidence explaining the slightly increase in absolute and relative kidney weights was evident for female rats. All male rats treated with 1000 mg/kg bw/day MIBK had positive Mallory’s Heidenhain (MH) staining similar to that seen in kidney sections of d-limonene treated male rats with respect to the disruption of the normal pattern of staining and the presence of crystal-like aggregates. The kidneys sections from MIBK-treated female rats were negative for MH-stained droplets in proximal tubule cytoplasm. Immunohistochemical staining of kidney sections for a2µ localized the protein to the renal cortex of control male rats. The staining in kidney sections from both MIBK and d-limonene treated male rats was more intense and occupied a greater area of the renal cortex than that of control rats. Due to uneven staining on the kidney sections, semi-quantitative grading of these sections was difficult. No positive staining for a2µ was observed in the kidneys from control or MIBK administered female rats. A statistically significant threefold increase in renal cell proliferation was reported in male rats administered MIBK, but not in female rats compared to their respective controls.

Executive summary:

Male F-344 rats were administered corn oil (vehicle control), d-limonene (positive control, 300mg/kg), or MIBK (1000mg/kg) and female F-344 rats corn oil (vehicle control) or MIBK for 10 consecutive days by oral gavage. Approximately 24h after the final dose the kidneys were excised and the left kidney prepared and evaluated for histological changes including protein (hyaline) droplet accumulation, immunohistochemical staining for alpha2µ-globulin (alpha2µ), and proliferating cell nuclear antigen (PCNA) to quantitate renal cell proliferation. The right kidney was prepared for quantitation of total protein and alpha2µ using an ELISA. MIBK elicited an increase in protein droplets, accumulation of alpha2µ, and renal cell proliferation in male, but not female rats, responses characteristic of alpha2µ-mediated nephropathy. MIBK produced identical histopathological changes in the male rat kidney when compared to d-limonene, an acknowledged inducer of alpha2µ-nephropathy except that the grade of severity tended to be slightly lower with MIBK. MIBK did not induce any effects in female rats. Therefore, renal histopathology, along with the other measures of alpha2µ accumulation, provides additional weight of evidence to support the inclusion of MIBK in the category of chemicals exerting renal effects through an alpha 2µ-nephropathy-mediated mode-of-action.

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Principles of method if other than guideline:
The objective of this study is to evaluate the ability of MIBK, under exposure conditions of the 2-year chronic bioassay, to induce specific measures of alpha2µ-nephropathy (alpha2µ-N) such as histological lesions associated with the accumulation of alpha2µ globulin, increased renal concentration of alpha2µ globulin, and sustained renal cell proliferation in the kidneys of male but not female rats. In addition, the ability of MIBK to bind reversibly to alpha2µ globulin was investigated, fulfilling another criterion that supports the alpha2µ-N MoA. Identification of the MoA for MIBK-induced renal toxicity and carcinogenesis is important for assessing the human health risks from exposure to MIBK.
GLP compliance:
yes
Remarks:
Both the inhalation MIBK study and the d-limonene oral dose study were conducted in accordance with U.S. EPA's GLP from animal receipt through euthanasia but not including dose formulation for d-limonene.
Type of method:
other: in vivo and in vitro
Endpoint addressed:
carcinogenicity
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratory (Kingston, NY)
- Age at reception:9 weeks
- Weight at study initiation: no data
- Housing: no data
- Diet (ad libitum): NTP-2000 diet (Zeigler Bros., Gardners, PA) {feed was not provided to rats during inhalation exposure periods)
- Water (ad libitum): reverse-osmosis-treated tap water (City of Durham, NC)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1 m3 stainless steel and glass inhalation cages (Hazelton H1000 Lab Products, Seaford, DE)
- Method of holding animals in test chamber: whole-body exposure
- Source and rate of air: no data
- Method of conditioning air: no data
- System of generating particulates/aerosols: a liquid metering pump (Fluid Metering Inc., FMI) was used to direct liquid MIBK into a J-tube generator.
The FMI pump was calibrated and set to the nominal flow rate expected for each exposure concentration. Nitrogen flowed upward through the J-tube in all concentration groups at a flow rate of approximately 25 L/min. The MIBK vapor flow was introduced counter current to the HEPA-filtered chamber airtlow to facilitate mixing of the MIBK vapor with dilution air.
- Temperature, humidity: 20-25°C, 30-70%
- Air flow rate: approximately 250 L/min
- Air change rate: 15 air changes each hour
- Method of particle size determination: not relevant for vapor
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: GC-FID
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test atmosphere in each chamber was analyzed using a Hewlett Packard Series lI 5890 gas chromatograph (Agitent Technologies, Inc., Palo Atlo, CA) equipped with a flame ionization detector (FID) and 10-port sampling valve (VIVI Valco Instruments, Houston, TX). The 10-port sampling valve rotated through a series of positions, which sampled from the exposure chambers and the control chamber. The gas chromatography analysis was timed so that the valve rotated through all 10 positions in 30 min during the exposure period.
Duration of treatment / exposure:
1 week (4 exposures) or 4 weeks (19 exposures, 5 days/week for 3 weeks, then 4 days/week the 4th week)
Frequency of treatment:
6 h/day
Post exposure period:
18 h following the last exposure
Remarks:
Doses / Concentrations:
450, 900 or 1800 ppm
Basis:
other: target conc.
Remarks:
Doses / Concentrations:
446.7±2.6, 902.3±7.3 or 1803± 16 ppm
Basis:
analytical conc.
male week 1
Remarks:
Doses / Concentrations:
452.3±6.5, 909.5± 15 or 1797±19
Basis:
analytical conc.
male week 4
Remarks:
Doses / Concentrations:
454.3 ±4.5, 929.0±2.3 or 1793 ±29 ppm
Basis:
analytical conc.
female week 1
Remarks:
Doses / Concentrations:
452.5±6.9, 908.6± 16 or 1798± 18 ppm
Basis:
analytical conc.
female week 4
No. of animals per sex per dose:
7-9
Control animals:
yes, sham-exposed
Details on study design:
Three and a half days prior to euthanasia, at both 1- and 4-weeks, rats were subcutaneously implanted with an osmotic pump containing 5-bromo-2-deoxyuridine (BrdU) to measure cell proliferation. At study termination, body weights were recorded and animals euthanized to harvest kidneys.
Examinations:
Rats in both the MIBK inhalation study and the d-limonene positive control study were euthanized with an intraperitoneal injection of sodium pentobarbital ( 100 mg/kg) and then exsanguinated from the abdominal aorta. The kidneys were removed and prepared for histopathology and biochemical endpoints.
Positive control:
d-Limonene is a chemical used as a positive control for measures of a2µ-N since it fulfills all of the criteria outlined by IARC (1999) to characterize this response. ln this positive control study, twelve male rats were randomly allocated to one of two dose groups. Rats were administered either corn oil (vehicle control) or o-limonene at a dose of 300 mg/kg/day (5 mL/kg) by oral gavage for 4 consecutive days and euthanized ~24 h following the last dose.
Details on results:
There was an exposure-related increase in all measures of a2µ nephropathy in male, but not female rat kidneys. The hyaline droplets present in male rat kidney stained positively for a2µ. The changes in HDA and a2µ concentration were comparable to d-limonene, an acknowledged inducer of a2µ nephropathy. In a separate in vitro study using a two-compartment vial equilibration model to assess the interaction between MIBK and a2µ, the dissociation constant (Kd) was estimated to be 1.27×10(-5)M. This Kd is within the range of other chemicals known to bind to a2µ and cause nephropathy.

Terminal body and kidney weights


There were no statistical differences in the final body weights of animals exposed to MIBK compared to concurrent controls. There was a significant increase in absolute kidney weights in male and female rats exposed to 900 or 1800 ppm MIBK for 1-and 4-weeks compared to the concurrent control along with an exposure-related increase at the 4-week exposure period.


 


Histopathology


A trace of intertubular mineralization was observed in the cortex of H&E stained kidney section from both male and female rats. Chronic progressive nephropathy (CPN) is a background lesion in control rats that is identifiable at 2-4 months of age in male rats of susceptible strains such as the F344 (Hard et al., 2013 ). In this study the mean grade of CPN in rats not exposed to MIBK was 0.38-0.50. Exacerbation of CPN was not observed after 1 week of MIBK exposure or administration of the positive control o-limonene. However, exacerbation of CPN was slightly increased in male rats exposed to 900 and 800 ppm MIBK for 4 weeks. CPN was not exacerbated in female rats following exposure to MIBK for either 1- or 4-weeks.


Accumulation of protein (hyaline) droplets associated with MIBK exposure was observed in proximal convoluted tubules of all MIBK-exposed male, but not female rats evaluated in both H&E (viewed under fluorescence) and MH-stained kidney sections. With increasing MIBK exposure concentration, in both1- and 4-weeks, there was increasing prominence of hyaline droplets in terms of size of droplet tracts, droplet pattern disruption observed with fluorescence microscopy as well as withincreasing intensity of autofluorescence. On a 0-16 scale, an exposure-related increase in HDA was observed in 1 week, ranging from grade 1 in the control male rats up to grade 10.75 in the high exposure male rat group (1800 ppm) (Fig. 3). The male rats exposed to MIBK for 4 weeks showed the same trend as the animals exposed for 1 week. HDA was also prominent in the o-limonene positive control group with a mean grade of 12 after 1 week of exposure. The sections of kidney stained with anti-a2µ antibody showed the protein droplets accumulating in male rats exposed to MIBK oradministered o-limonene were positive for a2µ, whereas there was less staining in control male rats kidney and no staining in female rats. Although there was an absence of well-formed granular casts in any exposure group, 6/8 males exposed to 1800 ppm MIBK for 4 weeks had solitary tubules at the junction of the outer and inner stripes of the outer medulla containing eosinophilic granular debris. These were consistent with precursors of granular casts described by Hard (2008).


 


Renal concentration of a2µ and total protein


The concentration of a2µ was measured in the kidney homogenate of male and female rats exposed to MIBK or male rats administered corn oil or the positive control d-limonene. MIBK caused an exposure related increase in concentration of a2µ in the male rats kidneys at both 1- and 4-weeks of exposure (Fig. 5A and C). Although, the magnitude of the increase was not as significant in the positive control d-limonene administered male rats (Fig. SA), it was statistically significant compared to the concurrent control at ail MIBK exposure concentrations. Total protein was not changed in male rats exposed to MIBK, but there was a significant increasefollowing o-limonene administration  (Fig. 5B and D). The concentration of a2µ in female rat kidneys was 250-fold lower compared to male rats. Although there was a significant decrease in the concentration of a2µ in female rats exposed to 1800ppm MIBK compared to the concurrent control, considering the low level of a2µ in the female kidneys it is most likely that this change is not biologically significant and further demonstrates that MIBK does not increase a2µ in the kidneys of female rats (Fig. 6A).


 


Renal cell proliferation


Following 1 week of MIBK exposure, there was a statistically significant increase in the percent of cells labeled with BrdU (LI) in male rats exposed to 450 ppm MIBK compared to the concurrent control, but not following 900 or 1800 ppm (Fig. 7). The increase in the LI was more apparent in male rats following 4 weeks of MIBK exposure with a significant increase at ail exposure concentrations compared to the concurrent controls. A significant exposure related increase in LI was observed at bath 1 and 4 weeks in male rats. Although there were no changes in the LI in the female rats between each exposure group and concurrent control at either 1- or 4-weeks of MIBK exposure, there was a significant exposure ­related increase following the 1-week exposure period. Considering the data at both exposure periods, this change does not appear to have any biological significance.


The counts of mitotic figures in cortical proximal tubule cells were approximately 10 times higher in males rats exposed to 1800ppm MIBK for 1- or 4-weeks compared to their concurrent controls (Table 4). All of the mitotic figures counted were in proximal convoluted tubules and many were in tubules containinghyaline protein droplets. There was no increase in mitotic counts in high-dose females at 1- or 4-weeks of exposure.


 


In vitro binding of MIBK to a2µ


Total protein concentration was determined to be 145.4 ±14.6 mg/g and 147.6 ± 7.8 mg/g total kidney in male and female rats, respectively. There was a concentration dependentdecrease in PC in male, but not female kidney tissue suggesting that the uptake of MIBK into male rat tissue is only dependent on solubility alone (Fig. 9). The PC of MIBK in male rat kidney tissue was decreased after the addition of oLO, a known ligand for a2µ, providing evidence that MIBK is displaced from binding to a2µ (Fig. 9). Table 5 provides the 2-compartment model parameter values achieved mathematically to describe the uptake of MIBK into the kidney tissue based on solubility and binding to a2µ. Using this model and the data presented in Fig. 9, aKdwas estimated in the range of 1.27 x 10-5 M to describe binding of MIBK to a2µ. This 2-compartment modeling approach provides an indirect method to assess chemical binding to a2µ with volatile chemicals and was used previously for methyl tertiary butyl ether (MTBE) (Poet and Borghoff, 1997).

Conclusions:
These studies provide the data necessary to demonstrate the ability of MIBK to induce exposure-related male rat specific changes in measures of a2µ-N (protein droplet accumulation, staining of the protein droplets for a2µ, sustained renal cell proliferation, and an increased concentration of a2µ along with providing evidence that MIBK binds reversibly to a2µ, the initiating event in a2µ-N. The strength of these data is supported by previous studies that have contributed to the characterization of MIBK as an inducer of a2µ-N (Dodd et al., 1982; Phillips et al., 1987; Nemec et al., 2004; Stout et al., 2008; Borghoff et al., 2009) meeting all of the IARC criteria (IARC, 1999) for characterization of this MoA. Together these studies provide the weight of evidence that the MIBK induced male rat renal tumors are not relevant to humans.
Executive summary:

Chronic exposure to methyl isobutyl ketone (MIBK) resulted in an increase in the incidence of renal tubule adenomas and occurrence of renal tubule carcinomas in male, but not female Fischer 344 rats. Since a number of chemicals have been shown to cause male rat renal tumors through the a2µ nephropathy-mediated mode of action, the objective of this study is to evaluate the ability of MIBK to induce measures of a2µ nephropathy including renal cell proliferation in male and female F344 rats following exposure to the same inhalation concentrations used in the National Toxicology Program (NTP) cancer bioassay (0, 450, 900, or 1800 ppm). Rats were exposed 6h/day for 1 or 4 weeks and kidneys excised approximately 18h post exposure to evaluate hyaline droplet accumulation (HDA), a2µ staining of hyaline droplets, renal cell proliferation, and to quantitate renal a2µ concentration. There was an exposure-related increase in all measures of a2µ nephropathy in male, but not female rat kidneys. The hyaline droplets present in male rat kidney stained positively for a2µ. The changes in HDA and a2µ concentration were comparable to d-limonene, an acknowledged inducer of a2µ nephropathy. In a separate in vitro study using a two-compartment vial equilibration model to assess the interaction between MIBK and a2µ, the dissociation constant (Kd) was estimated to be 1.27×10(-5)M. This Kd is within the range of other chemicals known to bind to a2µ and cause nephropathy. Together, the exposure-related increase in measures of a2µ nephropathy, sustained increase in renal cell proliferation along with an indication of reversible binding of MIBK to a2µ, support the inclusion of MIBK in the category of chemicals exerting renal effects through a protein droplet a2µ nephropathy-mediated mode of action (MoA).

Description of key information

A study in mice indicated that methyl isobutyl ketone (MIBK) may be an agonist for the constitutive androstane receptor (CAR) in liver mice.  This was evaluated following inhalation exposure to 1800 ppm MIBK for 7 days.  The study authors noted that this mechanism of action for liver carcinogenicity is not relevant to humans. In vivo and in vitro mechanistic studies support the proposed mechanism of action (CAR mediated) as not being relevant for humans. 
A study in rats, demonstrated that MIBK may exert effects on the male rat kidney via an alpha-2u-globulin mechanism. As well, this mecanism of action for kidney carcinogenicity, which is species and male specific, is not relevant to humans.

Additional information

Studies on the mode of action (MoA) for carcinogenicity


1. IARC criteria for MIBK causing kidney tumours through an a2µ-globulin-associated response in male rats


The development of kidney tumors in male rats in association with chemically induceda2µ-globulin nephropathy is mechanism that is not considered to be a predictor of carcinogenic risk to humans by the IARC or the US EPA (US EPA, 1991; Hard et al., 1993; Swenberg & Lehman-McKeeman, 1999). The lack of relevance of the a2µ-globulin mechanism for the evaluation of carcinogenic risk is based on the absence of the production of an analogous protein in humans. Strict scientific criteria have been outlined to establish the role ofa2µ-globulin-associated nephropathy in renal carcinogenesis in male rats (Swenberg & Lehman-McKeeman, 1999), and were used to determine the plausibility of ana2µ-globulin associated nephropathy based on a studies that have been carried out with subchronic and chronic exposures to methyl isobutyl ketone.


1. Lack of genotoxic activity (agent and/or metabolite) based on an overall evaluation of in-vitro and in-vivo data


A battery of genotoxicity assays with MIBK published in O’Donoghue et al. (1988) yielded mostly negative responses. MIBK did not induce revertant point mutations in five Salmonella tester strains (TA98, TA100, TA1535, TA1537, and TA1538), either in the presence or absence of Aroclor 1254-induced rat liver microsomal enzymatic activation. Mutant frequencies were also not affected in the L5178Y TK+/- mouse lymphoma mutagenesis assay in the presence of Aroclor-induced rat liver S-9. No dose-response relationship was observed in cultures exposed to MIBK in the absence of exogenous metabolic activation, although mutation frequency was significantly elevated in three out of six of the MIBK-treated cultures in the absence of S-9 metabolic activation. MIBK was also negative in the unscheduled DNA synthesis assay in rat primary hepatocytes cultures and in the in vivo micronucleus cytogenetic assay in mice administered MIBK intraperitoneally at 0.73 mL/kg (the dose level selected as the LD20 on the basis of a preliminary toxicity study). The following additional set of genotoxicity assays also yielded negative results: reverse mutation assays in five strains of Salmonella typhimurium and three strains of Escherichia coli and gene mutations in Saccharomyces cerevisiae, all both in the presence and absence of exogenous metabolic activation and a structural chromosome damage assay in cultured rat liver cells (Brooks et al., 1988).


Two of MIBK’ metabolites - 4-hydroxymethyl isobutyl ketone (Diacetone alcohol, DAA, CAS no. 123-42-2) and 4-methyl-2-pentanol (methylisobutyl carbinol, MIBC, CAS no. 108-11-2) – that are found in male Sprague-Dawley rat liver, serum and lung after exposure to methyl isobutyl ketone (Gingell et al., 2003; Duguay and Plaa, 1993) have also been evaluated for genotoxicity.


Diacetone alcohol was not mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2uvrA, with or without an exogenous metabolic activation system (MHW, 1997a). Two other Salmonella typhimurium studies indicated the negative results with or without metabolic activation (Priston et al., 1983; Brook et al., 1988). Mitotic recombination study in Saccharomyces cerevisiae showed the negative results with or without metabolic activation (Brooks et al., 1988). Genotoxicity of diacetone alcohol was studied by chromosomal aberration test in cultured Chinese hamster lung (CHL/IU) cells (MHW, 1997b) and RL4 rat liver cells (Brook et al., 1988). Structural chromosomal aberrations and polyploidy were not induced.  Diacetone alcohol did not induce mutations at the TK (Thymidine Kinase) locus in L5178Y mouse lymphoma cells with or without an exogenous metabolic activation system(Sire, 2010a).


MIBC was not mutagenic in bacterial reverse mutation assays (Shimizu et al., 1985; Clare, 1984) and mammalian cells (L5178Y) in vitro with or without metabolic activation (Sire 2010b). In a mammalian cell cytogenetic assay in rat liver cells, MIBC was negative with and without metabolic activation (Clare, 1984).


Thus, this criterion is met.


2. Male rat specificity for nephropathy and renal tumorigenicity induction of the characteristic sequence of histopathological changes in shorter-term studies, of which protein droplet accumulation is obligatory


Groups of six male and six female rats were exposed for 6 hrs/day, 5 days/week, for 9 days to measured concentrations of 0, 101, 501, or 1996 ppm (0, 410, 2053, or 8178 mg/m3) MIBK (Dodd et al., 1982). Groups were evaluated for changes in clinical signs, body weight, organ weights (liver, lungs, kidneys, and testes), ophthalmology, gross pathology, and histopathology. The only exposure-related effects observed were periocular wetness in rats exposed to 8178 mg/m3, increased relative liver weights in male rats at 2053 and 8178 mg/m3 and in female rats, increased kidney weights in male rats, and hyaline droplet degeneration in kidneys of male ratsexposed to 2053 or 8178 mg/m3, with epithelial regeneration of proximal convoluted tubules in the high-exposure group. No effects of any kind were observed in the 410 mg/m3 exposure group.


In a 90-day inhalation study in rats, groups of 14 male and 14 female Fischer 344 rats were exposed to measured mean concentrations of 0, 50, 252, and 1002 ppm (0, 205, 1033, and 4106 mg/m3) MIBK for 6 hrs/day, 5 days/week, for 14 weeks and sacrificed the animals following their final exposure day (Dodd and Eisler, 1983; Phillips et al., 1987). The following endpoints were evaluated: clinical signs, body weights, organ weights (kidneys, heart, liver, lungs, and testes), urinalysis, haematology, serum chemistry (including glucose and hepatic enzyme levels), complete gross pathology, targeted histopathology (nasal cavity, trachea, liver, kidneys, and lungs) in all animals and complete histopathology in control and high-exposure groups.  Urine glucose was significantly increased in male rats at 1033 mg/m3 (+37%) and 4106 mg/m3 (+55%) and in female rats at 4106 mg/m3 (+26%). Significantly increased urine protein (+28%) was also observed in male rats at 4106 mg/m3.  The only renal histological lesion observed was hyaline droplet formation in all male rats; the severity of the lesion generally increased with exposure level. In conclusion, other than the male rat kidney effect, exposure of male and female rats to MIBK at levels up to 1000 ppm for 14 weeks was without significant toxicological effect.


In a study (Mulligan, 1986) groups of 30 male and 30 female Sprague-Dawley rats were administered MIBK by gavage in corn oil at daily dose levels of 0 (vehicle control), 50, 250, or 1000 mg/kg-day for 13 consecutive weeks and evaluated for exposure-related changes in body weight, food consumption, mortality, clinical signs, ophthalmological parameters, and terminal organ weights (heart, liver, spleen, brain, kidney, gonads, adrenals, thyroid, and parathyroid).  The following changes suggestive of adverse kidney effects were observed at 1000 mg/kg-day: increased terminal absolute and relative kidney weights (from 25 to 34% in males and from 20 to 22% in females) as compared to controls, increased blood-urea-nitrogen (BUN) in males (+37%, interim), increased serum potassium in males (+34%, terminal), decreased serum glucose in males (-27%, terminal), and a reported increase in urinary protein and ketones in males and females at terminal sacrifice (summary data were not provided).  Histological examination of kidney tissues revealed an increased incidence of male rats with mild nephropathy (multifocally distributed swollen or hyperchromatic and flattened renal cortical tubular epithelial cells) at 1000 mg/kg-day (16/20) as compared to controls (4/20) but no increase in such lesions in females.


These studies indicate the induction of hyaline protein droplets in shorter-term studies.a2µ-globulin protein droplets were also specifically identified and characterized further in the studies of Borghoff et al. (2009 and 2015).


Thus, this criterion is met.


 


3. Identification of the protein accumulating in tubule cells asa2µ-globulin


The effects of MIBK exposure on Fischer 344 rat kidneys were assessedunder exposure conditions used in the 2-year MIBK chronic bioassay (Borghoff et al., 2015). Rats were exposed 6h/day for 1 or 4 weeks and kidneys were excised approximately 18h post exposure to evaluate hyaline droplet accumulation (HDA), a2µ staining of hyaline droplets and to quantitate renal a2µ concentration. There was an exposure-related increase in all measures of a2µ nephropathy in male, but not female rat kidneys. The hyaline droplets present in male rat kidney stained positively for a2µ. The changes in HDA and a2µ concentration were comparable to d-limonene, an acknowledged inducer of a2µ nephropathy Although this study was of short duration, it was designed to provide additional support to characterize MIBK as an inducer ofa-nephropathy, a MoA associated with a low incidence of kidney tumors in male, but not female rats.


The effects of MIBK oral exposure on Fischer 344 rat kidneys were assessed (Borhoff et al., 2009). Male and female rats were administered corn oil, 1000 mg/kg bw/day MIBK, or d-limonene (positive control) for 10 consecutive days by oral gavage (4/sex/group). Rats were euthanized 24 hours after the last dose, and the left kidney evaluated for histological changes in hyaline droplet accumulation, a-2µ-nephropathy, and proliferating cell nuclear antigen.The right kidney was assessed for total protein and a2µ-globulin using an enzyme-linked immunosorbent assay (ELISA).There were no changes in body weight gain, terminal body weights, or total kidney weight. The kidney/body weight ratio was increased slightly in male and female rats compared to controls. Changes observed in MIBK-treated male rats were similar or slightly milder than those observed in d-limonene-treated rats. Findings included mild to moderate hyaline droplet accumulation, positive Mallory’s Heidenhain staining, more intense alpha-2u-globulin staining compared to untreated controls, and a statistically significant increase in renal cell proliferation. There were no changes noted in any of the females administered MIBK. These results provide support that MIBK exerts renal effects in male rats through an alpha-2u-mediated mechanism.


Thus, this criterion is met.


4. Reversible binding of the chemical or metabolite toa2µ-globulin


In anin vitrostudy using a two-compartment vial equilibration model to assess the interaction between MIBK anda2µ, the dissociation constant (Kd) was estimated to be 1.27×10(-5)M. This Kd is within the range of other chemicals known to bind toa2µ and cause nephropathy(Borghoff et al., 2015).


The last criterion is the reversible binding of the chemical or metabolite toa2µ-globulin, which was not shown direct in any of the studies. One study showed reversibility of the adverse effects in the kidney after withdrawal of MIBK. The Wright-Patterson Air Force Base Aerospace Medical Research Laboratory (MacEwenet al., 1971) conducted a subchronic inhalation toxicity study in male Wistar albino rats that were exposed to 410 mg/m3MIBK vapour [100 ppm] for 90 days in an altitude chamber. The untreated control group was maintained in a separate altitude chamber. Statistically significant increases in liver and kidney weights and organ-to-body weight ratios for these tissues were noted in exposed rats. Microscopic examination of the kidneys revealed hyaline droplet degeneration of the proximal tubules (with occasional foci of tubular necrosis) in all of the exposed rats, including those that were removed from the inhalation chamber after 15, 22, 28, 71 and 85 days. The authors noted a trend towards a linear progression of hyaline droplet degeneration during exposure, but this pattern was not seen in all treatment groups. Moreover, the hyaline droplets appeared to increase in size with time. This observation was thought to have resulted from the coalescence of smaller droplets. Microscopic examination of rat kidneys removed after 15 days of exposure indicated a gradual reversion of tubular damage with time. Kidney damage was completely reversed in rats observed up to 60 days after exposure. Recovery MIBK-induced kidney lesions was also noted in rats that were serially killed to study reversibility after 90 days of exposure. However, recovery was not as rapid as that noted in animals exposed for shorter periods. A weakness of the study was the exclusion of female rats. The study showed the reversibility of effects that could be attributed toa2µ-globulin nephropathy.


Thus, this criterion is met.


5. Induction of sustained increased cell proliferation in the renal cortex


The effects of MIBK exposure on Fischer 344 rat kidneys were assessedunder exposure conditions used in the 2-year MIBK chronic bioassay (Borghoff et al., 2015). Male and female rats were exposed 6h/day for 1 or 4 weeks and were subcutaneously implanted with an osmotic pump containing 5-bromo-2-deoxyuridine (BrdU). Kidneys were excised approximately 18h post exposure to measure cell proliferation by immunohistochemical staining for BrdU.A significant exposure related increase in cells labeled with BrdU (LI) was observed at 4 weeks in male rats. The counts of mitotic figures in cortical proximal tubule cells were approximately 10 times higher in males rats exposed to 1800ppm MIBK for 1- or 4-weeks compared to their concurrent controls.


Thus, this criterion is met.


6. Similarities in dose–response relationship of the tumour outcome with the histopathological end-points (protein droplets, a2µ-globulin accumulation, cell proliferation


The histological endpoints identified (i.e., protein droplets,aaccumulation and even cell proliferation) are responses that occur early in the development ofa-nephropathy. A dose-response between these early lesions and renal tumors is difficult to demonstrate especially when the incidence of tumors is low and in the case of MIBK the two mid doses show a similar tumor response. As presented in theIARC MIBK evaluation (IARC, 2012), there was a correlation with linear mineralization and incidence of tumors that provided support for the association betweena-nephropathy and male rat renal tumors. ln the study of Borghoff et al. (2015), early measures ofa-nephropathy shown that there is an exposure-related correlation between renalaconcentration and HDA (Fig. 10A) as well as HDA and the labelling index (LI) in the renal cortex of male rats (Fig. 10B), supporting an association between these events.


 


Thus, this criterion is met.


Conclusion


All of the IARC criteria for characterization of the MoA are met. Together these studies provide the weight of evidence that the MIBK induced male rat renal tumors are not relevant to humans.


 


Additional references


Clare MG (1984). Toxicity of Chemical Solvents: Genotoxicity Studies With Methyl Isobutyl Carbinol. Testing laboratory: Shell Toxicology Laboratory (Tunstall), Sittingbourne Research Centre, Sittingbourne, Kent, ME9 8AG, England. Report no.: SBGR.83.381. Owner company: Royal Dutch Shell plc. Report date: 1984-01-09.


Hard GC, Rodgers IS, Baetcke KP et al. (1993) Hazard evaluation of chemicals that cause accumulation of alpha 2µ-globulin, hyaline droplet nephropathy, and tubule neoplasia in the kidneys of male rats.Environ Health Perspect., 99:313-49.


IARC (International Agency for Research on Cancer) Monographs, 2012. Some chemicals present in industrial and consumer products, food, and drinking water. Methyl lsobutyl Ketone 101, 305.


MHW (1997b). Reverse Mutation Tests Using Diacetone Alcohol on Bacteria. Testing laboratory: The Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa, 257, Japan. Owner company: Ministry of Health, Labor and Welfare, Japan.


MHW (1997c). Chromosomal Aberration Tests Using Diacetone Alcohol on Cultured Chinese Hamster Cells. Testing laboratory: Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa, 257, Japan. Owner company: Ministry of Health, Labor and Welfare, Japan.


Shimizu H, Suzuki Y, Takemura N, Goto S, & Matsushita H (1985). The Results of Microbial Mutation Test for Forty-Three Industrial Chemicals. Jpn. J. Ind. Health., Vol. 27, pp 400-419.


Sire G (2010a). Diacetone alcohol - In vitro mammalian cell gene mutation test in L5178Y TK+/- mouse lymphoma cells.Testing laboratory: CIT (Centre International de Toxicologie) - BP 563 - 27005 Evreux CEDEX - France.Report no.: 36344 MLY. Owner company: Arkema France. Report date: 2010-06-15.


Sire G (2010b). Methyl isobutylcarbinol - In vitro mammalian cell gene mutation test in L5178Y TK+/- mouse lymphoma cells.Testing laboratory: CIT (Centre Internation de Toxicologie), BP 563 - 27005 Evreux - France.Report no.: 36343 MLY. Owner company: Arkema France.


Swenberg JA, Lehman-McKeeman LD, 1999. a2-Urinary globulin-associated nephropathy as a mechanism of renal tubule cell carcinogenesis in male rats. ln:Capen CC, Oybing E, Rice JM, Wilbourn JO (Eds.), Species Differences in Thyroid, Kidney and Urinary Bladder Carcinogenesis. International Agency for Research on Cancer, Lyon, pp. 95-118 (IARC publications no. 147).


United States Environmental Protection Agency (U.S EPA), 1991. Alpha2µ globulin: Association with Chemically Induced Renal Toxicity and Neoplasia in the Male Rat (Risk Assessment Forum). U.S. EPA, Washington D.C (EPA/625/3-91 /019F. (NTIS PB92143668)).


  


 


2.Mouse liver tumors


In the NTP bioassay (2007), significant positive trends were reported for hepatocellular adenomas in male and female mice; the increases were statistically significant at 1800 ppm in both males and females. A significant increase in multiple hepatocellular adenomas were also observed in females at 900 and 1800 ppm and in males at 1800 ppm. Hepatocellular carcinoma was elevated in females at 1800 ppm. While the increase was not statistically significant, it exceeded the historical control range. Combined adenomas and carcinomas were significantly increased in females at 900 and 1800 ppm an in males at 1800 ppm. Stout et al. (2008) note that the histologic appearance of the hepatocellular lesions was consistent with those that develop spontaneously in control mice.


Previous investigations of rodents exposed to MIBK suggest a phenobarbital (PB)-like signature of changes in the liver-increased enzyme activity and hepatocellular proliferation that is transient and not sustained, starting after 3-4 days and lasting as long as 14-28 days of treatment but not longer (Kolaja et al., 1996; Whysner et al., 1996).


Regarding the mouse liver tumors, the IARC Working Group noted that “there was no evidence that the tumors arose from a cytotoxic-regenerative cell proliferation mechanism as no overt toxicity occurred in the livers of exposed mice.” The Working Group further explained that “only weak evidence exists that the tumors arose through a receptor-mediated mechanism, resulting from the induction of enzymes (CYP1A1 and CYP2B) that have been considered to be typical targets of the aryl hydrocarbon receptor and the constitutive activated receptor, respectively (Nebert et al., 2000; Zelko & Negishi, 2000).“ As a consequence, they concluded that the strength of evidence that male and female liver tumors arose through a nuclear receptor mechanism is weak.


In a non-guideline and non-GLP study, the effects of methyl isobutyl ketone (MIBK) on the mouse liver were evaluated (Geter, 2009). Male B6C3F1 mice were implanted with 5-bromo-2’deoxyuridine (BrdU) pumps and then exposed to 0 or 1800 ppm (n=6/group) of MIBK via whole-body inhalation for 6 hours/day for 7 days. In-life assessments included clinical signs and body weights. Mice were euthanized and assessed for clinical chemistry, gene expression analysis of the upper third of the left liver lobe, liver histopathological examination and BrdU proliferation analysis, and liver enzyme activity. There were no treatment-related effects noted for clinical signs, body weights, liver weights, or clinical chemistry assessments. Treatment-related findings included very slight hepatocytes hypertrophy with increased cytoplasic eosinophilia in the centrilobular/midzonal regions of the hepatic lobule which were consistent with increased smooth endoplasmic reticulum and induction of cytochrome P450 enzymes. CYP2B10 transcript levels increased 4-fold and CYP4A10 decreased 5.56-fold. This was verified by increased CYP2B10 enzyme activity (PROD) and hepatocyte proliferation. These responses are commonly observed following activation of constitutive androstane receptor (CAR) and indicate that MIBK may be an agonist ligand for CAR in mice and share a similar mode of action to that of Phenobarbital in mice. The study authors noted that this mechanism of action is not relevant to humans.


To further investigate the MoA for MIBK-induced murine liver tumors, male and female B6C3F1, C57BL/6, and CAR/PXR Knockout (KO) mice were exposed to either 0 or 1800 ppm MIBK for 6 h/d, 5 d/w for a total of 10 days (Hughes et al., 2015). On day 1, mice were implanted with osmotic mini-pumps containing 5-Bromo-2-deoxyuridine (BrdU) 1 h following exposure and humanely euthanized 1 to 3 h following the final exposure. B6C3F1 and FC57BL/6 mice had statistically significant increases in liver weights compared to controls that corresponded with hepatocellular hypertrophy and increased mitotic figures. Hepatocellular proliferation data indicated induction of S-phase DNA synthesis in B6C3F1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to control, no increase was observed in MIBK exposed CAR/PXR KO mice. Liver gene expression changes indicated a maximally-induced Cyp2b10 (CAR-associated) transcript and a slight increase in Cyp3a11(PXR-associated) transcript in B6C3F1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to controls, but not in Cyp1a1 (AhR associated) or Cyp4a10 (PPAR-a-associated) transcripts. Car/Pxr KO mice showed no evidence of activation of AhR, CAR, PXR or PPAR-a nuclear receptors via their associated transcripts.


MIBK induced hepatic effects are consistent with a phenobarbital-like MOA where the initiating events are activation of the CAR and PXR nuclear receptors leading to rodent liver tumors.


 


To investigate the potential mode of action further mechanistic studies were performed. An in vivo study in CAR/PXR knockout mice (Hughes et al., 2016), and in vitro mechanistic studies in human (Dowers, 2022) and mouse hepatocytes (Dowers, 2021) are available.  The in vivo study demonstrated a hepatic response in wild-type (WT) B6C3F1 and C57BL/6 mice exposed to 1800 ppm MIBK by inhalation (6 hours/day; 5 days/week for 2 weeks) consistent with CAR/PXR activation.  Increased liver weight was associated with centrilobular/midzonal hepatocyte hypertrophy, vacuolisation, increased mitotic figures and increased intensity of immunostaining for BrdU.  Analysis of liver mRNA transcription showed a marked increase in the transcription of Cyp2b10 (CAR-associated) mRNA; there was a slight effect on Cyp3a11 (PXR-associated) mRNA; but no effects  on the transcription of mRNA associayed with AhR (Cyp 1a1) or PPAR-alpha (Cyp4a10).  In CAR/PXR knockout (KO) C57BL/6 mice, the histopathological response was less marked and did not include an increase in the number of mitotic figures.  Immunostaining for BrdU in KO mice did not reveal any effects of exposure to MIBK.  There were no effects on gene transcription in KO mice.  The results of this study therefore indicate that the hepatic response to MIBK exposure is mediated through activation of CAR and , to a lesser extent, PXR.


The in vitro study in mouse hepatocytes indicates that MIBK is a weak CAR activator in male C57BL/6 mouse primary hepatocytes in vitro.  There was no evidence of PXR, AhR or PPAR-alpha activation in this study.  The activation of CAR was not associated with a proliferative response; however, it is noted that the CAR-mediated response was relatively weak.  The results of the in vitro study in human hepatocytes study do not indicate the potential of MIBK to activate the CAR, PXR, AhR or PPAR-alpha receptors in primary human hepatocytes.


The results of the mouse in vitro study is therefore supportive of the in vivo study, in indicating that MIBK is an inducer of CAR in the mouse liver.  The evidence for PXR activation was less marked in the mouse in vivo and was absent in the mouse in vitro.  Consequently, the liver carcinogenesis reported in the B6C3F1 mouse of both sexes can also be concluded to be CAR/PXR-mediated, a mechanism of action (MoA) not of relevance to humans.


Additional references


Kolaja KL et al. (1996) Subchronic effects of dieldrin and phenobarbital on hepatic DNA synthesis in mice and rats. Fundam Appl Toxicol 29:219-228.


Nebert DW et al. (2000) Role of the aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control, and apoptosis. Biochem Pharmacol 59:65–85.


Whysner J et al. Phenobarbital mechanistic data and risk assessment: enzyme induction, enhanced cell proliferation, and tumor promotion. Pharmacol Ther 71:153-191 (1996).


Zelko I and Negishi M. (2000) Phenobarbital-elicited activation of nuclear receptor CAR in induction of cytochrome P450 genes. Biochem Biophys Res Commun 277:1–6.