Methyl isobutyl ketone-induced hepatocellular carcinogenesis in B6C3F1 mice: A constitutive androstane receptor (CAR)-mediated mode of action.

In a National Toxicology Program (NTP) chronic inhalation study with methyl isobutyl ketone (MIBK), increases in hepatocellular adenomas and hepatocellular adenomas and carcinomas (combined) were observed in male and female B6C3F1 mice at 1800 ppm. A DNA reactive Mode-of-Action (MOA) for this liver tumor response is not supported by the evidence as MIBK and its major metabolites lack genotoxicity in both in vitro and in vivo studies. Constitutive androstane receptor (CAR) nuclear receptor-mediated activation has been hypothesized as the MOA for MIBK-induced mouse liver tumorigenesis. 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/day, 5 days/week 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 1-3 h following the final exposure. B6C3F1 and C57BL/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, and 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-α-associated) transcripts. CAR/PXR KO mice exposed to 1800 ppm MIBK showed no evidence of activation of AhR, CAR, PXR or PPAR-α 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 and resultant hepatocellular proliferation leading to rodent liver tumors.

Toxicity and carcinogenicity of methyl isobutyl ketone in F344N rats and B6C3F1 mice following 2-year inhalation exposure.

Methyl isobutyl ketone (MIBK) is primarily used as a denaturant for rubbing alcohol, as a solvent and in the manufacture of methyl amyl alcohol. Inhalation of vapors is the most likely route of exposure in the work place. In order to evaluate the potential of MIBK to induce toxic and carcinogenic effects following chronic exposure, groups of 50 male and 50 female F344/N rats and B6C3F1 mice were exposed to MIBK at concentrations of 0, 450, 900, or 1800ppm by inhalation, 6h/day, 5 days per week for 2 years. Survival was decreased in male rats at 1800ppm. Body weight gains were decreased in male rats at 900 and 1800ppm and in female mice at 1800ppm. The primary targets of MIBK toxicity and carcinogenicity were the kidney in rats and the liver in mice. In male rats, there was increased mineralization of the renal papilla at all exposure concentrations. The incidence of chronic progressive nephropathy (CPN) was increased at 1800ppm and the severity was increased in all exposed groups. There were also increases in renal tubule hyperplasia at all exposure concentrations, and in adenoma and adenoma or carcinoma (combined) at 1800ppm; these lesions are thought to represent a continuum in the progression of proliferative lesions in renal tubule epithelium. These increases may have resulted from the increased severity of CPN, either through alpha2micro-globulin-dependent or -independent mechanisms. An increase in mononuclear cell leukemia at 1800ppm was an uncertain finding. Adrenal medulla hyperplasia was increased at 1800ppm, and there was a positive trend for increases in benign or malignant pheochromocytomas (combined). In female rats, there were increases in the incidence of CPN in all exposure concentrations and in the severity at 1800ppm, indicating that CPN was increased by mechanisms in addition to those related to alpha2micro-globulin. There were renal mesenchymal tumors, which have not been observed in historical control animals, in two female rats at 1800ppm. The relationship of these tumors to exposure to MIBK was uncertain. Hepatocellular adenomas, and adenoma or carcinoma (combined) were increased in male and female mice exposed to 1800ppm. There were also treatment-related increases in multiple adenomas in both sexes.

Toxicology and carcinogenesis studies of methyl isobutyl ketone (Cas No. 108-10-1) in F344/N rats and B6C3F1 mice (inhalation studies).

UNLABELLED: Methyl isobutyl ketone is used as a denaturant for rubbing alcohol; as a solvent for paints, varnishes, nitrocellulose, lacquers, and protective coatings; in industrial extraction processes; in dry-cleaning preparations; and in the synthesis of methyl isobutyl carbinol. Methyl isobutyl ketone was nominated for study by the National Cancer Institute and the United States Environmental Protection Agency because of its widespread use, the high potential for worker exposure due to its many industrial applications, and its high production volume. Male and female F344/N rats and B6C3F1 mice were exposed to methyl isobutyl ketone (greater than 99% pure) by inhalation for 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium. 2-YEAR STUDY IN RATS: Groups of 50 males and 50 females were exposed to methyl isobutyl ketone at concentrations of 0, 450, 900, or 1,800 ppm by inhalation, 6 hours plus T(90) (12 minutes) per day, 5 days per week for 104 weeks. Survival of males exposed to 1,800 ppm was significantly less than that of the chamber controls. The mean body weights of the 900 and 1,800 ppm males were less than those of the chamber controls after weeks 97 and 89, respectively. In the standard evaluation of the kidney, there were slightly increased incidences of renal tubule adenoma and renal tubule adenoma or carcinoma (combined) in males exposed to 900 or 1,800 ppm, and renal tubule carcinoma in males exposed to 1,800 ppm. The incidences of renal tubule hyperplasia were also significantly increased in the 450 and 1,800 ppm males, and the severities were greater than in the chamber controls. Chronic nephropathy occurred in all males exposed to 1,800 ppm and in 70% to 88% of exposed females, and the severity was increased in 1,800 ppm males. The incidences of transitional epithelial hyperplasia of the renal pelvis in males exposed to 900 or 1,800 ppm and mineralization of the renal papilla in all groups of exposed males were significantly increased. In addition, two female rats exposed to 1,800 ppm had renal mesenchymal tumors. In the extended evaluation of the kidney, renal tubule adenomas and renal tubule hyperplasia occurred in all groups of exposed male rats. In the combined single and step section analysis, the incidences of renal tubule adenoma and renal tubule adenoma or carcinoma (combined) were significantly increased in males exposed to 1,800 ppm. The incidences of renal tubule hyperplasia were also significantly increased in all exposed groups of males. There was a positive trend in the incidences of mononuclear cell leukemia in males, and the incidence in the 1,800 ppm group was significantly increased. The incidence of adrenal medulla hyperplasia in the 1,800 ppm males was significantly increased. 2-YEAR STUDY IN MICE: Groups of 50 males and 50 females were exposed to methyl isobutyl ketone at concentrations of 0, 450, 900, or 1,800 ppm by inhalation, 6 hours plus T(90) (12 minutes) per day, 5 days per week for 105 weeks. Survival of males and females was similar to that of the chamber controls. The mean body weights of females exposed to 1,800 ppm were less than those of the chamber controls after week 17. The incidences of hepatocellular adenoma and hepatocellular adenoma or carcinoma (combined) were significantly increased in males and females exposed to 1,800 ppm. The incidences of eosinophilic foci were significantly increased in 450 and 1,800 ppm females. GENETIC TOXICOLOGY: Methyl isobutyl ketone was not mutagenic in Salmonella typhimurium strains TA97, TA98, TA100, or TA1535 when tested with and without hamster or rat liver metabolic activation enzymes. CONCLUSIONS: Under the conditions of these 2-year studies, there was some evidence of carcinogenic activity of methyl isobutyl ketone in male F344/N rats based on increased incidences of renal tubule neoplasms. Increased incidences of mononuclear cell leukemia in 1,800 ppm male F344/N rats may have been related to methyl isobutyl ketone exposure. There was equivocal evidence of carcinogenic activity of methyl isobutyl ketone in female F344/N rats based on the occurrence of renal mesenchymal tumors in the 1,800 ppm group. There was some evidence of carcinogenic activity of methyl isobutyl ketone in male and female B6C3F1 mice based on increased incidences of liver neoplasms. Exposure to methyl isobutyl ketone resulted in nonneoplastic lesions of the kidney characteristic of alpha2u-globulin accumulation in male rats and nephropathy in female rats.

Methyl isobutyl ketone exposure-related increases in specific measures of α2u-globulin (α2u) nephropathy in male rats along with in vitro evidence of reversible protein binding.

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 α2u nephropathy-mediated mode of action, the objective of this study is to evaluate the ability of MIBK to induce measures of α2u 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 1800ppm). Rats were exposed 6h/day for 1 or 4 weeks and kidneys excised approximately 18h post exposure to evaluate hyaline droplet accumulation (HDA), α2u staining of hyaline droplets, renal cell proliferation, and to quantitate renal α2u concentration. There was an exposure-related increase in all measures of α2u nephropathy in male, but not female rat kidneys. The hyaline droplets present in male rat kidney stained positively for α2u. The changes in HDA and α2u concentration were comparable to d-limonene, an acknowledged inducer of α2u nephropathy. In a separate in vitro study using a two-compartment vial equilibration model to assess the interaction between MIBK and α2u, 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 α2u and cause nephropathy. Together, the exposure-related increase in measures of α2u nephropathy, sustained increase in renal cell proliferation along with an indication of reversible binding of MIBK to α2u, support the inclusion of MIBK in the category of chemicals exerting renal effects through a protein droplet α2u nephropathy-mediated mode of action (MoA).

Alterations of fast axoplasmic transport in experimental methyl n-butyl ketone neuropathy.

Methyl n-butyl ketone (MBK) is known to produce a giant axonal neuropathy in man and experimental animals characterized pathologically by a gradual increase in the number of neurofilaments which become associated with focal areas of axonal swelling and thinning of the myelin sheath. Fast axoplasmic transport was studied in rats exposed to MBK. In 10 severely paralyzed rats exposed to MBK there was a significant impediment of fast axoplasmic transport following dorsal root ganglion injections (x +/- S.D. = 283.2 +/- 20.34 mm/day) compared to normal controls (417.6 +/- 23.78 mm/day). In rats undergoing injections into the ventral horn of the spinal cord there was a gradual impairment of the mean down flow rate for transport of [3H]leucine which correlated with the severity of the MBK induced neuropathy. Quantitative morphological determinations showed that the total number of neurotubules per unit cross-sectional myelin area and the number of neurotubules associated with mitochondria in swollen axons was unchanged from normal. The total number of mitochondria in randomly sampled axons varied significantly from controls but the absolute number of mitochondria associated with neurotubules was unchanged from normal. The results of these studies suggest that the impediment of fast axoplasmic transport may be related to the increased neurofilaments producing focal areas of axonal blockage.

Mechanisms of neurotoxicity as studied in tissue culture systems.

This presentation focused on describing the unique aspects of nervous tissue which make it susceptible to toxicological insult. It reviewed the published literature of compounds that have been studied in tissue culture of nerve tissue and compared the data in vivo toxicity studies with that obtained from the tissue culture system. Further, neuronal activity as a determinant of neurotoxicity was discussed as a basic mechanism to understand toxicological outcomes.

Neurotoxic interactions of industrially used ketones.

Occupationally workers are most often exposed to a mixture of solvents. Exposure limits are, however, usually set separately for single solvents. So we reviewed the present knowledge about possible neurotoxic interactions of the industrially most used ketones acetone (ACE), methyl ethyl ketone (MEK) and methyl isobutyl ketone (MIBK) with solvents in general. A literature search from the last 25 years (1974-1998) revealed 54 original publications describing neurotoxic monitoring after combined exposure (experimental and occupational) involving the mentioned ketones. Animal exposure was described in 27 reports, exposure involving human volunteers in 12 reports, and occupational surveys constituted 15 reports. Of the 54 papers, 25 dealt with potentiation by ACE, MEK or MIBK of n-hexane or 2,5-hexanedione (2,5-HD) induced neurotoxicity. Possible synergistic interactions of the ketones were reported in 12 of the 29 remaining works. Only two studies reported neurotoxic potentiation after acute short-term combined exposure to human volunteers. Possible neurotoxic potentiation by the ketones after occupational mixed exposure without the involvement of n-hexane or 2,5-HD, were reported in 8 papers. Some studies reported a different outcome of metabolic interactions based on animal or volunteer exposure, compared to more long-term occupational exposure. We conclude that the widespread use of the rule of additivity often underestimates the effect when dealing with combined exposure to industrially used ketones. We also conclude that the results of combined exposure obtained in animals or human volunteers cannot necessarily be extrapolated to occupational situations. More research is needed in particular concerning the most frequently occurring mixtures comprising ketones and aromatic solvents such as acetone (ACE) and styrene as well as methyl isobutyl ketone (MIBK) and toluene.

Olfactory adaptation, threshold shift and recovery at low levels of exposure to methyl isobutyl ketone (MIBK).

The anatomic position of olfactory receptors renders them vulnerable to airborne pollutants. Chamber inhalation studies have shown dose-dependent olfactory adaptation and temporary olfactory threshold perception shift for particular inhaled substances. The present study was undertaken to examine olfactory perception threshold (OPT) and adaptation of healthy subjects exposed for 7h to MIBK. Volunteers (n = 4) were exposed in an inhalation chamber to MIBK at concentrations of 20 ppm and 40 ppm. For each of 6 exposure days, OPT for MIBK and PM-Carbinol were determined once before exposure and at 3 successive intervals following exit. Exhaled air samples were taken at regular intervals during and after exposure. Perceived odor intensity and symptoms were ascertained through a questionnaire filled out hourly. On all days, post-exposure OPT-MIBK at chamber exit was significantly higher than pre-exposure, representing a ninefold increase in concentration; recovery was dose-dependent and not complete 95 min. after exit. No threshold shift was observed for OPT-PM-Carbinol. Perceived odor intensity was high when entering the chamber, but diminished with time, stabilizing after approximately 2 hours. Symptoms of nose, eye or throat irritation and headache were present in some subjects. The findings of this study suggest that at these levels of MIBK, there is olfactory adaptation during exposure and a transient OPT shift for the inhaled substance. Persons exposed professionally or environmentally to certain organic solvents may suffer temporary smell loss which hinders odor detection.

Hexacarbon neuropathy: tracking a toxin.

This paper will present the progression of hexacarbon studies which followed the investigation of circumstances surrounding the occurrence of peripheral neuropathy in an occupational setting. The identification of methyl n-butyl ketone as the neurotoxin led to systematic studies of the biotransformation and mechanism of neurotoxicity. A consistent line of evidence showed that the neurotoxic potential of hexacarbons is directly related to the formation of the gamma-diketone metabolite, 2,5-hexanedione. The precise nature of the chemical interaction at the neurofilament is the subject of continuing investigation.

Developmental neurotoxicology of in utero exposure to industrial solvents in experimental animals.

In summary, relatively few solvents have been examined for developmental neurotoxicology. Although most of the studies have not been replicated, the majority of the solvents tested have produced significant differences from controls. Many used inhalation, which is often the most likely route of occupational or environmental exposure. The majority have extended the exposure for much of gestation of rats. The extensive usage of solvents and the proportion of those tested which have produced positive effects (although admittedly some not at environmentally-relevant exposure concentrations), make a strong case for additional testing of industrial solvents for developmental neurotoxicology.

The effect of repeated methyl iso-butyl ketone vapor exposure on schedule-controlled operant behavior in rats.

Methyl iso-butyl ketone (MiBK), a commercial solvent, was selected by the US Environmental Protection Agency (US EPA) for testing under the Multi-Substance Rule for the Testing of Neurotoxicity (US EPA, 1993) using schedule-controlled operant behavior (SCOB) to determine if subchronic exposure to MiBK vapor had the potential to alter behavior as an indicator of neurotoxicity. Food-restricted and ad libitum-fed Sprague-Dawley male rats were exposed to 0, 250, 750, or 1500 ppm MiBK for 6 h/day, 5 d/wk for 13 weeks. SCOB testing of food-restricted animals, using a multiple fixed ratio (FR)/fixed interval (FI) schedule (FR20:FI120), was conducted prior to each exposure to maintain the operant behavior; the data from Weeks -1, 4, 8, and 13 were evaluated for evidence of neurotoxicity. SCOB testing was also evaluated for two weeks following the cessation of exposures. Ad libitum-fed animals were included to assess systemic effects using routine indicators such as changes in body weight, food consumption, and organ weight. No significant differences were seen in fixed-ratio run rate, FR pause duration, fixed-interval response rate, and index of curvature values at any concentration. Animals exposed to 750 and 1500 ppm MiBK exhibited clinical signs associated with transient reduced activity levels, but only during exposure. No signs of reduced activity were observed immediately after exposure for either group. No other treatment-related abnormalities were observed during exposure. Food-restricted animals did not demonstrate any increased or decreased sensitivity to the CNS depressive effects of MiBK relative to the ad libitum-fed animals. No treatment-related body weight differences were observed within either the food-restricted groups or the ad libitum-fed groups, although body weights of the former were clearly depressed compared with those of the latter. Relative and absolute liver, and relative kidney weights were significantly greater for the 750 and 1500 ppm ad libitum-fed animals. No differences in kidney weight were observed for food-restricted animals, but absolute and/or relative liver weights were significantly higher for all the treated food-restricted groups. The results of this study indicate that repetitive exposures to high concentrations of MiBK vapors do not result in adverse effects on operant behavior in the rat.

Comparative metabolism of methyl isobutyl carbinol and methyl isobutyl ketone in male rats.

Methyl isobutyl carbinol (MIBC) is an oxygenated solvent that is metabolized to methylisobutyl ketone (MIBK) and then to 4-hydroxymethyl-4-methyl-2-pentanone (HMP). Plasma levels of MIBC, MIBK and HMP were determined up to 12 h after a single oral 5 mmol/kg dose of MIBC or MIBK to male rats. The major material in the plasma in both cases was HMP, with similar areas-under-the-curve (AUC) and C(max) at 9 h after dosing. MIBK plasma levels and AUC were also comparable after MIBK or MIBC administration. MIBC AUC was only about 6% of the total material in the blood after MIBC, and insignificant after MIBK administration. No other metabolites were detected in the plasma under the analytical conditions used. The extent of metabolism of MIBC to MIBK, by comparing combined AUCs for MIBK and HMP, was at least 73%. The limited systemic toxicity data for MIBC are consistent with those for MIBK, which has been well studied. The metabolic equivalency of MIBC with MIBK indicates that MIBC will have a low potential for toxicity similar to that of MIBK, and reduces the need for additional animal studies.

Mechanisms in 2-hexanone potentiation of chloroform hepatotoxicity.

2-Hexanone (2-Hx) is known to potentiate chloroform (CHCl3) hepatotoxicity in part by increasing the bioactivation of CHCl3 to phosgene (COCl2). Treatment of rats with 2-Hx + CHCl3 in vivo did not initiate peroxidation of hepatic fatty acids as determined by formation of conjugated dienes or depletion of unsaturated fatty acids, or as determined by production of malondialdehyde (MDA) in vitro. A 5-fold decrease in the specific activity of succinate-dependent cytochrome c reductase in liver from rats treated in vivo with corn oil (vehicle) + CHCl3 and in rats treated with 2-Hx + CHCl3 indicated that a mechanism independent of CHCl3 bioactivation may add to the hepatotoxic effects which result from the metabolism of chloroform to phosgene.

An electrophysiological study of 2-hexanone and 2,5-hexanedione neurotoxicity in rats.

n-Hexane and its metabolites are neurotoxic to animals and man. Studies have revealed a progressive neuropathy which affects the distal regions of motor and sensory peripheral nerves. This paper describes efforts to determine whether 2-hexanone or 2,5-hexanedione is more neurotoxic than 2-hexanone and that it first affects the distal axon. Concentrations of 20 mM produced no effects after 3 weeks but 40 mM increased distal latency after 2 weeks.

Renal and hepatic interactions between 2-hexanone and carbon tetrachloride in F-344 rats.

Fisher-344 rats were pretreated with 2-hexanone (HX) and challenged with carbon tetrachloride (CCl4) in a replicated 3 X 4 factorial experiment to determine if HX potentiated CCL4-induced renal and hepatic damage. Rats given both HX and CCl4 demonstrated more severe hepatic injury at 24 and 48 h than did controls. However, in contrast to our experience with chloroform (CHCl3), CCl4-induced renal injury in HX-pretreated rats was only slightly greater than in vehicle-pretreated controls.

Gonadotoxic effects of 2-hexanone and 1,2-dibromo-3-chloropropane on the enzymatic activity of rat testicular 17 alpha-hydroxylase/C17,20-lyase.

In this study the gonadotoxic effects of 2-hexanone administered as a pretreatment and/or 1,2-dibromo-3-chloropropane administered as a challenge on the activity of several key steroidogenic enzymes in the rat testis were examined. Despite the absence of an effect when either treatment was administered individually, the pretreatment/challenge combination inhibited the steroidogenic capacity of the rat testis. The specific activity of testicular 17 alpha-hydroxylase was significantly reduced (P less than 0.05), with respect to the control, following the pretreatment/challenge combination. There were no significant differences between the control and the individual treatments. The inhibition of 17 alpha-hydroxylase activity occurred in the absence of significant differences in testis weight and testicular protein content. This inhibition of testicular steroidogenic enzyme activity was specific as the activity of another testicular enzyme, namely C17,20-lyase, was not affected by any treatment. The decline in rat testicular 17 alpha-hydroxylase activity 18 h after the 1,2-dibromo-3-chloropropane challenge precedes the reported alterations in the seminiferous epithelium following this same treatment. The results of the present study indicate that the steroidogenic cell of the testis, i.e. the Leydig cell, is a potential site for the primary toxic effects of these agents in the rat testis.

Induction of chromosome loss in yeast by combined treatment with neurotoxic hexacarbons and monoketones.

The neurotoxic hexacarbon compounds n-hexane, 2-hexanone and 2,5-hexanedione were tested in combination with acetone and methyl ethyl ketone for the potential to induce chromosome loss in strain D61.M of Saccharomyces cerevisiae. n-Hexane and 2-hexanone, alone or in combination, induced only marginally positive chromosome loss, whereas the metabolite and presumed proximal genetically active agent 2,5-hexanedione was strongly positive when tested alone and in combination. These observations are discussed in relation to the reported potentiation of the neurotoxic effects of these hexacarbons when exposure results from combinations with other solvents, e.g., acetone and methyl ethyl ketone. Treatments that result in neurotoxicity in experimental animals and humans and those that result in chromosome loss in a yeast genetic test system may be correlated by their activity on a common intracellular target.

An investigation into the short term and medium term health impacts of personal incapacitant sprays. A follow up of patients reported to the National Poisons Information Service (London).

OBJECTIVES: The aim of this study is to describe the pattern of ill health after personal incapacitant spray (PIS) exposures reported to the National Poisons Information Service-London (NPIS-L) and the Chemical Incident Response Service and to evaluate the relation between sub-categories of PIS exposure and adverse health effects. METHODS: Case series study of patients reported to the NPIS-L, by attending medical personnel during the period 16 January to 31 September 1998. Data collected by questionnaire sent to these medical personnel. RESULTS: Several "adverse" symptoms, particularly dermatitis and blisters were reported for cases exposed to police PIS. These cases were more frequent than in those people exposed to non-police PIS. Adverse effects occurring more than six hours after exposure were also observed, which is in conflict with the recorded immediate, short lived, and self limiting symptoms that PIS are designed to cause. Most patients with persisting symptoms required further treatment. CONCLUSIONS: These findings suggest that the formulation of CS (o-chlorobenzylidine malononitrile) with MiBK (methyl iso-butyl ketone) used by the police is more harmful that has been previously assumed. If confirmed then the continued use of this formulation should be reviewed because of longer duration of adverse effects. Less concentrated formulations may reduce the severity or persistence of the adverse effects.