The short-term inhalation toxicity of MIBK has been previously characterized; however, no studies investigating the chronic toxicity and carcinogenicity of MIBK have been reported. The objective of the present studies was to characterize the toxicity and carcinogenicity of MIBK in F344N rats and B6C3F1 mice following exposure to 0, 450, 900 or 1800 ppm for two years by inhalation. The primary findings of these studies are presented in this report; a more detailed report can be found in NTP TR 538 (NTP, 2007
Exposure concentrations for the present 2-year studies in rats and mice were selected following the review of previously conducted inhalation toxicity studies (Phillips et al., 1987
). Based on the toxicity data from these studies, it was anticipated that 2000 ppm would likely exceed the maximum tolerated dose for a 2-year exposure period. Therefore, 1800 ppm was selected as the highest exposure concentration for the present studies in both rats and mice. The lower exposure concentrations were spaced by half to examine the dose-response of toxic or carcinogenic effects of MIBK.
In the present study, exposure of male rats to MIBK resulted in decreased survival at 1800 ppm and decreased body weight at 900 and 1800 ppm. These effects were not observed in female rats, or male mice. There were decreased body weights in female mice at 1800 ppm. The major target organ sites of toxicity and carcinogenicity were the kidney in rats and the liver in mice.
CPN was observed in almost all male rats including the chamber controls. However, there were treatment related significant increases in both the incidence (1800 ppm) and severity (all exposed groups). Although CPN is one of the most commonly recognized spontaneous lesions in the rat (Seely et al., 2002
), this condition can be exacerbated by chemical exposure, leading to increased incidences and average severities (Lock and Hard, 2004
). CPN as a syndrome is more prevalent and severe in male rats. However, there were also significant increases in both the incidence (all exposed groups) and severity (1800 ppm) of CPN in females. Hyperplasia of the transitional epithelium lining the renal pelvis was increased in exposed males, and the increases were significant at 900 and 1800 ppm. Such hyperplasia frequently accompanies severe CPN (Montgomery and Seely, 1990
), and the increased incidences in the current study may reflect the exacerbated CPN. Linear mineralization of the epithelium of collecting ducts in the renal papilla, which frequently accompanies CPN, was significantly increased in all exposed groups of males.
Because renal tubule hyperplasia, adenoma, and carcinoma represent a continuum in the progression of proliferative lesions in the kidney and because significant increases in hyperplasia and marginal increases in adenoma, carcinoma, and adenoma or carcinoma (combined) were identified by the standard single-section evaluation of the kidney in males, step sections were evaluated in an extended evaluation. Additional hyperplasias in all exposed groups and adenomas in all groups were identified in the step section analysis; additional carcinomas were not identified. When the results of the single and step sections were combined, there were significant increases in renal tubule hyperplasia at all exposure concentrations and in adenoma and adenoma or carcinoma (combined) at 1800 ppm. These lesions were not observed in females; however, renal mesenchymal tumors were identified in two female rats in the 1800 ppm exposure group. This very rare tumor has not been observed in male or female control animals fed NTP 2000 diet in NTP studies (all routes of administration). In treated F344/N rats fed NTP 2000 diet, mesenchymal tumors were identified in only one male and three female rats in three 2-year studies including the current study. The occurrence of this neoplasm in only two female rats makes the relationship to MIBK exposure uncertain.
The spectrum of kidney lesions described in the literature after short-term exposures to MIBK (Phillips, et al., 1987
; Nemec, et al., 2004
) supports the observation that the kidney is the primary target in rats. In the 3-month study of MIBK (Phillips, et al. 1987
), there were dose-related increases in the severity of hyaline droplet formation and regeneration of PCT cells in male rats, although the highest dose tested in the 13-week study was 1,000 ppm. In the present study, there were increases in the incidence and severity of CPN, linear mineralization in the renal papilla, and renal tubule hyperplasia in male rats. Minimal hyaline droplet accumulation was observed in two 900 ppm and two 1800 ppm male rats that died relatively early in the study; hyaline droplet formation diminishes with age and is unlikely to be detectable in rats at the end of a 2-year study. These lesions are suggestive of α2u-globulin nephropathy, a syndrome specific to male rats.
α2u-Globulin neophropathy is thought to be a possible mechanism of xenobiotic-induced renal carcinogenesis (Montgomery and Seely, 1990
); the pathophysiology, diagnostic characteristics, and relevance of α2u-globulin neophropathy in human risk assessment have been extensively discussed in the literature (Swenberg et al., 1989
; Borghoff et al., 1990
; USEPA, 1991
; IARC, 1999
). The proposed sequence of events involved in the induction of α2u-globulin neophropathy includes binding of a chemical to the male rat specific protein α2u-globulin, accumulation of the complex in phagolysosomes of renal proximal tubule cells (hyaline droplets), and a cycle of cytotoxicity, apoptotic death and compensatory cell proliferation, that if chronic, may lead to the promotion of neoplasia. Alternatively, it has been proposed that α2u-globulin may serve as a vector to increase the delivery of a toxicant or protoxicant to proximal tubule cells, leading to an elevated chemical concentration in the male rat kidney (Melnick, 1992
). Further studies, would be necessary to more definitively identify MIBK as an inducer of α2u-globulin nephropathy. These studies would include a thorough characterization of renal histopathology, characterization of the binding affinity of MIBK with α2u-globulin, and quantitation of cell proliferation, at exposure concentrations of 2000 ppm or greater, with inclusion of doses used in the present study.
Doi and coworkers recently reviewed selected NTP studies of compounds that induced α2u-globulin nephropathy; studies examined in this review were d-limonene, decalin, Stoddard Solvent IIC, and propylene glycol mono-t-butyl ether (Doi, et al., 2007
). The authors identified lesions generally observed in prechronic studies, which included α2u-globulin accumulation, cell proliferation, hyaline droplet accumulation, tubular regeneration, and granular cast formation, and lesions generally observed in chronic studies, which included CPN, mineralization, and renal tubule hyperplasia. The authors concluded that exacerbated CPN and linear mineralization in the renal papilla, indicators of sustained damage, were the best predictors of the tumor outcome, but that the pathologic presentation of α2u-globulin nephropathy was variable and that no one event or set of pathologic events clearly predicted tumor response.
Increases in cell turnover, associated with CPN arising from α2u-globulin dependent or independent mechanisms, are recognized kidney tumor risk factors (Hard, 1998
; Hard et al., 1997
; Swenberg et al., 1989
). There does not appear to be a counterpart of CPN or α2u-glubulin nephropathy in humans. In an evaluation of the renal histopathological changes occurring in ethylbenze-treated rats from NTP-sponsored studies (NTP, 1999
), Hard concluded that tumor development was due to chemically-induced exacerbated CPN (Hard, 2002
); these effects occurred in male and female rats. Increases in both exacerbated CPN and renal tubule tumors have been noted in several other NTP studies, including coumarin (NTP, 1993
), primidone (NTP, 2000
) and benzophenone (NTP, 2006
). Thus, the increase in the severity of the CPN in the present study, whether dependent on or independent of α2u-globulin, likely contributed to the increase in renal tubule tumors. α2u-Globulin dependent mechanisms did not appear to be involved in the renal effects observed in any of these studies. Conversely, although there were increases both the incidence and severity of CPN in female rats, the association between MIBK exposure and renal tumor induction was uncertain, and there was no evidence of renal tubule tumor induction, in females. The exacerbated CPN in females was not due to α2u-globulin nephropathy, as female rats produce little if any hepatic α2u-globulin and thus do not develop α2u-globulin nephropathy (MacInnes et al., 1986
; Chatterjee et al., 1989
; Lehman-McKeeman and Caudill, 1992
There were findings in tissues other than the kidney in male rats. Although there was a significant increase in mononuclear cell leukemia at 1800 ppm, the strength of the response made the finding uncertain. There were increases in adrenal medulla hyperplasia, which were significant at 1800 ppm. There was also a positive trend for increases in benign or malignant pheochromocytomas (combined); however increases were neither statistically significant nor exceeded the historical control range. Nemec et al. (2004)
reported significant changes in adrenal gland weights of female Sprague-Dawley rats exposed to 2,000 ppm MIBK for 70 days, which were the F0 generation of a two generation reproductive study.
The liver was the sole target tissue in male and female B6C3F1 mice in the present study. Eosinophilic foci were increased in females at 450 and 1800 ppm; incidences in exposed males were similar to controls. Hepatic foci are more frequently observed in mice treated with hepatocarcinogens than untreated controls, and although there is evidence linking these lesions to the development of hepatocellular neoplasms, their exact role in hepatocarcinogenesis is still uncertain (Harada et al., 1999
). In general, these lesions precede the development of hepatic neoplasms and may increase in incidence and multiplicity with time and administration of liver carcinogens. However, while some foci progress to neoplasia, others regress when the inciting carcinogenic stimulus is removed.
Induction of hepatocellular neoplasms following MIBK treatment was similar between male and female mice. In both sexes, increases in hepatocellular adenomas and adenoma or carcinomas (combined) were significant and exceeded historical control ranges at 1800 ppm. There was also increased multiplicity of hepatocellular adenomas at 900 ppm in females and 1800 ppm in males and females. There were no significant increases in carcinomas in either sex, although the incidence was elevated in females at 1800 ppm. Although hepatocellular adenoma is the most frequent spontaneous liver neoplasm in B6C3F1 mice, the combination of increased hepatocellular neoplasms and increased multiplicity in exposed males and females supports the carcinogenic activity of MIBK in the liver. The similarities in exposure-responses between males and females suggest that similar mechanisms are responsible for the induction of hepatocellular tumors in male and female mice, or at least are independent of the sex of the animal.
Findings reported in prechronic studies provide support for the liver as the target organ of MIBK-related toxicity in mice. Effects on the liver in prechronic studies included increased liver weights and centrilobular hypertrophy (Phillips et al., 1987
, Nemec et al. 2004
). Although the significance of hepatocellular hypertrophy in the liver carcinogenic response is not completely understood, it was identified as the best single predictor of liver cancer in a recent survey of 111 NTP studies over a 10-year period (Allen et al., 2004
). Although it has been considered an adaptive response to excessive metabolic load (Schulte-Hermann, 1974
), hepatocellular hypertrophy was not observed during histologic evaluation of mice in the present study. Induction of P450 enzymes by MIBK may have contributed to the observed lesions in the liver in mice.
In conclusion, following two-year inhalation exposure, the targets of toxicity and carcinogenicity of MIBK were the kidney in rats and the liver in mice. In male rats, the observed CPN and increase in renal tubular tumors following MIBK exposure may have resulted from an α2u-globulin-related mechanism; however, since exacerbated CPN was also observed in female rats, additional mechanisms were likely involved. Increases in mononuclear cell leukemias in male rats and the occurrence of two renal mesenchymal tumors in female rats were uncertain findings. Increases in hepatocellular tumors and tumor multiplicity were similar between male and female mice and MIBK was considered to be a hepatocarcinogen in both sexes.