The highest exposure concentrations selected for the 2-year studies were 2,500-ppm for male rats, 5,000-ppm for female rats, and 1,250-ppm for male and female mice. These same exposure concentrations and species combinations in the 14-week toxicity studies exerted minimal effects on survival, hematology, clinical chemistry, organ weights, and histopathology; the final body weights of these animals relative to controls were: 95% (male rats); 94% (female rats); 93% (male mice); and 88% (female mice) (Chan et al. 2006
; NTP 2004). These exposure concentrations, however, markedly reduced body-weight gains in rats and mice during the course of the 2-year studies; the effects on body weight were seen as early as week 14 in the rat study. Feed consumption was lower only in 5,000-ppm female rats. The neurobehavioral effects observed in 5,000-ppm female rats probably influenced their food intake. The body weight effects observed in rats and mice were likely attributable to the toxicity of 4MI at the highest exposure concentrations. MacKenzie et al. (1992) reported that male and female F344/N rats given caramel color IV (which contained 110 mg 4MI per kilogram body weight) in drinking water at 10 g/kg for 2 years had significantly lower body weights, but no accompanying histopathology. Tierney (1979)
reported that B6C3F1 mice given caramel color IV at up to 63 g/kg body weight per day in drinking water for 4 weeks also had significantly reduced body weight gains. In these studies, the body weight effects were attributed to reduced fluid intake. The reduced body weight gain observed in the present 2-year studies may be partly due to reduced water intake; however, fluid intake was not measured in the current studies.
During the 2-year study period, female rats in the 2,500- and 5,000-ppm groups showed numerous clinical findings associated with 4MI administration, whereas male rats did not. These treatment-related clinical findings included clonic seizures, unusual stance or gait, and excessive activity manifested as either hyperactivity or excitability. No microscopic lesions were observed in nervous tissues that correlated with the observed behavioral effects. The reason why female F344/N rats are more sensitive to the neurobehavioral effects of 4MI than male F344/N rats is unclear. The neurobehavioral effects displayed by female rats are consistent with those observed in farm animals fed ammoniated hay (Wiggins 1956
; Nishie et al. 1970
; Morgan and Edwards 1986a
; Nielsen et al. 1993
; Weiss et al. 1986
; Perdok and Leng 1987
). In male albino mice, a single dose of 4MI induced tremors, restlessness, running, sialorrhea, opisthotonus, Straub tail, and tonic extensor seizure (Nishie et al. 1969
). The median convulsant dose (CD50) of 4MI estimated for male albino mice by the authors was 155 mg/kg intraperitoneally and 360 mg/kg orally. In the current 2-year dosed feed study, the 1,250-ppm groups of male and female B6C3F1 mice received 4MI equivalent to 170 mg/kg body weight per day and exhibited no convulsions. In the 14-week toxicity studies of 4MI in feed (Chan et al., 2006
), the highest exposure concentration groups of male and female mice (10,000-ppm) received estimated doses of 1,840 and 3,180 mg/kg per day, respectively, and exhibited no convulsions. It is probable that the dosed feed route of administration delivered much less 4MI at any time point compared to the bolus gavage effect shown in a previous study of Nishie et al. (1969)
Mononuclear cell leukemia in F344/N rats constitutes a common background lesion, and the control rate in the current study was similar to that in historical controls; the increased incidence of leukemia in 5,000-ppm female rats slightly exceeded the historical range in feed study controls and, therefore, may be attributed to the effects of 4MI.
The liver was a target organ for 4MI toxicity in rats. Increases in the incidences and severities of several nonneoplastic hepatic lesions occurred in both sexes; these included histiocytosis, chronic inflammation, hepatocytic focal fatty change, and eosinophilic and mixed cell foci of hepatocytes. Cytoplasmic vacuolization of hepatocytes was also observed in male and female rats in the 14-week toxicity study (Chan et al. 2006
; NTP 2004a
). These lesions may be related to altered lipid metabolism and hepatic injury. The hepatic effects were consistent with increases in activities of serum alanine aminotransferase, sorbitol dehydrogenase, and alkaline phosphatase activities, as well as concentrations of bile acid reported in the 14-week toxicity study (Chan et al., 2006
; NTP, 2004a
Incidences of thyroid gland follicle cyst in 2,500-ppm male rats and thyroid gland follicle mineralization in 5,000-ppm female rats were increased. In addition, the increased incidence of thyroid gland follicular cyst in 1,250-ppm female mice was statistically significant. These cysts are commonly found in aging mice, but the increased incidences may be related to 4MI exposure.
In the 14-week toxicity studies (Chan et al., 2006
; NTP 2004a
), the 4MI treated male and female rats and mice exhibited no specific changes in serum triiodothyronine (T3), total thyroxine (T4), or thyroid stimulating hormone (TSH) levels that could be attributed to 4MI exposure. There were also no changes observed in thyroid gland histopathology at terminal sacrifice in the 14-week toxicity studies compared with controls. In the 2-year dosed feed studies of 2-methylimidazole (2MI) in rats and mice, increased incidences of thyroid gland follicular cell neoplasm was observed (NTP, 2004b
). The thyroid gland carcinogenesis induced by 2MI was probably due to increased UDP-glucuronyl transferase metabolism of T4, which in turn stimulated TSH synthesis and release, leading to neoplastic development in the thyroid gland. Sanders et al. (1998)
reported that 2MI enhanced whereas 4MI inhibited hepatic UDP-glucuronyl transferase activity. The inhibitory effects of 4MI on UDP-glucuronyl transferase probably did not affect serum T4 and TSH levels, and as a result exerted no stimulatory effect on TSH synthesis and the thyroid gland. Thus, no neoplastic changes were observed in the thyroid gland.
The incidence of prostate gland inflammation in 2,500-ppm male rats was significantly increased. Increased incidences of chronic focal lung inflammation, heart cardiomyopathy, and focal pancreatic acinus atrophy were also observed in all exposed groups of female rats. The cause of these increases was not clear.
Dose-related decreases in the incidences of benign, complex, or malignant adrenal medulla pheochromocytoma (combined) in male rats, pituitary gland adenoma in the pars distalis in both male and female rats, and clitoral gland adenoma, mammary gland fibroadenoma, and uterine stromal polyp in female rats were probably related to loss of body weight resulting from exposure concentration-related body weight loss. However, using the equations in Haseman et al. (1997)
to predict the number of neoplasms that can be explained by body weight (Haseman et al. 1997
), there appears a greater decrease in pituitary gland neoplasms in 5,000-ppm female rats than can be attributed to body weight differences alone. Likewise, there is a greater decrease in mammary gland neoplasms in all exposed groups of female rats than can be attributed to body weight differences alone. The effect of 4MI in reducing the incidences of these lesions could not be identified.
The incidences of alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in 1,250-ppm male and 625- and 1,250-ppm female mice; these increases were exposure concentration-related. The incidence of lung alveolar epithelial hyperplasia was significantly increased in 1,250-ppm female mice compared with controls. Hyperplasia of the alveolar epithelium is thought to be a precursor to neoplastic development. Interestingly, 4MI had no effect on the respiratory epithelium in the 14-week toxicity study at concentrations as high as 10,000-ppm (NTP, 2004a
). Clara cells in the terminal bronchiolar epithelium constitute a cell type from which alveolar/bronchiolar neoplasms are thought to arise. This cytochrome P450-containing cell type is capable of xenobiotic metabolism, whereas other cell types in the lung have little or no cytochrome P450. 4MI is structurally similar to 2- and 3-methylfuran. Both alkyl furans are metabolized in the Clara cell to reactive species, which may account for their pulmonary toxicity. Oxidative metabolism of heterocycles like 4MI has been reviewed by Dalvie et al. (2002)
. They suggested that oxidative metabolism of imidazoles would lead to at least two reactive intermediates, an epoxide and dicarbonyl compound, and pyruvaldehyde. The general scheme can be adapted to 4MI () (Dalvie et al. 2002
). Whether these intermediates are formed or are responsible for the development of alveolar/bronchiolar neoplasms is not known at this time. It should be pointed out that it is unlikely that an alkylating intermediate is involved in mouse lung carcinogenesis in view of the genetic toxicity-study findings that 4MI is not mutagenic in Salmonella typhimurium and does not induce micronuclei in mouse peripheral blood erythrocytes or rat and mouse bone marrow cells. The mechanism of action of 4MI in mouse lung tumorigenesis is not clear.
Under the condition of the present studies 4MI induced alveolar/bronchiolar adenoma and carcinoma in male and female mice. 4MI also induced clonic seizures and mononuclear cell leukemia in female rats, and hepatic histiocytosis, hepatocellular eosinophilic and mixed cell foci in male and female rats. The related compound methimazole was reported to induce toxic changes in the olfactory epithelium of rats and mice (Bergman and Brittebo 1999
; Genter et al. 1995
; Jeffry et al. 2006
). The nasal cavities of mice and rats exposed to 4MI were checked histologically, but no changes were noted. No other treatment-related changes were noted in any of the organs examined.