Long-term in vivo carcinogenicity study of nalidixic acid in CDF1 mice

Nalidixic acid (NA), a drug used for the treatment of urinary-tract infections, was administered in the diet to groups of 51 male and 51 female CDF1 mice at concentrations of 0, 0.08 and 0.16% for 76 wk. All the surviving animals were killed at wk 85 after 9 wk on the basal diet. Survival of the treated male and female mice was similar to that of the corresponding controls. The body weights were slightly lower in high- and low-dose males and in high-dose females than in the controls. All groups showed relatively high incidences of tumours of the small intestine, lung and haematopoietic organs in both sexes, of the liver and Harderian gland in males and of the uterus in females. However, there were no statistically significant differences between NA-treated and control mice of either sex in the incidences of tumours in any organs. It was therefore concluded that, under the conditions of this study, NA showed no carcinogenic potential in either male or female CDF1 mice.     

Use of statistics when examining lifetime studies in rodents to detect carcinogenicity

The lifetime assay for carcinogenicity that subjects groups of 50 animals per sex per dose to three doses and a control is examined for its statistical properties. Using the standard formulation of tests of hypothesis, it is shown that there is a 20-50% chance of having a false positive and that it is possible to define a "weak carcinogen" in terms of the degree of effect that would produce a false negative less than 5% of the time. Whether hypothesis testing is a proper use of statistics in this context is questioned, and alternatives are proposed.     

Urolithiasis and bladder carcinogenicity of melamine in rodents

Melamine (2,4,6-triamino-s-triazine) was administered in the diet to F344 rats or B6C3F₁ mice for 13 weeks (subchronic) or for 103 weeks (chronic) to determine its toxicologic profile, including carcinogenic potential in the chronic study. The dose levels of melamine in the subchronic studies ranged from 750 to 18,000 ppm for rats, and 6000 to 18,000 ppm for mice. In the chronic studies the dose levels of melamine were 2250 or 4500 ppm for male rats and mice of each sex, and 4500 or 9000 ppm for female rats. In these studies, compound-related lesions were observed in the urinary tract. Most noticeable was the development of uroliths (urinary bladder stones), which occurred at a greater frequency in males than females of either species. Increased incidences of urinary bladder stones and hyperplasia of the bladder epithelium were observed at 13 weeks in male rats fed diets containing melamine. In the chronic study, transitional-cell carcinomas in the urinary bladder of male rats occurred at a significantly (p ≤ 0.016) higher incidence in the 4500 ppm (high dose) group ($\text{8}\text{49}$) than in the controls ($\text{0}\text{45}$). Seven of the eight male rats with transitional-cell carcinomas of the urinary bladder also had bladder stones. There was a statistically significant association (p ≤ 0.001) between bladder stones and bladder tumors in male rats fed melamine (4500 ppm). Urinary bladder tumors were not observed in the low-dose (2250 ppm) male rat group, while bladder stones were observed in one rat in this group. In the female rat chronic study, chronic inflammation of the kidney was observed at an increased incidence (relative to controls) in both the low (4500 ppm) and high (9000 ppm) dose groups. Ulceration of the bladder epithelium was observed in male and female mice in the 13-week study. The distribution of these toxic lesions was not correlated statistically with the distribution of urinary bladder stones. Acute and chronic inflammation and epithelial hyperplasia of the urinary bladder were found in increased incidence in dosed male mice (2250 and 4500 ppm) in the chronic study. In addition, a high incidence of urinary bladder stones was observed in dosed male mice relative to controls. However, there was no evidence of bladder tumor development in this species.     

No evidence of carcinogenicity for L-ascorbic acid (vitamin C) in rodents

Carcinogenesis studies of L-ascorbic acid were conducted by offering diets containing 0, 25,000 or 50,000 ppm L-ascorbic acid to groups of 50 F344/N rats and 50 B6C3F1 mice of each sex for 103 wk. Survival of dosed and control female rats and of dosed and control female mice were comparable. Survival of high-dose male rats was slightly greater than that of the controls, whereas survival of high-dose male mice was significantly greater than that of the controls. There was no observed differences in neoplasms between treated and control groups that were considered related to L-ascorbic acid. In female rats, several lesions usually seen in aged animals showed a dose-related decline. Under the conditions of these studies, L-ascorbic acid given at 2.5% or 5.0% in the diet for 103 wk was not toxic or carcinogenic for male and female F344/N rats or for male and female B6C3F1 mice.     

Toxicity and carcinogenicity of nickel compounds

The toxicity and carcinogenicity of nickel compounds are considered in three broad categories: (1) systemic toxicology, (2) molecular toxicology, and (3) carcinogenicity. The systemic toxicity of nickel compounds is examined based upon human and animal studies. The major organs affected are discussed in three categories: (1) kidney, (2) immune system, and (3) other organs. The second area of concentration is molecular toxicology, which will include a discussion of the chemistry of nickel, its binding to small and large molecular weight ligands, and, finally, its cellular effects. The third major area involves a discussion of the carcinogenicity and genotoxicity of nickel compounds. This section focuses on mechanisms, using studies conducted in vivo and in vitro. It also includes a discussion of the assessment of the carcinogenicity of nickel compounds.     

Methylated arsenicals: the implications of metabolism and carcinogenicity studies in rodents to human risk assessment

Monomethylarsonic acid (MMA(V)) and dimethylarsinic acid (DMA(V)) are active ingredients in pesticidal products used mainly for weed control. MMA(V) and DMA(V) are also metabolites of inorganic arsenic, formed intracellularly, primarily in liver cells in a metabolic process of repeated reductions and oxidative methylations. Inorganic arsenic is a known human carcinogen, inducing tumors of the skin, urinary bladder, and lung. However, a good animal model has not yet been found. Although the metabolic process of inorganic arsenic appears to enhance the excretion of arsenic from the body, it also involves formation of methylated compounds of trivalent arsenic as intermediates. Trivalent arsenicals (whether inorganic or organic) are highly reactive compounds that can cause cytotoxicity and indirect genotoxicity in vitro. DMA(V) was found to be a bladder carcinogen only in rats and only when administered in the diet or drinking water at high doses. It was negative in a two-year bioassay in mice. MMA(V) was negative in 2-year bioassays in rats and mice. The mode of action for DMA(V)-induced bladder cancer in rats appears to not involve DNA reactivity, but rather involves cytotoxicity with consequent regenerative proliferation, ultimately leading to the formation of carcinoma. This critical review responds to the question of whether DMA(V)-induced bladder cancer in rats can be extrapolated to humans, based on detailed comparisons between inorganic and organic arsenicals, including their metabolism and disposition in various animal species. The further metabolism and disposition of MMA(V) and DMA(V) formed endogenously during the metabolism of inorganic arsenic is different from the metabolism and disposition of MMA(V) and DMA(V) from exogenous exposure. The trivalent arsenicals that are cytotoxic and indirectly genotoxic in vitro are hardly formed in an organism exposed to MMA(V) or DMA(V) because of poor cellular uptake and limited metabolism of the ingested compounds. Furthermore, the evidence strongly supports a nonlinear dose-response relationship for the biologic processes involved in the carcinogenicity of arsenicals. Based on an overall review of the evidence, using a margin-of-exposure approach for MMA(V) and DMA(V) risk assessment is appropriate. At anticipated environmental exposures to MMA(V) and DMA(V), there is not likely to be a carcinogenic risk to humans.     

Comparison of human exposures to selected chemicals with thresholds from NTP carcinogenicity studies in rodents

The National Toxicology Program (NTP) Technical Reports online database was reviewed to find chemicals that were reported to show clear evidence of carcinogenicity in the NTP rodent studies and for which data on human exposure could be found. Six representative compounds were selected. Three volatile compounds: ethyl benzene, perchloroethylene, and methylene chloride; two drugs in current use: phenytoin and primidone; and one naturally occurring, widely used, flavor: allyl isothiocyanate, were selected. The carcinogenicity data from each of the NTP Technical Reports were plotted using the Rozman scale to determine the threshold for carcinogenicity from the rodent studies. The human exposures for each chemical were calculated and compared with that threshold. The thresholds for carcinogenicity of the three volatile compounds were several orders of magnitude above the levels present in ambient air in the USA. The Threshold Limit Value (TLV) of the American Conference of Governmental Industrial Hygienists (ACGIH) for these three compounds varied between several orders of magnitude below the carcinogenicity threshold to being at the threshold. The maximum recommended doses of both drugs were at the carcinogenicity threshold. The estimated mean daily human consumption of the natural flavor was less than 100 x below the carcinogenicity threshold. This method of comparison between human exposure and animal carcinogenicity studies is more objective and informative than those in current use.     

Toxicity and carcinogenicity of chromium compounds in humans

Chromium is a human carcinogen primarily by inhalation exposure in occupational settings. Although lung cancer has been established as a consequence of hexavalent chromium exposure in smokers and nonsmokers, some cancers of other tissues of the gastrointestinal and central nervous systems have also been noted. Except for a few reports from China, little is known about the health risks of environmental exposures to chromium. Likewise, there has been a lack of epidemiological studies of human exposure to hexavalent Cr by drinking water or ingestion, and it has been suggested that humans can perhaps tolerate hexavalent Cr at higher levels than the current drinking water standard of 50 ppb. This review highlights the most recent data on the induction of skin tumors in mice by chronic drinking-water exposure to hexavalent chromium in combination with solar ultraviolet light. This experimental system represents an important new animal model for chromate-induced cancers by ingestion of drinking water, and it suggests by extrapolation that chromate can likely be considered a human carcinogen by ingestion as well. The potential use of this animal model for future risk assessment is discussed.     

Toxicity and carcinogenicity of furan in human diet

Furan is formed during commercial or domestic thermal treatment of food. The initial surveys of furan concentrations in heat-treated foods, published by European and US authorities, revealed the presence of relatively high furan levels in coffee, sauces, and soups. Importantly, furan is consistently found in commercial ready-to-eat baby foods. Furan induces hepatocellular tumors in rats and mice and bile duct tumors in rats with a high incidence. Epidemiological studies are not available. It is assumed that cis-2-butene-1,4-dial, the reactive metabolite of furan, is the causative agent leading to toxicity and carcinogenicity. Based on this data, furan is classified as a possible human carcinogen. The initial exposure estimates revealed a relatively small margin (~2,000) between human exposure and those furan doses, which induce liver tumors in experimental animals. As this may give rise for concern, in this review, the currently available toxicological and mechanistic data of furan are summarized and discussed with regard to its applicability in assessing the risk of furan in human diet.     

Chronic studies evaluating the carcinogenicity of monomethylarsonic acid in rats and mice

Monomethylarsonic acid (MMA) was administered in the diet of male and female Fischer F344 rats and B6C3F1 mice in 2-year feeding studies according to US EPA guidelines. Rats were treated with 50, 400, or 1300 ppm MMA and mice were treated with 10, 50, 200, or 400 ppm MMA based on preliminary short-term studies. The highest dose in the male and female rat groups was reduced to 1000 ppm during week 53 and then further reduced to 800 ppm during week 60 due to high mortality in the male rats. There was no treatment-related mortality in the mice. The primary target organ for MMA-induced toxicity in rats and mice was the large intestine. Toxicity was more severe in rats compared to mice and in male rats compared to female rats. The maximum tolerated dose for chronic dietary administration of MMA in rats and mice was assessed as 400 ppm, and the no effect level with regard to intestinal toxicity was assessed as 50 ppm for rats and female mice and 200 ppm for male mice. There were no treatment-related neoplastic effects detected in either the rat or the mouse.     

Toxicity and carcinogenicity of riddelliine in rats and mice

These investigations of riddelliine analyzed potential carcinogenesis and the utility of the female-rat/male-mouse design in bioassays and dose-response. Groups of 50 Fischer rats and B6C3F1 mice were gavage-administered riddelliine 5 days per week for 105 weeks. The dose levels for male rats were 0 or 1.0 mg/kg body weight; female rats 0, 0.01, 0.033, 0.1, 0.33, or 1.0 mg/kg; male mice 0, 0.1, 0.3, or 1.0, 3.0 mg/kg; and female mice 0 or 3.0 mg/kg. The dose groups were purposely designed to evaluate the dose-response relationship only in female rats and male mice. In rats, liver hemangiosarcoma, hepatocellular adenoma, and mononuclear cell leukemia were significantly increased in the 1.0 mg/kg male and female dose groups. Non-neoplastic lesions occurred in the liver and kidney of male and female rats. In mice, hemangiosarcomas increased significantly in the liver of males in the 3.0 mg/kg dose group. Alveolar/bronchiolar neoplasms in the 3.0 mg/kg dose group of female mice were significantly increased. Hepatocellular neoplasms were significantly decreased in the 1.0 mg/kg dose group of male and 3.0 mg/kg dose groups of male and female mice. Non-neoplastic lesions occurred in the liver and kidney of male and female, and lung and arteries of female mice. These studies demonstrate toxicity and carcinogenicity of riddelliine in rats and mice, and a dose-response relationship in female rats and male mice under the experimental conditions employed.     

Issues in the design and interpretation of chronic toxicity and carcinogenicity studies in rodents: approaches to dose selection

For more than three decades chronic studies in rodents have been the benchmark for assessing the potential long-term toxicity, and particularly the carcinogenicity, of chemicals. With doses typically administered for about 2 years (18 months to lifetime), the rodent bioassay has been an integral component of testing protocols for food additives, pesticides, pharmaceuticals, industrial chemicals, and all manner of byproducts and environmental contaminants. Over time, the data from these studies have been used to address an increasing diversity of questions related to the assessment of human health risks, adding complexity to study design and interpretation. An earlier ILSI RSI working group developed a set of principles for the selection of doses for chronic rodent studies (ILSI, 1997). The present report builds on that work, examining some of the issues that arise and offering new perspectives and approaches for putting the principles into practice. Dose selection is considered both from the prospective viewpoint of the choosing of dose levels for a study and from the retrospective interpretation of study results in light of the doses used. A main theme of this report is that the purposes and objectives of chronic rodent studies vary and should be clearly defined in advance. Dose placement, then, should be optimized to achieve study objectives. For practical reasons, most chronic studies today must be designed to address multiple objectives, often requiring trade-offs and innovative approaches in study design. A systematic approach to dose selection should begin with recognition that the design of chronic studies occurs in the context of a careful assessment of the accumulated scientific information on the test substance, the relevant risk management questions, priorities and mandates, and the practical limitations and constraints on available resources. A stepwise process is described. The aim is to increase insofar as possible the utility of an expensive and time-consuming experiment. The kinds of data that are most commonly needed for dose selection and for understanding the dose-related results of chronic rodent studies, particularly carcinogenicity studies, are discussed as "design/interpretation factors." They comprise both the inherent characteristics of the test substance and indicators of biological damage, perturbation or stress among the experimental animals. They may be primary toxicity endpoints, predictors or indicators of appropriate dose selection, or indicators of conditions to be avoided in dose selection. The application and interpretation of design/interpretation factors is conditioned by the study objectives-what is considered desirable will depend on the strategy for choice of doses that is being followed. The challenge is to select doses that accommodate all of the issues raised by the relevant design/interpretation factors. Three case studies are presented here that illustrate the interplay between study objectives and the design and selection of doses for chronic rodent studies. These examples also highlight issues associated with multiple plausible modes of action, multiple pathways for biotransformation of the chemical, extraneous high-dose effects, the use of modeling in dose selection, and the implications of human exposure levels. Finally, looking to the future, the report explores seven potential paradigm shifts for risk assessment that will significantly impact the design and interpretation of toxicity and carcinogenicity studies.     

Toxicity and carcinogenicity of the water disinfection byproduct, dibromoacetic acid, in rats and mice

Dibromoacetic acid (DBA) is a water disinfection byproduct formed by the reaction of chlorine oxidizing compounds with natural organic matter in water containing bromide. Male and female F344/N rats and B6C3F₁ mice were exposed to DBA in drinking water for 2 weeks (N = 5), 3 months (N = 10), or 2 years (N = 50). Concentrations of DBA in drinking water were 0, 125, 250, 500, 1000, and 2000 mg/L in the 2-week and 3-month studies, and 0, 50, 500, and 1000 mg/L in the 2-year studies. Toxic effects of DBA in the prechronic studies were detected in the liver (hepatocellular cytoplasmic vacuolization in rats and mice) and testes (delayed spermiation and atypical residual bodies in male rats and mice, and atrophy of the germinal epithelium in rats). In the 2-year studies, neoplasms were induced at multiple sites in rats and mice exposed to DBA; these included mononuclear cell leukemia and abdominal cavity mesothliomas in rats, and neoplasms of the liver (hepatocellular adenoma or carcinoma and hepatoblastoma) and lung (alveolar adenoma or carcinoma) in mice. The increase in incidence of hepatocellular neoplasms in male mice was significant even at the lowest exposure concentration of 50 mg/L, which is equivalent to an average daily dose of approximately 4 mg/kg. These studies provide critical information for future re-evaluations of health-based drinking water standards for haloacetic acids.     

Genotoxicity and carcinogenicity studies of antihistamines

This review provides a compendium of the results of genotoxicity and carcinogenicity assays performed on marketed antihistamines. Of the 70 drugs examined, 29 (41.4%) have at least one genotoxicity and/or carcinogenicity test result: 12 tested positive in at least one genotoxicity assay, six in at least one carcinogenicity assay, and four gave a positive response in both at least one genotoxicity assay and at least one carcinogenicity assay. Of 19 drugs with both genotoxicity and carcinogenicity data, eight were neither genotoxic nor carcinogenic, two were carcinogenic in at least one sex of mice or rats but tested negative in genotoxicity assays, five tested positive in at least one genotoxicity assay but were non-carcinogenic, and four gave a positive response in at least one genotoxicity assay and in at least one carcinogenicity assay. Only 12 (17.1%) of the 70 drugs examined have all data required by present guidelines for testing of pharmaceuticals, but it should be considered that a large fraction of them were developed and marketed prior the present regulatory climate.