James Huff

Use of Historical Control Data in Carcinogenicity Studies in Rodents

This paper considers the use of historical control data in the evaluation of tumor incidences from carcinogenicity studies in rodents. Although the most appropriate control group for interpretative purposes is always the concurrent control, there are instances in which the use of historical control information can aid an investigator in the overall evaluation of tumor incidence data. One example is rare tumors; another is a tumor that shows a marginally significant result relative to concurrent controls. However, before historical control data can be used in a formal testing framework, a number of important issues must first be considered. The nomenclature conventions and diagnostic criteria for each study should be identical to insure unambiguous identification of all relevant tumors in the historical control database. Criteria should be established that will aid in determining whether a particular study should be included in the database. This will assure a homogeneous set of studies upon which to base statistical comparisons. Since study-to-study variability in tumor rates may exceed what would be expected by chance alone, these sources of variability should be identified and controlled. Finally, statistical procedures should be employed that adjust for extra-binomial variability. This paper also summarizes tumor incidence data from untreated Fischer 344 rats and B6C3F1 mice in the National Toxicology Program (NTP) historical control database. All studies in the database are of two years duration, and all neoplasms occurring with a frequency of 0.5% or more are reported.

The carcinogenicity of dietary di(2‐ethylhexyl) phthalate (DEHP) in fischer 344 rats and B6C3F1 mice

Groups of 50 male and female Fischer 344 rats and male and female B6C3Fl mice were fed diets containing 6000 or 12,000 (rats) or 3000 or 6000 (mice) mg di(2‐ethylhexyl) phthalate (DEHP)/kg feed for 103 consecutive wk. Concurrent controls (50 of each sex and species) were fed diet without the addition of DEHP. Treatment with DEHP did not affect survival rates for rats or mice, nor did it alter the amount of food consumed. Mean body weight gains of treated male rats (6000 and 12,000 mg/kg), female rats (12,000 mg/kg), and female mice (3000 and 6000 mg/kg) were less than those of the corresponding controls. Seminiferous tubular degeneration and hypertrophy of cells in the anterior pituitary were observed in male rats at 12,000 mg/kg, and chronic inflammation of the kidney and seminiferous tubular degeneration were observed in male mice at 6000 mg/kg. Neither clinical signs of toxicity nor nonneoplastic lesions were detected in the other treated groups at incidences greater than in the corresponding controls....

Cadmium-induced Cancers in Animals and in Humans

Abstract Discovered in the early 1800s, the use of cadmium and various cadmium salts started to become industrially important near the close of the 19th century, rapidly thereafter began to flourish, yet has diminished more recently. Most cadmium used in the United States is a byproduct from the smelting of zinc, lead, or copper ores, and is used to manufacture batteries. Carcinogenic activity of cadmium was discovered first in animals and only subsequently in humans. Cadmium and cadmium compounds have been classified as known human carcinogens by the International Agency for Research on Cancer and the National Toxicology Program based on epidemiologic studies showing a causal association with lung cancer, and possibly prostate cancer, and studies in experimental animals, demonstrating that cadmium causes tumors at multiple tissue sites, by various routes of exposure, and in several species and strains. Epidemiologic studies published since these evaluations suggest that cadmium is also associated with cancers of the breast, kidney, pancreas, and urinary bladder. The basic metal cationic portion of cadmium is responsible for both toxic and carcinogenic activity, and the mechanism of carcinogenicity appears to be multifactorial. Available information about the carcinogenicity of cadmium and cadmium compounds is reviewed, evaluated, and discussed.

Benzene-, catechol-, hydroquinone- and phenol-induced cell transformation, gene mutations, chromosome aberrations, aneuploidy, sister chromatid exchanges and unscheduled DNA synthesis in Syrian hamster embryo cells.

Benzene is a human carcinogen present naturally in petroleum and gasoline. For the simultaneous assessment of benzene-induced carcinogenicity and mutagenicity, benzene and its principal metabolites, phenol, catechol and hydroquinone were examined for their ability to induce cell transformation and genotoxic effects using the same mammalian cells in culture. Each of the four compounds induced morphological transformation of Syrian hamster embryo (SHE) cells. Catechol was the most potent, inducing transformation at concentrations of 1-30 microM, followed by hydroquinone (3-30 microM), phenol (10-100 microM) and benzene (only at 100 microM). Gene mutations at two loci in SHE cells were induced by all four compounds, with catechol being the most potent; both ouabain-resistant and 6-thioguanine-resistant mutant frequencies were increased. Chromosomal aberrations in SHE cells were especially induced by catechol, lesser by hydroquinone, and to a marginal extent by phenol at only the 100 microM concentration, whereas sister chromatid exchanges in SHE cells occurred with hydroquinone (1-30 microM), catechol (10-30 microM) and phenol (1000-3000 microM). Aneuploidy in the near diploid range of SHE cells was significantly induced by benzene and catechol. All three metabolites induced unscheduled DNA synthesis in SHE cells, whereas benzene did not. This is the first report that the cell transforming activity and mutagenicity of benzene and its metabolites were assessed with the same mammalian cells in culture. The results provide evidence that benzene and several of its metabolites are cell transforming and genotoxic to cultured mammalian cells.

Fundamental flaws of hormesis for public health decisions.

Hormesis (defined operationally as low-dose stimulation, high-dose inhibition) is often used to promote the notion that while high-level exposures to toxic chemicals could be detrimental to human health, low-level exposures would be beneficial. Some proponents claim hormesis is an adaptive, generalizable phenomenon and argue that the default assumption for risk assessments should be that toxic chemicals induce stimulatory (i.e., “beneficial”) effects at low exposures. In many cases, nonmonotonic dose–response curves are called hormetic responses even in the absence of any mechanistic characterization of that response. Use of the term “hormesis,” with its associated descriptors, distracts from the broader and more important questions regarding the frequency and interpretation of nonmonotonic dose responses in biological systems. A better understanding of the biological basis and consequences of nonmonotonic dose–response curves is warranted for evaluating human health risks. The assumption that hormesis is generally adaptive is an oversimplification of complex biological processes. Even if certain low-dose effects were sometimes considered beneficial, this should not influence regulatory decisions to allow increased environmental exposures to toxic and carcinogenic agents, given factors such as interindividual differences in susceptibility and multiplicity in exposures. In this commentary we evaluate the hormesis hypothesis and potential adverse consequences of incorporating low-dose beneficial effects into public health decisions.

Results from 86 two‐year carcinogenicity studies conducted by the national toxicology program

Five categories of evidence of carcinogenicity in rats and mice were used to group interpretative results on 86 chemicals studied in recent carcinogenicity tests carried out by the National Toxicology Program (NTP). Of these studies, 50% (43/86) were regarded as showing carcinogenic effects, 42% (36/86) gave no evidence of carcinogenicity, 6% (5/86) showed equivocal evidence of carcinogenicity, and 2% (2/86) were regarded as inadequate experiments. The liver was the most frequent site of cancer in male and female Fischer-344 rats and in male and female B6C3F1 mice. Male rats appeared more sensitive than female rats to the induction of neoplasia, while for mice the females seemed more responsive. The routes of administration yielding the highest percentage (80-83%) of positive studies were gavage and inhalation; approximately one-third of the feed, drinking water, and dermal studies showed carcinogenic effects. In feeding studies, overall survival in dosed and control groups were similar, while the majority of gavage studies showed significantly reduced survival in one or more dosed groups relative to the corresponding controls. The overall percentage of studies showing carcinogenic effects (50%) agrees closely with the rate reported by other investigators for nearly 200 earlier carcinogenicity experiments conducted by the National Cancer Institute.

Sources of Variability in Rodent Carcinogenicity Studies

A number of factors may influence tumor rates in rodent carcinogenicity studies, including the animal room environment, genetic differences, food consumption/weight gain, survival/age of the animals, identification of gross lesions, pathology sampling procedures and preparation of the histology slides, and histopathologic diagnosis. The relative importance of these factors is evaluated, making use of laboratory animal carcinogenicity data from the National Toxicology Program and from other sources. An investigator must be aware of these potentially confounding factors, so that appropriate measures can be taken to reduce or eliminate their impact on the interpretation of study results. Certain potential sources of within-study variability can be controlled by appropriate experimental design and by proper conduct according to standard operating procedures. The effect of certain factors influencing tumor prevalence may be magnified when variability from study to study is considered, and thus it may be difficult to formulate a biologically meaningful statistical analysis that uses historical control data in a formal testing framework.

Long‐Term Chemical Carcinogenesis Bioassays Predict Human Cancer Hazards: Issues, Controversies, and Uncertainties

Abstract: Long‐term carcinogenesis bioassays are the most valued and predictive means for identifying potential carcinogenic hazards of various agents to humans. Agents may be chemicals, chemical mixtures, multiple chemicals, combinations of chemicals, residues and contaminants, commercial products and formulations, and various exposure circumstances. Life‐styles, dietary factors, and occupational exposure circumstances are very difficult, but not totally impossible, to evaluate experimentally. Historically, the first chemical bioassay took place in the early part of this century: Yamagiwa and Ichikawa 1 in 1915, showed that coal tar applied experimentally to rabbit ears caused skin carcinomas. Since then, nearly 1500–2000 bioassays of one sort or another have been carried out. Importantly, however, some of these bioassays must be considered inadequate for judging the absence of carcinogenicity, since there were various limitations on the way they were performed: too few animals, too short a duration, too low exposure concentrations, too limited pathology, as examples. Thus, each bioassay must be critically evaluated, especially those reported to be negative, because “false negatives” are certainly more hazardous to human health than are “false positives”. Likewise, one must be careful not to discount bioassay results simply because a target organ in rodents may not have a direct counterpart in humans (e.g., Zymbal glands 2 ), or because an organ site in rodents may not be a major site of cancers in humans (e.g., mouse liver). The design and conduct of a bioassay is not simple, however, and one must be fully aware of possible pitfalls as well as viable and often necessary alternatives. Similarly, evaluating results and interpreting findings must be approached with the utmost objectivity and consistency. These and other select issues, controversies, and uncertainties possibly encountered in long‐term bioassays are covered in this paper. One fact remains abundantly clear: for every known human carcinogen that has been tested adequately in laboratory animals, the findings of carcinogenicity are concordant.

Carcinogenicity of p-chloroaniline in rats and mice

p-Chloroaniline (PCA), a dye intermediate, was evaluated for potential long-term toxicity and carcinogenicity. Groups of 50 F344/N rats of each sex were given by gavage PCA hydrochloride in deionized water at doses of 0, 2, 6 or 18 mg/kg body weight, 5 days/wk for 103 wk. Groups of 50 male and female B6C3F1 mice of each sex were given 0, 3, 10 or 30 mg/kg on the same schedule. In general, body weights and survival were unaffected by PCA administration. In rats the group given 18 mg/kg had mild haemolytic anaemia and slight increases in methaemoglobin at various times during the study. Fibrosis of the spleen was significantly increased in all PCA-treated groups of male rats and in the 18-mg/kg group of female rats. Sarcomas of the spleen occurred in male rats, their incidence being 0/49, 1/50, 3/50 and 38/50 in control low-, mid- and high-dose groups, respectively. There was a slightly increased incidence of pheochromocytomas of the adrenal gland in both male and female rats. Dosed groups of male mice had increased incidences of hepatocellular adenomas or carcinomas (11/50, 21/49, 20/50 and 21/50 in controls, low- mid- and high-dose groups, respectively). Haemangiosarcomas of the liver or spleen were also increased in the high-dose group (incidences of 4/50, 4/49, 1/50 and 10/50 in controls, low-, mid- and high-dose groups, respectively). In conclusion, PCA was carcinogenic in male rats and male mice.

Toxicity and carcinogenicity of hydroquinone in F344/N rats and B6C3F1 mice

Toxicology and carcinogenesis studies were conducted by administering hydroquinone (more than 99% pure) by gavage to groups of F344/N rats and B6C3F1 mice of each sex for 14 days, 13 wk or 2 yr. 14-day studies were conducted by administering hydroquinone in corn oil to rats at doses ranging from 63 to 1000 mg/kg body weight and to mice at doses ranging from 31 to 500 mg/kg, 5 days/wk. In the 13-wk studies, doses for rats and mice ranged from 25 to 400 mg/kg. At those doses showing some indication of toxicity in the 14-day and 13-wk studies, the central nervous system, forestomach and liver were identified as target organs in both species and renal toxicity was observed in rats. Based on these results, 2-yr studies were conducted by administering 0, 25 or 50 mg hydroquinone/kg in deionized water by gavage to groups of 65 rats of each sex, 5 days/wk. Groups of 65 mice of each sex were given 0, 50 or 100 mg/kg on the same schedule. 10 rats and 10 mice from each group were killed and evaluated after 15 months. Mean body weights of high-dose male rats and high-dose mice were approx. 5-14% lower than those of controls during the second half of the study. No differences in survival were observed between dosed and control groups of rats or mice. Nearly all male rats and most female rats in all vehicle control and exposed groups had nephropathy, which was judged to be more severe in high-dose male rats. Hyperplasia of the renal pelvic transitional epithelium and renal cortical cysts were increased in male rats. Tubular cell hyperplasia of the kidney was seen in two high-dose male rats, and renal tubular adenomas were seen in 4/55 low-dose and 8/55 high-dose male rats; none was seen in vehicle controls or in female rats. Mononuclear cell leukaemia in female rats occurred with increased incidences in the dosed groups (vehicle control, 9/55; low dose, 15/55; high dose, 22/55). Compound-related lesions observed in the liver of high-dose male mice included anisokaryosis, syncytial alteration and basophilic foci. The incidences of hepatocellular neoplasms, primarily adenomas, were increased in dosed female mice (3/55; 16/55; 13/55). Follicular cell hyperplasia of the thyroid gland was increased in dosed mice.(ABSTRACT TRUNCATED AT 400 WORDS)