© Oxford University Press 2006.
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Researchers Exploring Faster Alternatives to 2-Year Test for Carcinogenicity
For nearly 40 years, scientists have relied on a costly, time-consuming test called the 2-year bioassay to screen chemicals for carcinogenicity. This assay exposes mice and rats of both sexes to a given substance at the maximum tolerated dose (and one or two lower doses) for the animal's lifetime—about 2 years. Chemicals that produce more tumors in test animals than control subjects are flagged as carcinogens, and this designation triggers regulatory measures to set human exposure limits with the goal of protecting the public's health.
Health agencies in the United States and abroad view the 2-year bioassay as a "gold standard" for carcinogen identification. Most human carcinogens listed by the Environmental Protection Agency in its Integrated Risk Information System were first identified with the assay, and the U.S. Food and Drug Administration requires it in premarket studies of drugs destined for long-term clinical use.
But despite its entrenched regulatory role, the assay's costs and time requirements have long been a source of contention. Each assay requires millions of dollars, years of planning, and extensive preliminary dosing studies. Bruce Ames, Ph.D., professor of biochemistry and molecular biology at the University of California at Berkeley, has found that more than half of all chemicals evaluated test positive in the 2-year bioassay. He attributes this to the maximum tolerated dose, which he claims overwhelms the body's natural detoxification mechanisms independently of a substance's carcinogenicity.
What's more, the assay's continued relevance to humans is becoming more dubious. A growing body of evidence shows that some chemicals produce cancer in rodents through species-specific mechanisms that are irrelevant to human physiology. Therefore, many in the scientific community suggest that screening resources should target biological endpoints that humans and rodents share in common.
The debate has intensified in light of a huge backlog of untested chemicals in global commerce and because of a growing need to expedite new drug approvals at the FDA, says James MacDonald, Ph.D., executive vice president for preclinical development at the Schering-Plough Research Institute in Kenilworth, N.J. According to MacDonald, the number of new drugs approved by the FDA fell from 53 in 1998 to just 17 this year.
Still Useful?
Samuel Cohen, M.D., Ph.D., chair of the Department of Pathology and Microbiology at the University of Nebraska Medical Center in Omaha, has led the charge among scientists that it is time to drop the 2-year bioassay. "It's outdated and no longer necessary," he insisted. "It has already served its purpose. Let's take what we've learned from it and move on."
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Other scientists concur in principle but concede that regulatory pressures force a slower pace of change. "I've never made a human risk decision based solely on the results of a 2-year bioassay, and in the 30 years I've been doing this, I rely on those data less and less," MacDonald said. "But as in all human endeavors, the devil is in the detail. Reduction to practice is the challenge."
Cohen was among the first to demonstrate that rodent carcinogenic pathways don't always mirror those of humans. In a seminal series of studies launched in 1974 while he was working at St. Vincent Hospital in Worcester, Mass., he showed that male rats get bladder cancer from saccharin—an artificial sweetener—through a rodent-specific mechanism induced only with extraordinarily high doses. It took years to convince other scientists of these findings and even longer to convince regulators and scientists with the National Toxicology Program (NTP) at the National Institutes of Health, which finally dropped saccharin from its list of human carcinogens in 2000. Today, most scientists agree with Cohen's finding: namely that saccharin causes cancer by binding to 
2u globulin, a protein with which it forms crystals that mechanically irritate rat bladder and kidney cells. Humans don't make this protein and thus cannot develop cancer via the same pathway.
Another example is phenobarbital, which is carcinogenic in rats because it raises levels of thyroid-stimulating hormone (TSH), which in turn triggers a cancerous proliferation of thyroid follicular cells. Human TSH doesn't react the same way to phenobarbital, so the public's risk of cancer from this chemical is negligible.
An Alternative
The increased knowledge about carcinogenic differences between rodents and humans coincides with an emerging view that cancer arises from fewer pathways than scientists once assumed. Cohen suggests that most of the meaningful, early-stage events in rodent carcinogenesis have already been identified and that, moreover, these events can be tested in a 13-week, in vivo assay, which obviates the need for the 2-year counterpart altogether. He first proposed the 13-week alternative in an article published in Carcinogenesis in 1984 and has since refined it in later papers, including a detailed framework described in the November 2003 issue of Critical Reviews in Toxicology.
According to his model, a 13-week assay—combined with structure–activity models of chemical effects—should focus on four endpoints: genotoxicity, immunosuppression, estrogenicity, and increased cell proliferation. Short-term assessments of these effects, Cohen says, should provide predictive signals that allow scientists to determine the likelihood of cancer in rodents, as well as the human relevance of the associated pathways. Four decades of testing with the 2-year bioassay have provided more than enough data to extrapolate long-term outcomes, he adds. "If I can pick up 2u globulin activity within 13 weeks, then I know the chemical will eventually cause cancer in male rats by generating bladder or kidney granules," he said. "I don't need another 2-year bioassay to tell me that."
A recent study co-authored by Robert Maronpot, Ph.D., chief of the Laboratory of Experimental Pathology at the National Institute of Environmental Health Sciences, substantiates this view. Published in Toxicologic Pathology in 2004, the study shows that pooled observations from studies done in less than 3 months of liver carcinogens in mice and rats predict the 2-year cancer response. Specifically, Maronpot and colleagues reviewed NTP data gathered over 10 years and found that subchronic lesions including hepatocellular necrosis, hypertrophy, and cytomegaly are associated with liver cancer at 2 years, particularly when combined with increased liver weight.
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But despite these findings, Maronpot didn't back Cohen's call to abandon the 2-year bioassay now. Rather, he described Cohen's 90-day alternative as a "reasonably good hypothesis" that still needs to be tested. "And that's what we're trying to do," he said. "If we can get good data for all the endpoints he's looking for, we might be able to get reasonably good prediction. But the regulatory and toxicology communities will need more tangible evidence that a 90-day study will be predictive enough."
A technical working group focused on cancer hazard identification, which operates under the umbrella of the Health and Environmental Sciences Institute (HESI) in Washington, D.C., could provide that evidence. Co-chaired by MacDonald, this group is exploring the hypothesis that carcinogenic signals can be identified in short-term studies exclusive of the 2-year bioassay. According to MacDonald, the group recently completed a study matching short-term signals with 2-year cancer outcomes for 17 chemicals in the NTP database. The data are currently being analyzed. While declining to comment specifically on the findings, MacDonald did say they were "not as simple as we would have liked." He added, "I don't think we're going to get an easy yes-or-no answer. We're planning our next experiment, which will look into what we might have missed and whether what we missed was important."
High-Throughput Advances
Meanwhile, scientists are continuing to explore other cancer-screening alternatives, particularly high-throughput screens based on genomic, proteomic, and metabolomic cancer biomarkers. Rusty Thomas, Ph.D., director of the Functional Genomics Program at the CIIT Centers for Health Research in Research Triangle Park, N.C., is currently screening a subset of NTP chemicals for biomarkers in rodents that might predict the 2-year assay's outcome. His most recent (unpublished) data suggest that genomic biomarkers are more predictive than those derived from metabolomic studies. However, Thomas was reluctant to concede that any molecular screen could substitute for the 2-year bioassay entirely. "What I believe is that these screens could help us to prioritize chemicals for long-term testing," he said. "That would produce monetary savings that we could devote toward mechanistic studies of a chemical's low-dose effects."
This is precisely the tack that the NTP proposes to take. In its new "Roadmap for the 21st Century," the NTP describes plans to use high-throughput screens as priority-setting tools for long-term carcinogenicity testing. Christopher Portier, Ph.D., the NTP's outgoing associate director, and one of the 2-year bioassay's strongest supporters, suggests that the screens will help scientists identify low-risk chemicals for further research. Meanwhile, Portier says, the 2-year bioassay will be increasingly applied to "anchoring studies" of a few select chemicals whose mechanistic features are common to a broader set of compounds.
William Farland, Ph.D., acting deputy administrator in EPA's Office of Research and Development, applauds that approach. "I think the ability to focus in on these archetypical chemicals will help us determine which need to go through the 2-year bioassay and where we can use more data to make preliminary judgments about carcinogenicity," he said. Nevertheless, the 2-year bioassay remains the most stringent test, Farland emphasized. "You might be able to make predictions about carcinogenesis based on a few preneoplastic endpoints, or molecular biomarkers," he suggested. "But how do we know when we've looked at enough of them?"
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As it stands now, more than 500 chemicals, including industrial chemicals, natural products, and drugs, have been tested in the 2-year bioassay since it first came into use. Bill Jameson, Ph.D., director for the Report on Carcinogens at the NTP, says the number of chemicals evaluated with the test is dropping, mainly because of budget constraints and the push for mechanistic studies. Whether these tests ultimately replace the 2-year bioassay remains to be seen.
The 2-year test does allow researchers to study potential long-term effects to chemical exposure—a measure that's not likely to come from the short-term or in vitro assays.
"How much does it cost to save a child with cancer and how much is it worth it to society to prevent that cancer in the first place?" Portier asked. "Where do you draw the line regarding what's too long, or too costly, or what requires too many animals? We're trying to identify things in the environment that could wind up making you very sick."
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