Commentary The Limits of Two-Year Bioassay Exposure Regimens for Identifying Chemical Carcinogens James Huff,1 Michael F. Jacobson,2 and Devra Lee Davis 3 1National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA; 2Center for Science in the Public Interest, Washington, DC, USA; 3Center for Environmental Oncology, University of Pittsburgh Cancer Institute, Department of Epidemiology, Graduate School of Public Health, Pittsburgh, Pennsylvania, USA BACKGROUND: Chemical carcinogenesis bioassays in animals have long been recognized and accepted as valid predictors of potential cancer hazards to humans. Most rodent bioassays begin several weeks after birth and expose animals to chemicals or other substances, including workplace and environmental pollutants, for 2 years. New ﬁndings indicate the need to extend the timing and duration of exposures used in the rodent bioassay. OBJECTIVES: In this Commentary, we propose that the sensitivity of chemical carcinogenesis bio- assays would be enhanced by exposing rodents beginning in utero and continuing for 30 months (130 weeks) or until their natural deaths at up to about 3 years. DISCUSSION: Studies of three chemicals of different structures and uses—aspartame, cadmium, and toluene—suggest that exposing experimental animals in utero and continuing exposure for 30 months or until their natural deaths increase the sensitivity of bioassays, avoid false-negative results, and strengthen the value and validity of results for regulatory agencies. CONCLUSIONS: Government agencies, drug companies, and the chemical industry should conduct and compare the results of 2-year bioassays of known carcinogens or chemicals for which there is equivocal evidence of carcinogenicity with longer-term studies, with and without in utero exposure. If studies longer than 2 years and/or with in utero exposure are found to better identify potential human carcinogens, then regulatory agencies should promptly revise their testing guidelines, which were established in the 1960s and early 1970s. Changing the timing and dosing of the animal bioassay would enhance protection of workers and consumers who are exposed to potentially dan- gerous workplace or home contaminants, pollutants, drugs, food additives, and other chemicals throughout their lives. KEY WORDS: animal cancer tests, aspartame, bioassay designs, bisphenol A, cadmium, carcinogenicity, chemical carcinogens, genistein, toluene, toxicology. Environ Health Perspect 116:1439–1442 (2008). doi:10.1289/ehp.10716 available via http://dx.doi.org/ [Online 30 June 2008] Additional research on molecular end points and other biological approaches has been recommended for evaluating the toxicity of chemicals (Committee on Toxicity Testing and Assessment of Environmental Agents 2007). However, currently and for the fore- seeable future, short- and long-term bioassays represent the most reliable way to evaluate and profile the toxicology of chemicals to which humans are exposed, especially for pre- dicting and preventing long-term adverse exposure effects, including cancer. We focus here on two potentially important means of increasing the sensitivity of carcinogenesis bioassays for identifying potentially carcino- genic food additives, pesticides, workplace chemicals and contaminants, pharmaceuti- cals, industrial chemicals, consumer products, and other natural and synthetic chemicals. Chemical carcinogenesis bioassays in ani- mals have long been recognized and accepted as valid predictors of potential cancer hazards to humans (Huff 1999, 2002; Rall 2000; Tomatis 2006; Tomatis and Huff 2002). The relevance of experimental bioassays to humans rests on four well-accepted observations: a) Experimental animals and humans are mam- mals sharing many basic genetic, pharmaco- logic, toxicologic, and carcinogenic responses; b) findings from independently conducted bioassays on the same chemicals are consistent; c) all known human carcinogens that have been tested adequately are also carcinogenic in animals and, almost without exception, share identical target sites; and d) nearly one-third of human carcinogens were first discovered to induce cancer in animals (e.g., 1,3-butadiene, diethylstilbestrol, dioxins, ethylene oxide, 2-naphthylamine, formaldehyde, vinyl chlo- ride), although most of these were not regu- lated until human evidence mounted. Thus, in light of the fact that animal bioassays predict human cancer risks, conducting more sensitive tests would better protect the public, and espe- cially workers, from involuntary exposure to animal carcinogens. Equivocal Carcinogenesis Bioassay Results Worldwide, most carcinogenesis bioassays expose rodents for ≤ 2 years, starting after birth and ending well before natural death, which typically occurs for rats at 3 years and for mice at 2.5 years. In fact, as examples out- lined below indicate, timing and duration of exposure and observation in chemical carcino- genesis bioassays may determine whether the results are positive or negative. Environmental Health Perspectives • VOLUME 116 | NUMBER 11 | November 2008 Aspartame. Two 3-year-long lifetime feeding studies using Sprague-Dawley rats, conducted by the European Ramazzini Foundation of Oncology and Environmental Sciences in Bologna, Italy, have raised ques- tions about the safety of the artificial sweet- ener aspartame (Abdo et al. 2007; Belpoggi et al. 2006; Huff and LaDou 2007; Soffritti et al. 2006, 2007). The first study began exposures at 8 weeks of age (postnatal study), whereas the second began exposures in utero. Exposures in both studies continued until the animals’ natural deaths, with the oldest rats living about 2.75–3.0 years. In the postnatal lifetime exposure study, aspartame caused mainly leukemias and lymphomas in males and females, peripheral nerve schwannomas in males, and possibly increases in total malignant tumors, kidney tumors, and brain tumors (Soffritti et al. 2006). The prenatal lifetime study showed increased incidences of lymphomas/leukemias in males and females, mammary gland cancers in females, and total tumor-bearing males (Soffritti et al. 2007). [The European Food Safety Authority and the U.S. Food and Drug Administration (FDA) dispute the conclusion of the first study and have not yet reviewed the second.] Previous 2-year feeding studies in Wistar rats did not detect increased tumor incidences (Ishii et al. 1981), although there was some debate about the occurrence of rare tumors of the brain, also observed in the Ramazzini studies, and there are general concerns regard- ing the questionable quality of other earlier negative studies (Davis 2007). Cadmium. Cadmium is another telling example of a chemical that was not shown to be carcinogenic in 2-year studies of Wistar rats (Löser 1980), but caused various tumors in the lung in a 31-month study of Wistar rats (Takenaka et al. 1983). According to Takenaka et al. (1983), “length of experimen- tal duration was the likely reason” their study, but not the other study, showed positive Address correspondence to M.F. Jacobson, Center for Science in the Public Interest, 1875 Connecticut Ave., Suite 300, Washington, DC 20009 USA. Telephone: (202) 777-8328. Fax: (202) 265-4954. E-mail: mjacobson@cspinet.org M.F.J. is employed by an advocacy organization that supports stronger testing protocols for chemicals in food. The other authors declare they have no competing ﬁnancial interests. Received 28 July 2007; accepted 30 June 2008. 1439