Longer Rodent Bioassay Fails to Address 2-Year Bioassay's Flaws

Environ Health Perspect. doi:10.1289/ehp.11964 available via http://dx.doi.org [Online 25 November 2008]

Referencing: The Limits of Two-Year Bioassay Exposure Regimens for Identifying Chemical Carcinogens

In their commentary, Huff et al. (2008) proposed that exposing experimental animals to test substances in utero and for 30 months or until their natural deaths increases the sensitivity of bioassays, avoids false negative results, and strengthens the value and validity of results. Instead, longer exposure results in increased numbers of spontaneously arising tumors, as well as increased cost and animal suffering, while failing to address the bioassay's fundamental flaws.

Although it is troubling when the bioassay produces false negative results, a far more pervasive problem is that of false positives. In our analysis of > 500 National Toxicology Program (NTP) bioassays [People for the Ethical Treatment of Animals (PETA) 2006], we found that more than half of the substances evaluated (259) produced evidence of carcinogenicity in at least one group of animals, but only about one-third of these (89) were subsequently classified as known or probable human carcinogens by the NTP itself. Even fewer of the substances, 40 and 16, respectively, were classified as carcinogens by the U.S. Environmental Protection Agency (EPA) and the International Agency for Research on Cancer (PETA 2006). This high false-positive rate is thought to be largely an indirect effect of increased cell proliferation in response to cell injury and death caused by the near toxic doses of test substances used in the bioassay (Gaylor 2005). Species-specific modes of action operating in rats or mice but not in humans, such as those mediated by 2µ-globulin, peroxisomes, and thyroid-stimulating hormone, also contribute to the high rate of false positives (Cohen 2004).

Huff et al. (2008) asserted that one of the "well-accepted observations" upon which the "relevance of experimental bioassays to humans" rests is that "findings from independently conducted bioassays on the same chemicals are consistent." In fact, in a comparison of 121 bioassays from the NTP database with those in the published scientific literature, Gottmann et al. (2001) found that the studies produced consistent results only 57% of the time.

Huff et al. (2008) cited questions about the safety of aspartame raised by 3-year bioassays conducted by the Ramazzini Foundation to support their conclusions. Although they noted that the European Food Safety Authority (FSA) and the U.S. Food and Drug Administration (FDA) dispute these studies' conclusions, the FSA's Committee on Carcinogenicity (COC 2006) observed that

In view of the inadequacies in design of the [Ramazzini Foundation] study and the use of rats with a high concurrent infection rate, the COC considered that no valid conclusions could be derived from it.

Further, the COC noted that groups of animals fed aspartame had lower body weights and thus lived longer, which may have compromised the results by leading to an apparent increase in spontaneously arising tumors. Considering that lower body weights are typically observed among animals in the bioassay's experimental groups, this is likely to generally confound the interpretation of longer bioassays.

We must stress that animals suffer during the bioassay: They live in the barren, stressful conditions of the laboratory—often including daily forced feeding or inhalation—and many also suffer from exposure to near toxic doses of test substances. These exposures often produce lethargy, anemia, diarrhea, weight loss, and other symptoms of sickness and distress. The proposal of Huff et al. (2008) to extend the length of the bioassay would obviously result in a proportional increase in this suffering.

Further, extending the bioassay runs counter to current trends in regulatory testing. Concern for the suffering of animals has caused regulatory agencies to review the usefulness of long-term studies, resulting in elimination of the 1-year dog toxicity test (U.S. EPA 2007) and an international effort to replace the two-generation reproductive toxicity test (Cooper et al. 2006). Huff et al.'s proposal thus clearly represents a step backward for toxicological science.

According to the NTP's own estimates, each bioassay requires 5 years to plan, conduct, and evaluate; 860 animals to be killed; and $2–$4 million. As a result, the NTP has conducted an average of only 12 bioassays/year over the past several decades. Considering that humans are thought to be exposed to approximately 80,000 environmental toxicants (Ward et al. 2003), it would take more than 32 millenia, 68 million animals, and $160 billion to test them all at this rate. Once again, extending the length of the bioassay would only increase these already ridiculous numbers.

The time has clearly come for antiquated animal tests such as the bioassay to be abandoned in favor of modern, human-relevant methods such as epidemiologic studies, high-throughput in vitro methods, and computational toxicology.

The authors are employed by People for the Ethical Treatment of Animals, an animal advocacy organization.

References

COC (Committee on Carcinogenicity of Chemicals in Food, Consumer Products and the Environment). 2006. Statement on a Carcinogenicity Study of Aspartame by the European Ramazzini Foundation. Available: http://www.advisorybodies.doh.gov.uk/pdfs/aspart.pdf [accessed 7 November 2008].

Cohen SM. 2004. Human carcinogenic risk evaluation: an alternative approach to the two-year rodent bioassay. Toxicol Sci 80:225–229.

Cooper RL, Lamb JC, Barlow SM, Bentley K, Brady AM, Doerrer NG, et al. 2006. A tiered approach to life stages testing for agricultural chemical safely assessment. Critical Rev Toxicol 36:69–98.

Gaylor DW. 2005. Are tumor incidence rates from chronic bioassays telling us what we need to know about carcinogens? Regul Toxicol Pharmacol 41:128–133.

Gottmann E, Kramer S, Pfahringer B, Helma C. 2001. Data quality in predictive toxicology: reproducibility of rodent carcinogenicity experiments. Environ Health Perspect 109:509–514.

Huff J, Jacobson MF, Davis DL. 2008. The limits of 2-year bioassay exposure regimens for identifying chemical carcinogens. Environ Health Perspect 116: 1439–1442.

PETA. 2006. Wasted Money, Wasted Lives: A Layperson's Guide to the Problems with Rodent Cancer Studies and the National Toxicology Program. Norfolk, VA: People for the Ethical Treatment of Animals. Available: http://www.stopanimaltests.com/pdfs/Wasted$$$.pdf [accessed 5 November 2008].

U.S. EPA. 2007. Pesticides; Data Requirements for Conventional Chemicals; Final Rule. Fed Reg 72: 60934–60988. Available: http://edocket.access.gpo.gov/2007/pdf/E7-20826.pdf [accessed 5 November 2008].

Ward EM, Schulte PA, Bayard S,  Blair A, Brandt-Rauf P, Butler MA, et al. 2003. Priorities for development of research methods in occupational cancer. Environ Health Perspect 111:1–12.

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Longer Rodent Bioassay: Huff et al. Respond

Environ Health Perspect. doi:10.1289/ehp.11964R available via http://dx.doi.org [Online 25 November 2008]

We appreciate and sympathize with Manuppello and Willett's concerns about the inappropriate use of animals in research. Significant advances in animal protection have been secured in the United States through standards that have been promulgated, and we hope that further progress will be made.

Drugs are developed today often using methods that evaluate impacts in vitro and take advantage of innovations in three-dimensional structure-activity modeling. Still, because in vitro and theoretical methods are imperfect and because relying on them alone could result in considerable harm to humans, drugs and other chemicals are studied—and are required to be studied—in animals before being tested on humans or introduced into the human environment.

Epidemiologic studies, high-throughput in vitro methods, and computational toxicology would be preferable to animal research if they provided sensitive, accurate measures of human risk. Unfortunately, epidemiology studies are insufficiently sensitive, in vitro methods are insufficiently predictive, and computational toxicology is insufficiently developed. Ideally, government would provide much greater funding to further develop those and other technologies to expedite less expensive and more accurate means of risk assessments and reduce the need for animal bioassays.

The fact that positive animal bioassays have not always been mirrored in positive human findings provides an ethical public policy challenge. If we must wait for human evidence of cancer before acting to prevent future cases, then we are conducting experiments on unwitting subjects without controls—especially if animal evidence already indicated a risk.

Current standards for concluding that an agent is a human carcinogen require statistically significant proof of sufficient numbers of cases of cancer in humans with measured or estimated exposures. Human cancers can take up to several decades to become evident in populations. Surveillance of the workplace and general environmental monitoring are not being widely conducted at this time. The last national survey of workplace carcinogens was conducted in the last century, and no new survey is planned at this time. Furthermore, except in cases where the cancer risks are enormous, such as tobacco smoking and workplace exposure to asbestos, linking a chemical outside the workplace, for example, in the diet, air, or water, to human cancers is virtually impossible.

In fact, the 2.5- to 3-year bioassays of the European Ramazzini Foundation of Oncology and Environmental Sciences on toluene, benzene, radiation, and aspartame (Soffritti et al. 2004, 2007) consistently indicate that the results of 2-year studies—the normal length of rat studies—underpredict potency/carcinogenicity and that true lifetime studies more accurately reflect cumulative impacts.

In addition, the paradigm-busting work of researchers on transgenerational effects, male-mediated teratogenesis, and the general critical impact of early developmental windows all strengthen the case for regarding 2-year postnatal bioassays as incomplete indications of toxicity/carcinogenicity (e.g., Dolinoy et al. 2007; Newbold and McLachlan 1982; Sonne et al. 2008; Swan et al. 2006).

In our commentary (Huff et al. 2008) we indicated that 2-year results tend to be biased toward the null hypothesis. In no way do we state or imply that a positive finding in a 2-year assay would be negated by a longer test, nor do we indicate the contrary, that the absence of a positive finding in a 2-year assay should be construed as proof that there is no impact.

The current typical bioassay has served society well, but it does have several flaws: It cannot provide adequate prediction for the growing proportion of the population that is now living well into their eighties and nineties; it is not designed to evaluate the impact of prenatal exposures on later life; rodents are not a perfect model for humans; and countless animals must be sacrificed to obtain admittedly imperfect results.

By focusing improved bioassays on high-volume chemicals for which there are a priori grounds for concern, current approaches promise to yield the greatest good with the least harm. Simultaneously, more research must be done to identify fast, sensitive, accurate, and economical means of identifying chemicals that might be harmful to humans.

M.F.J. is employed by an advocacy organization that supports stronger testing requirements for chemicals in food. The other authors declare they have no competing financial interests.

References

Dolinoy DC, Huang D, Jirtle RL. 2007. Maternal nutrient supplementation counteracts bisphenol A-induced DNA hypomethylation in early development. Proc Natl Acad Sci USA 104:13056–13061.

Huff J, Jacobson MF, Davis DL. 2008. The limits of 2-year bioassay exposure regimens for identifying chemical carcinogens. Environ Health Perspect 116: 1439–1442.

Newbold RR, McLachlan JA. 1982. Vaginal adenosis and adenocarcinoma in mice exposed prenatally or neonatally to diethylstilbestrol. Cancer Res 42: 2003–2011.

Soffritti M, Belpoggi F, Padovani M, Lauriola M, Degli Esposti D, Minardi F. 2004. Life-time carcinogenicity bioassays of toluene given by stomach tube to Sprague-Dawley rats. Eur J Oncol 9:91–102.

Soffritti M, Belpoggi F, Tibaldi E, Esposti DD, Lauriola M. 2007. Life-span exposure to low doses of aspartame beginning during prenatal life increases cancer effects in rats. Environ Health Perspect 115:1293–1297.

Sonne SB, Kristensen DM, Novotny GW, Olesen IA, Nielsen JE, Skakkebaek NE, et al. 2008. Testicular dysgenesis syndrome and the origin of carcinoma in situ testis. Int J Androl 31(2):275–287

Swan SH. 2006. Prenatal phthalate exposure and anogenital distance in male infants. Environ Health Perspect 114:A88–A89.