Bioassay bashing is bad science. (Correspondence).

The Spheres of Influence, "Assessing Assays" (Schmidt 2002), in the May 2002 issue of EHP criticizes the National Toxicology Program rodent bioassay (NTPRB) without discussing its importance in regulation and public health. The Spheres article (Schmidt 2002) does not express concern for the validity of alternative transgenic methods proposed to replace the NTPRB for detecting carcinogens. By focusing on the limitations of the NTPRB without mentioning limitations of transgenic alternatives, Spheres creates the impression that transgenics are superior. Attempting to supplant the rodent bioassay with various mutation tests, DNA repair tests, cell transformation tests, and many others is a history of failure (Johnson 2000, 2001; Johnson and Snell 1986; Rall et al. 1987). The Spheres article (Schmidt 2002) does not provide any evidence to persuade us that transgenics will change that history.

To illustrate bias, Spheres (Schmidt 2002) states that

   Rodent models used to test potential carcinogens
   are by their nature "wrong" because they merely
   simulate the response of the real target species--humans.

However, the Spheres article (Schmidt 2002) gives no evidence to explain why rodent models are "wrong," or why by similar reasoning transgenics would not also be wrong. NTPRB results sometimes provide and precede the same indication of carcinogenicity found in humans (Huff, 1993; IARC, 2002; Tomatis, 1979). These responses in rodents have proven to be accurate, that is, not "wrong."

Also, according to a statement attributed to James MacDonald at Shering Plow Research Institute (Kenilworth, NJ), conventional rodent models predict human cancer no better than "a flip of the coin." This statement is disingenuous. Although the NTPRB has identified human carcinogens, no one knows for sure with what accuracy it will predict future human carcinogens. If the "coin flip" notion were true, then it would be just as true for transgenics. However, no reasonable person would suggest replacing established methods to identify carcinogens with "coin flips." Importantly, few human carcinogens have been tested thoroughly in any model, and very few known, human noncarcinogens are available to evaluate the negative responses in the model systems.

The Spheres article (Schmidt 2002) concludes with a quotation by Samuel Cohen: "My hope is that this is the first step toward getting rid of the two-year bioassay altogether." Obviously, Cohen ignores the fact that the rodent bioassay is an accepted regulatory standard and before we "get rid of it," an alternative method must be developed and validated.

Validation is a rigorous scientific process whereby the performance of a model is compared against the performance of accepted methods. Validation is governed by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM 2002). Although some people may express a preference for transgenic alternatives, no new method will gain regulatory acceptance without ICCVAM. In the case of rodent carcinogenesis tests, until proposed replacements are validated, regulatory agencies will be expected to use the standard bioassay for identifying carcinogenic agents.

The NTPRB represents a serious effort to evaluate agents by essentially one standard protocol: two species (usually Fischer rats and B6C3[F.sub.1] mice), both sexes, exposures lasting for two-thirds of the lifetime, and multiple doses including the maximum tolerated amount (Bucher 2000; Chhabra et al. 1990; Fung et al. 1995; Haseman et al. 2001; Huff 1999a, 1999b; Huff and Haseman 1991). Virtually all tissues are examined for tumors. This standard protocol has been adopted throughout the world.

Although criticism has been leveled against the bioassay (Ames and Gold 1990, 1997; Johnson 1999, 2000), some of it has been misguided (Tomatis et al. 2001). While questions of exposure levels and numbers of animals have been raised (Johnson 2002), these issues are not necessarily relevant for all of the > 500 rodent studies that have been done. For example, methylene chloride was tested at concentrations to which workers are exposed; butadiene at levels 150 times below the Occupational Safety and Health Administration standard (Huff et al. 1985; Melnick and Huff 1992); benzene at concentrations near those found in gasoline (Huff et al. 1989); dibromoethane at or below concentrations found in grain silos (Huff 1983b); tetranitromethane at levels found in the munitions industry (Bucher et al. 1991); and dibromochloropropane at levels found in manufacturing plants (Huff 1983a). Several other chemicals may fit into this category (Huff 1999a, 1999b; NTP 2002).

When transgenic models are contemplated as replacements for standard rodent systems, many of the same uncertainties arise, and effects of dose, numbers of animals per dose group, duration, routes of administration, and genotype are generally unknown. In the case of transgenics, however, we also need to know to what extent the power of detection may be altered compared to that provided by the NTPRB. For example, would a suggested 6-month study using 15 transgenic mice per dose per group be adequate to detect a rare, late-developing or weak tumor response we find in the standard NTPRB protocol? Even for the NTPRB, duration of exposure might be lengthened to increase sensitivity (Haseman et al. 2001; Maltoni 1995), especially for late-appearing carcinogenic effects (Soffretti et al. 2002).

For transgenic models, standards for practically all experimental variables remain under debate, and few comparisons between the NTPRB and transgenics have been made using the same chemicals. We have much less experience with transgenic models than we have with the NTPRB. Background control tumor rates in transgenic systems are only now becoming sufficiently well known for comparative evaluation. A transgene may alter responsiveness to different classes of chemicals. Thus, a transgene may make a system more sensitive for detecting one chemical class or tumor type, and less sensitive for another. Also, statistical false positives and false negatives, investigated in the NTPRB (Haseman and Elwell 1996), have not been explored in transgenics.

The question of how to predict human carcinogens more effectively would benefit from fundamental research. The number of genes contributing to increases or decreases in susceptibility to the carcinogenic effect of one chemical or another is unknown. In order for one or a few transgenic models to be able to accurately predict which chemicals will be carcinogenic and which will not, the number of genes that actually determine susceptibility to chemical carcinogens in human populations must necessarily be quite small. However, the number and variety of genes could be large, and different genes are likely involved in carcinogenicity of different chemicals. Conceivably, one-half or more of the genome could be involved in carcinogenic responses. Until we better appreciate the number and kind of genotypes involved in carcinogenic responses, our understanding of what a carcinogen is will remain incomplete and how we manage carcinogenic risks in our environment uncertain. The need for fundamental research is not a reason to change existing prevention strategies; however, results of that research could lead to future changes. Advocates of transgenic models do not seem to realize the importance of fundamental research and thus may repeat errors made in development of conventional rodent assays.

Transgenics are not a panacea for avoiding uncertainties associated with rodent bioassays (Johnson 2001). Because of transgene-specific and chemical-specific enhanced sensitivity, negative results in transgenics may only mean that the "correct" transgene was not used. Positive results may only reflect hypersensitivity not found in humans. Further, choices of particular transgenes are virtually limitless. Therefore, particular models, such as the mouse embryonic zetaglobin promoter fused to activated v-Ha-ras (Tg.AC), may or may not show the appropriate response to a given chemical. Furthermore, having additional responses from other similarly uncertain indicators will not help differentiate carcinogens from noncarcinogens.

Regardless of some uncertainties, the NTPRB is the accepted regulatory standard currently being used to protect worker safety and public health from carcinogens (e.g., Huff 1999a, 1999b; Maltoni 1995; Rall 2000; Tomatis et al. 2001; Tomatis and Huff 2001, 2002). Transgenic models must demonstrate equal or better performance before they can be accepted as replacements.

We thank the reviewers for their valued comments and suggestions.

Frank M. Johnson
E-mail: [e-mail]

James Fluff
National Institute of Environmental
Health Sciences
Research Triangle Park, North Carolina
E-mail: [e-mail]

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