Assessment of human cancer risk from environmental carcinogen exposures remains a challenging and inexact prospect. According to the March 29, 2005 Guidelines for Carcinogen Risk Assessment
), cancer risk is estimated using a human case-specific dose-response model, if such a model is available (1
). In this instance, the effective exposure leading to a specified level of risk may be interpolated directly from the dose-response curve. Alternatively, sufficient information on mode of action and critical parameters may allow development of a biologically-based model to be used in dose-risk characterization on an agent-specific basis. In the absence of either such model, dose-response risk evaluations are derived through extrapolation of experimental animal data from a high dose-response region, which has been measured experimentally, to a low dose-response region of interest, which has not been measured. The uncertainties introduced by these extrapolations remains a point of major concern. According to the 1996 Risk Assessment Workshop overview, “The major problem in cancer risk assessment [will] continue to be the inadequacy of available [low-dose] data on which to conduct more risk assessments.”
The magnitude of the problem is outlined in . Owing to costs and other limitations, dose-response data from a typical rodent carcinogen bioassay study are generally derived from a few hundred experimental animals, and thus are statistically limited to observations covering only about one order of magnitude, between 10% and 100% incidence. The EPA conservative approach models these high-incidence data to estimate the effective dose of that carcinogen to achieve 10% incidence (ED10), and to generate a lower confidence limit on this dose estimate (LED10) as a conservative point of departure for risk extrapolation. If the carcinogen is non-genotoxic or otherwise expected to produce a non-linear response, a Margin of Exposure procedure is applied to assign an acceptable human exposure. For genotoxic carcinogens, the conservative default assumption is that carcinogen-related cancer risk will extrapolate linearly toward zero exposure, that is, will vary in direct proportion to dose, below the LED10 estimate. Risk assessments based on this procedure can involve extrapolations several orders of magnitude below the actual experimental data. For example, estimation of the lifetime dose leading to one exposure-related (i.e. above-background) cancer case per million individuals (ED10−6) would require extrapolation to five orders of magnitude below the experimentally derived LED10 estimate ().
Figure 1 Hypothetical illustration of the magnitude of extrapolation necessary to evaluate acceptable human exposure based on typical rodent bioassay data, and the design goal of the present studies to extend the range of available tumor data. Typical rodent bioassay (more ...)
Although there have been two previous large-scale rodent studies that might address this need (2
), neither study provided treatment-related tumor data below 1% incidence. In any such low-incidence study, the lower limits for examining carcinogen-related, or background-corrected tumor risk will be bounded by several factors, among the most important being background target organ cancer rate. Hence the use of as many as 4080 animals in the BIBRA study (3
) to examine N-nitrosodiethylamine (DEN) dose-response for hepatocarcinogenicity in Colworth rats provided insufficient statistical power to examine carcinogen-related tumor response below 5% because that is the approximate background cancer rate for that organ in that rat strain. The potential for large-scale studies is also severely limited by financial constraints (animal purchase and husbandry costs, carcinogen cost and availability, pathology costs) and a need for dedicated infrastructures capable of housing many thousands of individuals simultaneously.
We are investigating the feasibility of providing experimental carcinogen dose-tumor response data extending substantially below the 5% or 1% levels, using a well-established aquatic animal carcinogenesis model that circumvents many of these limitations. The species used in this study, the rainbow trout (Oncorhyncus mykiss
), has historic background liver and stomach cancer rates near 0.1% in our facility, can be reared in the tens of thousands at extraordinarily low per diem and personnel costs, requires an infrastructure far less complex and of modest size compared to rodent requirements, and has well established pathologies and protocols for carcinogenesis experimentation (5
). Based on these attributes, we have designed and completed the first of two four-part studies to determine if the rainbow trout would be capable of providing robust cancer dose-response data extending down to or below its historical background rate of 0.1% in liver.
Much attention also has been given to the possibility that readily quantifiable biomarkers of cancer risk, such as initial target organ carcinogen-DNA adduction (8
), might provide accessible measures of eventual tumor outcome at exposure levels below those that can yield routinely measurable tumor response, with protocols requiring less time, cost, and infrastructure than an equivalent tumorigenesis bioassay. An obvious limitation to this approach, however, is the need for at least some experimental data to validate or assess biomarker-tumor dose-response correlations, down to measured ultra-low tumor response levels. We have incorporated one such biomarker assessment, qualitative and quantitative target organ DBP-DNA adduct measurements, into our study design to explore low-incidence biomarker correlations. We also assessed the adduction and tumorigenesis properties of several putative DBP intermediary metabolites, cell proliferation during exposure, and oncogenic Ki-ras mutational profiles in tumors at the end of the bioassay period to better understand dose-related mechanisms of tumorigenesis in this model.
DBP was selected as the test compound for this ED001
study due to its potency and widespread distribution in the environment. DBP is considered to be the most powerful naturally occurring carcinogen of the polycyclic aromatic hydrocarbon (PAH) class when tested in animal models of cancer (11
). It is effective in multiple species (rat, mouse, trout, medaka), elicits tumors in multiple target organs (mammary gland, skin, liver, lymphoid system, lung in rodents; liver, stomach, swim-bladder in fish), and produces tumors via several routes of exposure (dietary, dermal, i.p. injection, transplacental) (11
). DBP is found in the environment in particulates formed by combustion of smoky coal (17
), in soil and sediment samples (18
), and in cigarette smoke condensate (19
). The human cancer risk posed by this potent genotoxic compound remains to be established.