Department of Pharmacology and Toxicology, School of Medicine, University of Louisville, Louisville, Kentucky 40292
Received February 26, 2002; accepted April 12, 2002
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ABSTRACT |
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Key Words: threshold; carcinogenicity; flavors; foods; FEMA; GRAS; methyleugenol.
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INTRODUCTION |
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The actual shape of the dose-response curve for an adverse effect of an agent in living beings can be characterized correctly only with a sufficient number of data points. Most of the carcinogenicity studies in animals have too few data points to properly define that shape, especially at the low doses to which humans are exposed. However, it has been well established, since the original paper by Gaddum (1945), that the dose should be on a logarithmic scale to effect a linear response. The scale proposed by Rozman et al.(1996), hereinafter referred to as the Rozman scale, is ideally suited for carcinogenicity studies for several reasons, one of which is that it displays all doses on the abscissa down to a single molecule, which is the smallest dose that can be administered. This allows the proper perspective for comparison between doses used in the animal studies, those to which humans are exposed, and the lowest dose that can be administered.
The compounds with carcinogenicity studies in rodents that have been approved by the FEMA (Flavor and Extract Manufacturers Association) expert panel as GRAS (Generally Recognized as Safe) and some structurally related compounds (FEMA, personal communication) were examined for dose response on the Rozman scale. It became apparent that as more data points were available from a carcinogenicity study in animals, the better the fit for a straight line showing a clear threshold. It is proposed that this empirical approach should be used in the absence of any convincing alternative.
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MATERIALS AND METHODS |
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The remaining 8 compounds were plotted on the Rozman scale. Carcinogenicity data were obtained from the cited original references. The number of tumors in the control group was subtracted from the number of tumors in each of the dosed groups. Without this correction, the shape of the curve would not change, but would only be displaced parallel to the right. Doses were converted from weight/kg/day or ppm to molecules/kg/day. The doses were those used for long-term carcinogenicity studies, typically 2-year or lifetime administration orally; in most cases they could be considered lifetime average daily doses. There were four compounds with increasing responses at two doses each (benzyl acetate, cinnamyl anthranilate, ethyl acrylate, and estragole); three compounds had increasing responses at three doses (citral, 2,4-hexadienal, and pyridine); one compound had increasing responses at four doses (methyl eugenol). Shown on each graph is the dose calculated for "eaters only" of that flavor in the United States (FEMA, personal communication). The use of cinnamyl anthranilate was discontinued in 1982, but the level consumed by humans prior to that time is shown. Also shown for methyl eugenol is the dose to eaters of this flavor from that naturally occurring in foods and for pesto eaters (Smith et al., in press).
For those with three or four data points the best fit for a linear response was calculated and drawn by SlideWrite software (Advanced Graphics Software, Inc., Encinitas, CA); the correlation coefficient was calculated by the SlideWrite program and is shown for each linear fit. A linear scale of percent tumors on the ordinate provided a better fit than either a logarithmic or probit scale. A straight line was arbitrarily drawn through those with only two data points.
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RESULTS |
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DISCUSSION |
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The Rozman scale was chosen for plotting the dose response for several reasons. Dose on a logarithmic scale is obvious because it is well established that most biological responses are related to the logarithm of the dose. This emanates ultimately from the fact that the chemical potential of a substance is related to the logarithm of the activity or concentration of a substance (Waddell and Bates, 1969). Gaddum (1945), much earlier, empirically recognized the practical significance of this relationship in pharmacology. The Rozman scale uses molecules instead of weight as a measure of the dose of the chemical; this is again an obvious advantage when comparing compounds with different molecular weights. Lastly, and most importantly, the scale is continuous to one molecule (100); this places all possible doses in perspective, which is essential when one wishes to consider the possibility that a single molecule might cause cancer.
When Avogadro proposed his constant for the number of molecules in one gram-mole, Lord Kelvin was so impressed by the enormity of the number that he made an interesting calculation to illustrate it. He said, suppose one could mark each of the molecules in a glass of water; then pour the contents of that glass into the ocean and mix that water uniformly in all the water on earth. If then one took a one-glass sample of that mixed water, there would be about 100 molecules from the original glass in water everywhere on earth (see Schrödinger, 1944). The message from this is that it is most unlikely that there is a zero concentration of any chemical in the body of any living organism. A femtogram (which is near the limit of detection with current technology) of a compound with a molecular weight of 100 still contains more than 6 million molecules.
Most of the older carcinogenicity studies in animals were designed merely to attempt to detect whether a substance is carcinogenic at any dose; indeed, many substances were carcinogenic only at or near the MTD. Furthermore, even if more than one dose produced tumors, the range of the percentage tumors from those doses usually was narrow, thus limiting confidence in the shape of the full dose-response curve. These have led to the speculation and confusion over how to extrapolate to much lower doses.
Now that more doses are being used in the range that produces tumors, there is an opportunity to evaluate the shape of that curve. It is quite striking that the NTP study of methyl eugenol found four doses ranging from 2 to 68% tumors and that linearity unquestionably is the best fit for these points (r = 0.999983). Neither logarithmic nor probit scales gave nearly as good a fit. This gives one much more confidence in extrapolating this straight line to its intersection with the abscissa. Further, the fact that all the other studies with three data points have r values very near unity, similar slopes, and intersect in the same dose range lends additional confidence to the linear interpretation. There is no apparent evidence from these plots to suggest that any other extrapolation to zero dose should be attempted; indeed, such an extrapolation from the 2% tumors for methyl eugenol to zero dose (which is not even on the abscissa) would look ridiculous.
The studies with benzyl acetate, cinnamyl anthranilate, ethyl acrylate, and estragole have only two data points and therefore the straight line drawn through these points is completely arbitrary. A line of any shape, of course, can be drawn through any two points. These studies are included for several reasons. One is that the slopes and intercepts are so similar to the studies with three or four points. Also, some of the data points are in the low percentage of tumors (below 16%), which is where many conventional extrapolations deviate exponentially to the left. These did not and therefore are in agreement with and support the studies with more data points.
The fact that all of these curves have similar slopes and intersections with the abscissa in the range of 1 x 1020 molecules might suggest that somewhere near this concentration is necessary to displace the system from normal equilibrium and toward neoplasia. Confirmation of this, of course, awaits a wider and more complete analysis of animal carcinogenicity studies. Such an analysis is being planned.
In conclusion, this analysis is interpreted to indicate that these flavoring agents have a clear threshold for carcinogenicity in animals that is well above the levels currently approved for use in foods; consequently, these animal studies should not be a cause for concern for carcinogenicity of these compounds in humans. Rather, the animal studies should be viewed as providing evidence for the safety of these compounds at current levels of human exposure.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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