* TERRA Inc., Denver, Colorado;
Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida; and
National Institute for Environmental Health Sciences, Research Triangle Park, North Carolina
Received May 11, 2000; accepted August 7, 2000
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ABSTRACT |
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Key Words: risk assessment; carcinogenicity; cancer bioassays; estimating cancer risks; stop-exposure studies.
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INTRODUCTION |
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Conventional cancer risk assessments are generally predicated on the assumption that cancer risk increases as a function of the cumulative carcinogen dose. For exposure to a carcinogen at a given rate, this means that the excess cancer risk is a linear function of exposure duration (e.g., exposure for 7 years is assumed to carry one-tenth the cancer risk of exposure for 70 years). This is analogous to the concept of toxicity as a linear function of concentration and time, known as Haber's Law, although the original work of Haber (1924) involved the acute lethality of gases rather than chemical carcinogenesis. Evidence to support the validity of this relationship in carcinogenicity comes chiefly from studies of antineoplastic agents inducing secondary cancers in humans and primary cancers in rodents (Dedrick and Morrison, 1992; Kaldor et al., 1988
).
Despite this evidence, there is increasing concern that cancer risk may not be a linear function of cumulative carcinogen dose, and that the use of cancer potency estimates from lifetime studies may not adequately estimate cancer risks from less-than-lifetime exposures (Chen et al., 1988; Crump and Howe, 1984
; Kodell et al., 1987
; Murdoch et al., 1992
; U.S. EPA, 1998
). In part, this concern arises from inconsistency between the cumulative dose concept and current mathematical models of cancer. For example, the two-stage model of carcinogenesis predicts different risks for early exposure to initiators than for late exposures (Murdoch et al., 1992
; Portier, 1987
). Also, empirical data to support this assumption are extremely limited (e.g., Aylward et al., 1996; Dankovic and Staynor, 1998). Given that there are many situations such as short-term occupational or accidental exposures where the use of models and potency estimates developed from lifetime cancer bioassays might lead to biased cancer risk estimates, it is imperative that this assumption be evaluated critically.
The cumulative dose assumption predicts that a short-term carcinogen exposure, when averaged over the life span of the animal or human, will yield risk equivalent to a continuous exposure given at this averaged dose (e.g., U.S. EPA, 1996b). For example, the risk of cancer in an animal given dose d of a possible carcinogen for 1 year and necropsied at 2 years is assumed to be equivalent to the risks for an animal given dose d/2 continuously for the 2 years and then necropsied. It is possible to test this assumption by comparing observations from short-term versus lifetime-exposure cancer studies for specific chemicals. Tumorigenicity data for short-term and lifetime exposures exist in the literature for many chemicals, but, in general, the studies are poorly matched. That is, few studies incorporate both short-term and lifetime exposures, and differences in species, strain, dosing regimen, histopathological criteria, and other critical variables make it difficult to compare observations following lifetime exposure to a chemical in one study with those following less-than-lifetime administration in another. There is one data set, however, that is uniquely well suited to this kind of analysisthe stop-exposure studies conducted by the NTP. In these studies, some animals are exposed to the test chemical for the standard 2-year period, while other animals of the same strain are exposed for a more limited time, followed by an exposure-free period for the remainder of the 2-year study. The animals are the same age at sacrifice and analyzed collectively for tumor incidence. The dosing rate (in mg/kg/day) for the stop-exposure animals is not always the same as for 2-year-treated animals, but except for dose and duration (the 2 determinants of cumulative dose), animals in these treatment groups are probably as closely matched as can be reasonably achieved.
The analysis reported here uses data from the NTP stop studies to test the hypothesis that short-term exposure to carcinogens, when compared to lifetime exposure, result in proportional decreases in cancer risk. Data for 11 chemicals were found to be suitable for these analyses, and tumor incidence as a function of dose and time was evaluated through comparison of observations in stop-exposure and 2-year-exposure treatment groups.
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MATERIALS AND METHODS |
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Statistical Methods
The NTP has been evaluating the statistical significance of tumor-incidence rates in their 2-year cancer bioassays for many years. The methods used have included Fisher's exact test, logistic regression, Peto's test, and the poly-3 trend test. In this reanalysis of NTP data, significance of a finding was based upon the judgement of the NTP peer-review panel as stated in the summary of the technical report for each chemical; no new analysis for significance was conducted. Cancer endpoints regarded by NTP as potentially significant are summarized for each of the 11 chemicals in Table 1.
The poly-3 adjusted (Portier and Bailer, 1989) cancer rates were fit to a Weibull model using maximum-likelihood estimation. The Weibull model has the form:
![]() | (1) |
![]() | (2) |
To test whether exposure groups with stopped exposure differ markedly from the continuous-exposure groups when dose is averaged over the lifetime of the animal, a likelihood ratio test was used. The test was applied by fitting the Weibull model to all of the data (continuous and stop-exposure groups) and the maximum value of the log-likelihood is obtained (L1). A modified Weibull model was fit to the same data, again using maximum likelihood estimation (L2 = maximum value of the log-likelihood) in which the stop-exposure groups each have an additional parameter of the form:
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For each tumor response observed in a bioassay, the dose yielding an excess risk of 1% (ED01) was calculated. This is defined as the value satisfying the equation:
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![]() | (5) |
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RESULTS |
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There were only 2 cases of a 2-fold or higher increase in the ED01 (kidney adenomas in the methyleugenol study, a 3.2-fold increase; and mononuclear cell leukemia in the o-nitroanisole study, a 6-fold increase shown in Table 2). Overall, 10 of the 47 groups resulted in an increase in the ED01.
Another way to evaluate dose averaging is to determine the length of time one should average the stop-exposure dose so that the observed response falls exactly on the line resulting from fitting the continuous-exposure data. The method used for the calculation of "equivalent averaging times" is given as equation (5). An equivalent averaging time of 104 weeks would indicate that short-term exposures, averaged over the duration of the chronic study (i.e., 104 weeks), produced results consistent with continuous lifetime exposures. To illustrate the concept, equivalent averaging times for heart hemangiosarcomas for the 13-week stop-exposure group given 1,3-butadiene was 57 weeks (Table 2
). This indicates that for this chemical the stop-exposure dose (625 ppm), averaged over 57 weeks, would yield results similar to continuous lifetime exposure. Expressing this in a different way, if the dose used for this group in the dose-response analysis was 625 ppm x (13 weeks/57 weeks) = 142.5 ppm, the observed response would perfectly match the predicted response from fitting the model (Equation 1
) to the continuous-exposure-only data.
Table 5 summarizes the equivalent averaging times for the tumors/sites where these could be estimated. It is clear from this table that most of the averaging times are less than the full 2 years. In some circumstances, the equivalent averaging time was quite comparable with the actual treatment duration. Examples include forestomach squamous cell papilloma or carcinoma from 27-week stop exposure to 18,000 ppm o-nitroanisole (equivalent averaging time: 32 weeks) and Zymbal's gland adenomas and carcinomas from 13-week exposure to 2,2-bis(bromomethyl)-1,3-propanediol (equivalent averaging time: 12 weeks). In these situations, to achieve comparable results between the stop-exposure and continuous exposure tumor rates, the most appropriate averaging time for the stop-exposure was the exposure duration itself. There were also cases where the equivalent averaging time was less than the actual treatment time, such as the tumor response seen with 65-week stop exposure to coumarin compared with results from either shorter or longer exposures to the same dose (equivalent averaging time: 7 weeks) and several endpoints for 1,3-butadiene. For most endpoints, however, the equivalent averaging times for the stop-exposure studies were longer than the actual exposure durations, but less than 104 weeks, suggesting that short-term exposures were generally more effective in producing tumors than continuous exposure studies would predict. In Table 5
, the median equivalent averaging time was less than 104 weeks for 12 of the 14 dose groups for which this comparison could be made. The median equivalent averaging time for all of the groups was 62 weeks.
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DISCUSSION |
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There is ample basis to suspect that short-term exposures may yield different cancer potency estimates from continuous-exposure studies, and that the use of slope factors developed from chronic bioassays to estimate cancer risks from short-term exposures may lead to errors. If a carcinogen affects a human or animal primarily in a particular life stage, a short-term exposure during that stage may be very effective in producing cancer, while the same short-term exposure during a different life stage may be ineffective. When compared to continuous exposure over a lifetime, the apparent potency of the short-term exposure may be greater or less than that of continuous exposure, depending upon when the short-term exposure occurs relative to the period of greatest susceptibility to that particular carcinogen (Crump and Howe, 1984). For example, carcinogens that selectively affect initial steps in the carcinogenesis process would be more effective when exposure occurs early in life, while carcinogens that affect latter stages (i.e., "progressors" or "completors") would have greatest effect when exposure occurs later in life. A short-term exposure during the most relevant life stage would result in a greater potency estimate compared with continuous exposure in which much of the dose may be administered during times when the carcinogen is relatively ineffective. Consistent with this, data available on perinatal carcinogen exposure suggest that the rate of exposure in early life may more closely describe the carcinogenic potency than average lifetime exposure (McConnell, 1992
; U.S. EPA, 1996a
). These studies did not address in utero exposure, making it difficult to reach general conclusions.
Continuous exposure could also produce, on the other hand, a higher estimate of cancer potency than short-term exposure, if the short-term exposure occurs during a period of relative invulnerability. Using various models of carcinogenesis, it has been predicted that use of cancer potency values based on lifetime exposures to derive risk estimates for short-term exposures could result in a several-fold under- or over-estimation of risk, depending upon the nature of the carcinogen and the timing of the short-term exposure (Chen et al., 1988; Kodell et al., 1987
; Murdoch et al., 1992
). These theoretical observations have never been systematically evaluated using actual data until now, although there are some examples of conclusions for individual chemicals (e.g., Drew et al., 1983; Melnick et al., 1990). Information with which to compare potency of carcinogens under conditions of short-term versus continuous exposure is quite limited. For the analysis presented here, data from NTP stop-exposure studies conducted concurrently with chronic bioassays were used because they are well matched in terms of a variety of tangible and intangible variables that could affect tumorigenicity. An indication of the direction and magnitude of differences in cancer potency between stop-exposures and continuous exposures was gained by estimating cancer potency with and without inclusion of stop-exposure data. Among the 11 chemicals included in this analysis, tumor response was significantly greater than expected by dose-averaging in one or more tumor sites in the stop-exposure groups for 6 of the compounds and less than expected for 2. Inclusion of stop-exposure data with continuous-exposure data resulted in a 2-fold or greater reduction in ED01 values for at least one endpoint, compared with ED01 values derived from continuous-exposure data alone, for 6 of the 11 carcinogens. Some of the carcinogens produced increased tumors at several sites (e.g., 12 sites for 2,2-bis(bromomethyl)-1,3-propanediol). When the positive tumor data for all sites from the 11 compounds are viewed collectively, inclusion of stop-exposure data more than doubled cancer potency (i.e., 2-fold decrease in the ED01) in roughly 50% of the cases. These observations suggest that the apparent cancer potency from short-term exposures was usually greater than that from continuous exposure.
The magnitude of the difference in cancer potency derived from stop-exposures versus continuous exposures is difficult to estimate precisely. Derivation of cancer potency estimates from individual stop-exposure studies was not attemptedusually only one dose was tested in each of the stop-exposure experiments, making accurate estimation of potency difficult. In most cases, inclusion of stop-exposure data in the calculation of the ED01 for a cancer endpoint resulted in less than a 10-fold change (see Table 4), suggesting that differences in potency between stop and continuous exposures were in the range of one order of magnitude. Seven tumors from 2,2-bis(bromomethyl)-1,3-propanediol and 2 tumors for 1,3-butadiene were exceptions to this. A greater than 50-fold decrease in ED01 values were found when stop-exposure data were included for 3 cancer endpoints for 2,2-bis(bromomethyl)-1,3-propanediol and for one cancer endpoint for 1,3-butadiene, suggesting differences in potency under stop-study conditions can be large.
For o-nitroanisole, 4 sites in the stop-exposure group had tumors, while no tumors were found in these sites in the chronic groups, and for pentachlorophenol there was no evidence of carcinogenicity in the chronic groups, while two sites in the stop-exposure group had tumors. Interpretation of these very large changes is complicated somewhat by the fact that stop-exposure studies for these particular chemicals were conducted with dosages greater than those used in the continuous-exposure studies, and that tumor response for the continuous doses was sometimes very low, making measures of proportionate change unstable. As such, these very large apparent differences in potency may not be reliable, or could be due to biological effects associated with high dosing rates in the stop-exposure studies that do not occur with lesser, continuous exposure (e.g., effects involving a threshold or saturable biological process). However, they clearly indicate the possibility that high, short-term exposures could pose a cancer hazard unrecognized in a continuous exposure setting.
The implications of these findings in terms of cancer risk assessment are illustrated by observations regarding equivalent averaging times for the stop-exposure studies (Table 5). For most chemicals and endpoints, comparable cancer potencies for stop- and continuous-exposure were observed when stop-exposure doses were averaged over less than a lifetime. In a number of cases, the equivalent averaging time approximated the actual exposure duration. This suggests that if cancer potency values derived from lifetime exposures are to be used to estimate cancer risks from short-term exposures, lifetime averaging of the short-term dose may be inappropriate for some carcinogens. Averaging over a lesser period, or in some cases no averaging of the dose at all (i.e., use of the dosing rate as it occurs during the exposure period), may be necessary to avoid underestimating risk for some chemicals.
Comparisons between stop-exposure and continuous-exposure are particularly interesting for 1,3-butadiene as noted previously by Melnick et al. (1990). For example, for heart hemangiosarcomas, exposure to 1,3-butadiene for 13, 26, 40, or 52 weeks produced a much larger tumor response when compared to continuous exposure for a lifetime at the same dosing rate, with a mean equivalent averaging time for the stop-exposure groups of only 34 weeks (Table 2). These observations suggest that only a short period of exposure is necessary to account for much or all of 1,3-butadiene carcinogenicity for this endpoint, and in fact for most cancer endpoints for this chemical. For several cancer endpoints for 1,3-butadiene, longer exposure appeared to actually decrease the tumor incidence. This possibility has been anticipated by some carcinogenesis models in which premalignant cells may be more vulnerable to toxicity from the chemical during exposures in the later stages of carcinogenesis (Portier, 1987
). In this situation, after an initial exposure event, continued exposure to the chemical would reduce, rather than increase, tumor response. Some epidemiology studies of cancer mortality among workers exposed to 1,3-butadiene have found greater incidence among those with shorter, rather than longer exposures (e.g., Divine et al., 1993), although this has not been observed consistently (e.g., Matanoski et al., 1990) and it is possible that the shorter exposures may have involved higher concentrations (Landrigan, 1993
). If this phenomenon truly exists, even for only a limited number of compounds, it will be a difficult but important challenge to develop tools to effectively address this in the risk assessment process.
In some cases, tumor incidence appeared to be decreased from high doses over a shorter period of time. For example, for mononuclear cell leukemia in rats exposed to o-nitroanisole at 6000 or 18,000 ppm for 27 weeks, tumor incidence was 16.8% and 0%, respectively, while rats exposed continuously for a lifetime to 666 ppm and 2000 ppm had a tumor incidence of 82.3% and 73.3%, respectively (Table 2). In this example, much of this decrease may be explained by the very low survival in the high-dose, stop-exposure groups.
In general, it is unclear why tumorigenicity from some chemicals appears to be a linear function of exposure duration while that from other chemicals does not. Presumably, the basis for this difference lies in their mechanisms of carcinogenicity, but there are few clues offered by the limited set of chemicals available for this analysis. The carcinogenicity of most of these 11 chemicals has received little study beyond simple bioassays, and their mechanisms of carcinogenicity are largely unknown (the clear exception being 1,3-butadiene). Boutwell (1964), using an initiator-promoter regimen to study mouse skin carcinogenesis, found tumor response to be a function of cumulative dose for the initiator (9,10-dimethyl-1,2-benzanthracene, DMBA) but not for the promoter (croton oil). If these observations could be generalized to exposure duration, then a linear relationship with tumor response would be most likely for chemicals that are genotoxic. Genotoxicity studies conducted by the NTP for these chemicals are summarized in Table 6. Among the chemicals that showed the best agreement between responses from continuous- and stop-exposure (1-amino-2,4-dibromoanthraquinone, coumarin, 3,4-dihydrocoumarin, oxazepam, and salicylazosulfapyridine; see Tables 3 and 4
), only 1-amino-2,4-dibromoanthraquinone and coumarin shown strong genotoxic potential. On the other hand, the only commonality among the six chemicals for which short-term and lifetime exposures gave greater than 2-fold differences in cancer potency estimates for at least one endpoint (viz., 1-amino-2,4-dibromoanthraquinone, 2,2-bis(bromomethyl)-1,3-propanediol, 1,3-butadiene, coumarin, o-nitroanisole, and methyleugenol; see Table 4
) is that all except methyleugenol were positive in at least one Salmonella mutagenicity assay and all except 1,3-butadiene, which was not tested, and 2,2-bis(bromomethyl)-1,3-propanediol were positive in the sister chromatid exchange assay in Chinese hamster ovary cells. From these observations, no obvious relationship can be deduced between genotoxicity and the influence of exposure duration on tumor response. The relationship between tumorigenicity and cumulative carcinogen dose does not appear to be a function of any particular target organ, as there are examples of both types of responses in major target organs such as kidney, liver, and intestine. Also, there are no clear correlates with chemical structure.
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NOTES |
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