Experimental design constraints on carcinogenic potency estimates

The multistage model is used by U.S. regulatory agencies to calculate estimates of the carcinogenic potency (beta) of chemicals; the data for these estimates are generally obtained from chronic rodent bioassays. Three quantities characterize each group tested in the chronic bioassay: the dose level, the sample size, and the number responding to the dose. The dose levels tested are fixed by conventional protocols; the typical National Toxicology Program (NTP) experimental design calls for use of the maximum tolerated dose (MTD), one-half and one-fourth MTD, plus a control group. Only rarely are doses even one order of magnitude less than the MTD utilized in chronic bioassays. This experimental design constraint on dose selection limits the possible values of beta that can arise from multistage model analyses of chronic bioassay data. Sample size is also constrained by the experimental design of the chronic bioassay; the typical sample size in NTP studies is 50 animals. Occasionally, fewer animals are used, but only rarely are more. Thus, the multistage model which theoretically has three variable quantities with which to estimate carcinogenic potency, has in practice only one: the incidence of treatment-related response. Even this can vary within only a narrow range determined by sample size, the control incidence, and the level of statistical significance desired. The net result of these design constraints is that carcinogenic potency estimates derived from multistage-model analyses of chronic bioassay data may vary within only a narrow range surrounding the inverse maximum dose tested. We have illustrated this by calculating the largest possible finite potency estimates that could have arisen from the experimental designs used to test 82 mouse carcinogens in chronic bioassays. On average these maximum potency estimates were within one order of magnitude of the inverse maximum dose tested. We thus conclude that the chronic rodent bioassay, in and of itself, is altogether inadequate as a data source for estimating the risk to humans from exposure to carcinogenic chemicals.     

The ranking of chemicals for carcinogenic potency

A relatively new approach to assessing the carcinogenic hazard that chemicals pose to man involves the development of criteria by which carcinogens can be ranked regarding potency. Several such systems have been proposed which can be grouped into dose-dependent, mechanistic, and response-dependent classification systems. In this communication, examples of each type of classification system are described and the utility of these systems in assessing the carcinogenic hazard of chemicals is explored. Each of the classification systems which have been proposed has certain problems associated with it. However, in view of the potential utility of this approach, further efforts to develop a classification system for ranking carcinogens appear warranted. Such a system for ranking carcinogens could possibly be used in conjunction with low-dose extrapolation in the regulatory process. Once a carcinogen has been ranked for potency this ranking could influence the low-dose extrapolation model used in estimating the human risk associated with exposure to that carcinogen. The more potent the carcinogen, the more conservative the lowdose extrapolation model used.     

Cobalt and its compounds: update on genotoxic and carcinogenic activities

Abstract

This article summarizes recent experimental and epidemiological data on the genotoxic and carcinogenic activities of cobalt compounds. Emphasis is on the respiratory system, but endogenous exposure from Co-containing alloys used in endoprostheses, and limited data on nanomaterials and oral exposures are also considered. Two groups of cobalt compounds are differentiated on the basis of their mechanisms of toxicity: (1) those essentially involving the solubilization of Co(II) ions, and (2) metallic materials for which both surface corrosion and release of Co(II) ions act in concert. For both groups, identified genotoxic and carcinogenic mechanisms are non-stochastic and thus expected to exhibit a threshold. Cobalt compounds should, therefore, be considered as genotoxic carcinogens with a practical threshold. Accumulating evidence indicates that chronic inhalation of cobalt compounds can induce respiratory tumors locally. No evidence of systemic carcinogenicity upon inhalation, oral or endogenous exposure is available. The scarce data available for Co-based nanosized materials does not allow deriving a specific mode of action or assessment for these species.     

The calculation and use of carcinogenic potency: A review

Six methods of estimating carcinogenic potency now in use to some extent by regulatory agencies are examined. It is concluded that none of these methods is adequate for regulatory decision making when used alone, as is usually the case, but that all are useful as a contribution to the whole risk assessment process. The needs for further development and improvement of these methods, where appropriate to human risk, are identified and discussed in view of the growing use of potency estimates in regulatory decision making.     

Quantitative factors in chemical carcinogenesis: variation in carcinogenic potency

The quantitative assessment of toxicological data on the carcinogenic potential of chemicals requires consideration of a number of factors, including mathematical models of the mechanism of carcinogenic action and pharmacokinetic models for the metabolic activation of the parent compound to its reactive metabolite. In this article, the use of such models in estimating carcinogenic potency and in predicting risks at low levels of exposure is discussed, along with other factors involved in the evaluation of carcinogen bioassay data. The Carcinogenic Potency Database (CPDB) established by Gold et al. (1984, Environ. Health Perspect. 58, 9-322) is used to illustrate the application of quantitative approaches to carcinogenic risk assessment and to examine the variation in the potency of chemical carcinogens. Based on an analysis of 585 experiments selected from the CPDB, the risk-specific (10(-6) doses (RSDs) obtained by linear extrapolation from the TD50 were generally within a factor of 5-10 of those derived from the linearized multistage model. The RSDs obtained by linear extrapolation from the TD50 are roughly log-normally distributed with a median of about 20-90 ng/kg/day, depending on the subset of the CPDB considered. This distribution has been used by Rulis (1986, in Food Protection Technology (C. W. Felix, Ed.), pp. 29-37, Lewis, Chelsea, MI) to explore the concept of a threshold of regulation for chemical carcinogens present in the environment at low levels.     

Small difference in carcinogenic potency between GBP nanomaterials and GBP micromaterials

Materials that can be described as respirable granular biodurable particles without known significant specific toxicity (GBP) show a common mode of toxicological action that is characterized by inflammation and carcinogenicity in chronic inhalation studies in the rat. This study was carried out to compare the carcinogenic potency of GBP nanomaterials (primary particle diameter 1-100 nm) to GBP micromaterials (primary particle diameter >100 nm) in a pooled approach. For this purpose, the positive GBP rat inhalation carcinogenicity studies have been evaluated. Inhalation studies on diesel engine emissions have also been included due to the fact that the mode of carcinogenic action is assumed to be the same. As it is currently not clear which dose metrics may best explain carcinogenic potency, different metrics have been considered. Cumulative exposure concentrations related to mass, surface area, and primary particle volume have been included as well as cumulative lung burden metrics related to mass, surface area, and primary particle volume. In total, 36 comparisons have been conducted. Including all dose metrics, GBP nanomaterials were 1.33- to 1.69-fold (mean values) and 1.88- to 3.54-fold (median values) more potent with respect to carcinogenicity than GBP micromaterials, respectively. Nine of these 36 comparisons showed statistical significance (p < 0.05, U test), all of which related to dose metrics based on particle mass. The maximum comparative potency factor obtained for one of these 9 dose metric comparisons based on particle mass was 4.71. The studies with diesel engine emissions did not have a major impact on the potency comparison. The average duration of the carcinogenicity studies with GBP nanomaterials was 4 months longer (median values 30 vs. 26 months) than the studies with GBP micromaterials, respectively. Tumor rates increase with age and lung tumors in the rat induced by GBP materials are known to appear late, that is, mainly after study durations longer than 24 months. Taking the different study durations into account, the real potency differences were estimated to be twofold lower than the relative potency factors identified. In conclusion, the chronic rat inhalation studies with GBP materials indicate that the difference in carcinogenic potency between GBP nanomaterials and GBP micromaterials is low can be described by a factor of 2-2.5 referring to the dose metrics mass concentration.     

Automated and reproducible read-across like models for predicting carcinogenic potency

Several qualitative (hazard-based) models for chronic toxicity prediction are available through commercial and freely available software, but in the context of risk assessment a quantitative value is mandatory in order to be able to apply a Margin of Exposure (predicted toxicity/exposure estimate) approach to interpret the data. Recently quantitative models for the prediction of the carcinogenic potency have been developed, opening some hopes in this area, but this promising approach is currently limited by the fact that the proposed programs are neither publically nor commercially available. In this article we describe how two models (one for mouse and one for rat) for the carcinogenic potency (TD₅₀) prediction have been developed, using lazar (Lazy Structure Activity Relationships), a procedure similar to read-across, but automated and reproducible. The models obtained have been compared with the recently published ones, resulting in a similar performance. Our aim is also to make the models freely available in the near future thought a user friendly internet web site.     

Quantitative assessment of a human carcinogenic potency for propylene oxide

The potential for causing carcinogenic and mutagenic effects is the main concern when assessing the risks associated with low-level exposures of humans to the industrially important epoxide, propylene oxide (PO). The available basic information used in estimation of carcinogenic risk has been reviewed. It is concluded that the published data from gavage studies in rodents are less appropriate and that observed cancer incidences from long-term inhalation should preferably be utilized for quantitative risk assessment. Furthermore, PO and ethylene oxide (EO) are directly acting alkylating agents which exhibit several similarities. Although data are less comprehensive for PO than for EO, PO appears to yield a rather uniform alkylation pattern in various tissues. Also, similarly to EO, PO is probably detoxified at a rate which does not vary widely in various mammalian species, including man. For these reasons, the surface-based extrapolation model for estimation of the human equivalent dose may not be appropriate, and the previously derived carcinogenic potency factors should be revised downward. Alternative risk estimates are provided. From the most relevant available studies, we propose a carcinogenic potency factor of 0.001 (mg/kg/day)-1 for PO in humans by inhalation.     

Autoxidative activation of the nematocide 1,3-dichloropropene to highly genotoxic and mutagenic derivatives: consideration of genotoxic/carcinogenic mechanisms

1,3-Dichloropropene (1,3-DCP) is used as a soil nematocide worldwide. Technical grade 1,3-DCP is genotoxic/mutagenic and carcinogenic. Talcott and King reported that mutagenic activity is lost after purification of 1,3-DCP samples via silica gel column chromatography. We found that mutagenicity and SOS repair in Escherichia coli, strain PM 21, are strongly reduced after purification via silica gel and that mutagenicity and induction of SOS repair depend on oxidative impurities and secondary products. Both isomers (E and Z) of 1,3-DCP are oxidized to 1,3-dichloropropene epoxide (1,3-DCP-Ox). The epoxide is subjected to rapid internal rearrangement to 2,3-dichloropropanal (2,3-DCPA), which spontaneously eliminates HCl and forms the extremely mutagenic, genotoxic, and carcinogenic 2-chloroacrolein (alpha-chloroacrolein) alpha-ClA. Thus, the genotoxic/mutagenic effects of unpurified 1,3-DCP samples mainly depend on alpha-ClA. The underlying genotoxic and mutagenic mechanism is formation of promutagenic exocyclic 1,N(2)-propanodeoxyguanosine adducts of alpha-ClA. Pure 1,3-DCP samples have only a very low S(N)1 reactivity as measured in trifluoroacetic acid solvolysis reactions, hydrolysis, and computed reactivities but possess a moderate S(N)2 reactivity as determined in alkylation tests with the nucleophiles 4-(p-nitrobenzyl)pyridine (NBP) and N-methyl-mercaptoimidazole (MMI). Evidently, the low S(N)1 reactivity is not sufficient to form necessary amounts of O(6)-alkylguanine DNA adducts required for back-mutation in Salmonella typhimurium strain TA1535. The S(N)2 reactivity may, however, lead to other DNA adducts, e.g., N7-guanine adducts, which can induce error prone repair in S. typhimurium strain TA100 and thus lead to back-mutation in this strain. Application of 1,3-DCP samples in agriculture must be considered as a mutagenic risk because the samples can be oxidized and form the extremely mutagenic alpha-ClA. As a consequence, it is questionable whether any stabilizers can prevent oxidation during application.     

Comparison of teratogenic and carcinogenic risks

Risk assessment for teratogens has received much less attention than carcinogens. This may be due in part to the lack of biologically based dose-response models for teratogens. Also, there appears to be a perception that cancer risks far exceed teratogenic risks for most chemicals. This perception may be due in large part to the stringent practice of using conservative procedures to restrict cancer risk estimates to one in 100,000 or lower whereas procedures used to restrict teratogens may result in much higher risks. The purpose of this paper is to compare estimates of teratogenic and carcinogenic risks for chemicals when both effects occur in the same animal species. Similar procedures are employed here to obtain teratogenic and carcinogenic risk estimates for nine chemicals. When a similar standard of risk is applied to both effects, the relative potency of teratogens to carcinogens varied from an order of magnitude lower to an order of magnitude higher for these nine chemicals.     

Practical and theoretical aspects of the DCB assay for carcinogenic and other genotoxic agents

The DNA-cell binding (DCB) assay grew from our early studies demonstrating that in the presence of carcinogenic chemicals, radioactively labeled nucleic acids attached strongly to isolated cellular membranes and to intact prokaryotic and eucaryotic cells. A survey of over 300 chemicals revealed that this test is capable of correctly predicting carcinogenic potential in more than 95% of cases. Recently we began using formaldehyde-treated Escherichia coli indicator cells and noted that the accuracy of prediction and the sensitivity of the test are even greater. For example, a class of noncarcinogenic, intercalating agents that showed as false positives in the original study, scored negative in this modified version of DCB test. This assay has several advantages over other rapid tests for genotoxicity, including simplicity, rapidity, economy, and reproducibility. As demonstrated by our studies on a number of diverse products, it is especially suitable for analyzing industrial and other environmental chemicals and their mixtures. The increased DNA-cell binding is due to a property shared by most, if not all, carcinogenic agents—their ability to produce macromolecular adducts between proteins and nucleic acids, as well as inter- and intramolecular complexes in DNA. The latter class of adducts is responsible for inducing chromosomal transposition-like events that we observed in a separate set of experiments using transformation of Bacillus subtilis for measuring distances between genetic markers. In these experiments markers were stably transferred to a different position on the chromosome as the result of DNA-exposure to an ultimate carcinogen, N-acetoxy N-acetyl-aminofluorene. If, as many investigators believe, the specific chromosomal translocations are the cause of neoplasia, the high predictive potential of the DCB test may be due to its capability to measure the production of specific macromolecular complexes.     

Extrapolation of the carcinogenic potency of fibers from rats to humans

In 1999 Berry published a model for mesothelioma incidence following fiber exposure. He concluded, that the influence of the solubility of fibers on the mesothelioma rate is 17 times higher in humans than in rats. This conclusion may be helpful for evaluating the carcinogenic risk from man-made vitreous fibers, but it had little influence on some recent discussions. It has been demonstrated using this model, that in an injection experiment with rats, fibers with elimination constants of 0.1/year and 1/year--which would approximately correspond to crocidolite and perhaps ceramic fibers--differ in their mesothelioma risk only by a ratio of 3.2:1. In contrast, for humans exposed continuously from age 20 to age 60 a risk ratio of 4,750:1 is obtained. This result may be helpful for the assessment of the human cancer risk e.g., from exposure to refractory ceramic fibers. However, uncertainty is large, since the life-span of rats is too low to measure the elimination rate of bio-persistent fibers sufficiently.     

Search for compounds that inhibit the genotoxic and carcinogenic effects of heterocyclic aromatic amines

Over the last 30 years approximately 160 reports have been published on dietary compounds that protect from the mutagenic and carcinogenic effects of heterocyclic aromatic amines (HAAs). In the first section of this review, the current state of knowledge is briefly summarized. Based on the evaluation of the available data, various protective mechanisms are described, and the use of different methodologies for the detection of protective effects is critically discussed. In most antimutagenicity studies (>70%) bacterial indicators (predominantly Salmonella strain TA98) were used, and about 600 individual compounds and complex mixtures have been identified that attenuate the effects of HAAs. The most frequently used in vivo method to detect protective effects are adduct measurements; anticarcinogenic dietary factors were identified by aberrant crypt foci assays and liver foci tests with rats. The mechanisms of protection include inactivation of HAAs and their metabolites by direct binding, inhibition of enzymes involved in the metabolic activation of the amines, induction of detoxifying enzymes, and interaction with DNA repair processes. The detection spectrum of conventional in vitro mutagenicity assays with metabolically incompetent indicator cells is limited. These procedures reflect only simple mechanisms such as direct binding of the HAAs to pyrroles and fibers. It has been shown that these compounds are also effective in rodents. More complex mechanisms, namely, interactions with metabolic activation reactions are not adequately represented in in vitro assays with exogenous enzyme homogenates, and false-negative as well as false-positive results may be obtained. More appropriate approaches for the detection of protective effects are recently developed test systems with metabolically competent cells such as the human Hep G2 line or primary hepatocytes. SCGE tests and DNA adduct measurements with laboratory rodents enable the detection of antigenotoxic effects in different organs, including those that are targets for tumor induction by the amines. Medium term assays based on aberrant crypt foci in colon and liver foci tests have been used to prove that certain compounds that prevented DNA damage by HAAs also reduced their carcinogenic effects. These experiments are costly and time consuming and, due to the weak induction capacity of the amines, only pronounced anticarcinogenic effects can be detected. Over the years, a large bulk of data on HAA protective compounds has accumulated, but only for a few (e.g., fibers, pyrroles, constituents of teas, and lactic acid bacteria) is there sufficient evidence to support the assumption that they are protective in humans as well.     

Development of quantitative structure-activity relationship (QSAR) models to predict the carcinogenic potency of chemicals I. Alternative toxicity measures as an estimator of carcinogenic potency

Determining the carcinogenicity and carcinogenic potency of new chemicals is both a labor-intensive and time-consuming process. In order to expedite the screening process, there is a need to identify alternative toxicity measures that may be used as surrogates for carcinogenic potency. Alternative toxicity measures for carcinogenic potency currently being used in the literature include lethal dose (dose that kills 50% of a study population [LD(50)]), lowest-observed-adverse-effect-level (LOAEL) and maximum tolerated dose (MTD). The purpose of this study was to investigate the correlation between tumor dose (TD(50)) and three alternative toxicity measures as an estimator of carcinogenic potency. A second aim of this study was to develop a Classification and Regression Tree (CART) between TD(50) and estimated/experimental predictor variables to predict the carcinogenic potency of new chemicals. Rat TD(50)s of 590 structurally diverse chemicals were obtained from the Cancer Potency Database, and the three alternative toxicity measures considered in this study were estimated using TOPKAT, a toxicity estimation software. Though poor correlations were obtained between carcinogenic potency and the three alternative toxicity (both experimental and TOPKAT) measures for the CPDB chemicals, a CART developed using experimental data with no missing values as predictor variables provided reasonable estimates of TD(50) for nine chemicals that were part of an external validation set. However, if experimental values for the three alternative measures, mutagenicity and logP are not available in the literature, then either the CART developed using missing experimental values or estimated values may be used for making a prediction.     

Using carcinogenic potency ranking to assign air contaminants to emission classes

Carcinogenic air contaminants are assigned to emission classes with different emission limits on the basis of their inhalation carcinogenic potency within the revised form of the German First General Administrative Regulation Pertaining to the Federal Emission Control Law (Technical Instructions on Air Quality Control-TA Luft). Accordingly, compounds with high carcinogenic potency are regulated more strictly than less potent substances. The data on carcinogenic properties are heterogeneous. Twenty-five substances or substance groups have been scrutinized and a procedure has been developed to rank these chemicals according to their carcinogenic potency. For 14 substances well-founded unit risk estimates were available to allow assignment of these air contaminants to emission classes. Unit risk estimates for bromoethane, 2-butanone oxime, and o-toluidine were derived using the ED(10)/LED(10) method based on animal studies. For several substances no qualified unit risk estimates or carcinogenicity studies were available to estimate carcinogenic potency after inhalation. Carcinogenic potency of these substances was approximated using two simple methods, T25 and CELmin.