Use of Acute Toxicity to Estimate Carcinogenic Risk

Data on the effects of human exposure to carcinogens are limited, so that estimation of the risks of carcinogens must be obtained indirectly. Current risk estimates are generally based on lifetime animal bioassays which are expensive and which take more than two years to complete. We here show how data on acute toxicity can be used to make a preliminary estimate of carcinogenic risk and give an idea of the uncertainty in that risk estimate. The estimates obtained are biased upwards, and so are useful for setting interim standards and determining whether further study is worthwhile. A general scheme which incorporates the use of such estimates is outlined, and it is shown by example how adoption of the procedures suggested could have prevented regulatory hiatus in the past.     

Comparison of Contact Site Cancer Potency Across Dose Routes: Case Study with Epichlorohydrin*

Risk assessment for airborne carcinogens is often limited by a lack of inhalation bioassay data. While extrapolation from oral-based cancer potency factors may be possible for some agents, this is not considered feasible for contact site carcinogens. The change in contact sites (oral: g.i. tract; inhalation: respiratory tract) when switching dose routes leads to possible differences in tissue sensitivity as well as chemical delivery. This research evaluates the feasibility to extrapolate across dose routes for a contact site carcinogen through a case study with epichlorohydrin (EPI). EPI cancer potency at contact sites is compared across three bioassays involving different dose routes (gavage, drinking water, inhalation) through the use of dosimetry models to adjust for EPI delivery to contact sites. Results indicate a large disparity (two orders of magnitude) in potency across the three routes of administration when expressed as the externally applied dose. However, when expressed as peak delivered dose, inhalation and oral potency estimates are similar and overall, the three potency estimates are within a factor of seven. The results suggest that contact site response to EPI is more dependent upon the rate than the route of delivery, with peak concentration the best way to extrapolate across dose routes. These results cannot be projected to other carcinogens without further study.     

Improving the Regulation of Carcinogens by Expediting Cancer Potency Estimation1

The statutory language of the Safe Drinking Water and Toxic Enforcement Act of 1986 (Proposition 65; California Health and Safety Code 25249.5 et seq.) encourages rapid adoption of “no significant risk levels” (NSRLs), intakes associated with estimated cancer risks of no more than 1 in 100,000. Derivation of an NSRL for a carcinogen listed under Proposition 65 requires the development of a cancer potency value. This paper discusses the methodology for the derivation of cancer potencies using an expedited procedure, and provides potency estimates for a number of agents listed as carcinogens under Proposition 65. To derive expedited potency values, default risk assessment methods are applied to data sets selected from an extensive tabulation of animal cancer bioassays according to criteria used by regulatory agencies. A subset of these expedited values is compared to values previously developed by regulatory agencies using conventional quantitative risk assessment and found to be in good agreement. Specific regulatory activities which could be facilitated by adopting similar expedited procedures are identified.     

Point Estimates of Cancer Risk at Low Doses

There has been considerable discussion regarding the conservativeness of low-dose cancer risk estimates based upon linear extrapolation from upper confidence limits. Various groups have expressed a need for best (point) estimates of cancer risk in order to improve risk/benefit decisions. Point estimates of carcinogenic potency obtained from maximum likelihood estimates of low-dose slope may be highly unstable, being sensitive both to the choice of the dose–response model and possibly to minimal perturbations of the data. For carcinogens that augment background carcinogenic processes and/or for mutagenic carcinogens, at low doses the tumor incidence versus target tissue dose is expected to be linear. Pharmacokinetic data may be needed to identify and adjust for exposure-dose nonlinearities. Based on the assumption that the dose response is linear over low doses, a stable point estimate for low-dose cancer risk is proposed. Since various models give similar estimates of risk down to levels of 1%, a stable estimate of the low-dose cancer slope is provided by ŝ = 0.01/ED01, where ED01 is the dose corresponding to an excess cancer risk of 1%. Thus, low-dose estimates of cancer risk are obtained by, risk = ŝ × dose. The proposed procedure is similar to one which has been utilized in the past by the Center for Food Safety and Applied Nutrition, Food and Drug Administration. The upper confidence limit, s , corresponding to this point estimate of low-dose slope is similar to the upper limit, q ₁ obtained from the generalized multistage model. The advantage of the proposed procedure is that ŝ provides stable estimates of low-dose carcinogenic potency, which are not unduly influenced by small perturbations of the tumor incidence rates, unlike ₁.     

Experimental Design of Bioassays for Screening and Low Dose Extrapolation

Relatively high doses of chemicals generally are employed in animal bioassays to detect potential carcinogens with relatively small numbers of animals. The problem investigated here is the development of experimental designs which are effective for high to low dose extrapolation for tumor incidence as well as for screening (detecting) carcinogens. Several experimental designs are compared over a wide range of different dose response curves. Linear extrapolation is used below the experimental data range to establish an upper bound on carcinogenic risk at low doses. The goal is to find experimental designs which minimize the upper bound on low dose risk estimates (i.e., maximize the allowable dose for a given level of risk). The maximum tolerated dose (MTD) is employed for screening purposes. Among the designs investigated, experiments with doses at the MTD, 1/2 MTD, 1/4 MTD, and controls generally provide relatively good data for low dose extrapolation with relatively good power for detecting carcinogens. For this design, equal numbers of animals per dose level perform as well as unequal allocations.     

Uncertainty in Cancer Risk Estimates

Several existing databases compiled by Gold et al.^(1–3) for carcinogenesis bioassays are examined to obtain estimates of the reproducibility of cancer rates across experiments, strains, and rodent species. A measure of carcinogenic potency is given by the TD₅₀ (daily dose that causes a tumor type in 50% of the exposed animals that otherwise would not develop the tumor in a standard lifetime). The lognormal distribution can be used to model the uncertainty of the estimates of potency (TD₅₀) and the ratio of TD₅₀'s between two species. For near-replicate bioassays, approximately 95% of the TD₅₀'s are estimated to be within a factor of 4 of the mean. Between strains, about 95% of the TD₅₀'s are estimated to be within a factor of 11 of their mean, and the pure genetic component of variability is accounted for by a factor of 6.8. Between rats and mice, about 95% of the TD₅₀'s are estimated to be within a factor of 32 of the mean, while between humans and experimental animals the factor is 110 for 20 chemicals reported by Allen et al.⁽⁴⁾ The common practice of basing cancer risk estimates on the most sensitive rodent species-strain-sex and using interspecies dose scaling based on body surface area appears to overestimate cancer rates for these 20 human carcinogens by about one order of magnitude on the average. Hence, for chemicals where the dose-response is nearly linear below experimental doses, cancer risk estimates based on animal data are not necessarily conservative and may range from a factor of 10 too low for human carcinogens up to a factor of 1000 too high for approximately 95% of the chemicals tested to date. These limits may need to be modified for specific chemicals where additional mechanistic or pharmacokinetic information may suggest alterations or where particularly sensitive subpopu-lations may be exposed. Supralinearity could lead to anticonservative estimates of cancer risk. Underestimating cancer risk by a specific factor has a much larger impact on the actual number of cancer cases than overestimates of smaller risks by the same factor. This paper does not address the uncertainties in high to low dose extrapolation. If the dose-response is sufficiently nonlinear at low doses to produce cancer risks near zero, then low-dose risk estimates based on linear extrapolation are likely to overestimate risk and the limits of uncertainty cannot be established.     

Extrapolation of Carcinogenicity Between Species: Qualitative and Quantitative Factors

Prediction of human cancer risk from the results of rodent bioassays requires two types of extrapolation: a qualitative extrapolation from short-lived rodent species to long-lived humans, and a quantitative extrapolation from near-toxic doses in the bioassay to low-level human exposures. Experimental evidence on the accuracy of prediction between closely related species tested under similar experimental conditions (rats, mice, and hamsters) indicates that: (1) if a chemical is positive in one species, it will be positive in the second species about 75% of the time; however, since about 50% of test chemicals are positive in each species, by chance alone one would expect a predictive value between species of about 50%. (2) If a chemical induces tumors in a particular target organ in one species, it will induce tumors in the same organ in the second species about 50% of the time. Similar predictive values are obtained in an analysis of prediction from humans to rats or from humans to mice for known human carcinogens. Limitations of bioassay data for use in quantitative extrapolation are discussed, including constraints on both estimates of carcinogenic potency and of the dose-response in experiments with only two doses and a control. Quantitative extrapolation should be based on an understanding of mechanisms of carcinogenesis, particularly mitogenic effects that are present at high and not low doses.     

On the Relationship Between Carcinogenicity and Acute Toxicity

Parodi et al. ⁽¹⁾ and Zeise et al. ⁽²⁾ found a surprising statistical correlation (or association) between acute toxicity and carcinogenic potency. In order to shed light on the questions of whether or not it is a causal correlation, and whether or not it is a statistical or tautological artifact, we have compared the correlations for the NCI/NTP data set with those for chemicals not in this set. Carcinogenic potencies were taken from the Gold et al. database. We find a weak correlation with an average value of TD₅₀/LD₅₀= 0.04 for the non-NCI data set, compared with TD₅₀/LD₅₀= 0.15 for the NCI data set. We conclude that it is not easy to distinguish types of carcinogens on the basis of whether or not they are acutely toxic.     

Extended Analysis and Evidence Integration of Chloroprene as a Human Carcinogen

β-Chloroprene is used in the production of polychloroprene, a synthetic rubber. In 2010, Environmental Protection Agency (EPA) published the Integrated Risk Information System “Toxicological Review of Chloroprene,” concluding that chloroprene was “likely to be carcinogenic to humans.” This was based on findings from a 1998 National Toxicology Program (NTP) study showing multiple tumors within and across animal species; results from occupational epidemiological studies; a proposed mutagenic mode of action; and structural similarities with 1,3-butadiene and vinyl chloride. Using mouse data from the NTP study and assuming a mutagenic mode of action, EPA calculated an inhalation unit risk (IUR) for chloroprene of 5 × 10⁻⁴ per µg/m³. This is among the highest IURs for chemicals classified by IARC or EPA as known or probable human carcinogens and orders of magnitude higher than the IURs for carcinogens such as vinyl chloride, benzene, and 1,3-butadiene. Due to differences in pharmacokinetics, mice appear to be uniquely responsive to chloroprene exposure compared to other animals, including humans, which is consistent with the lack of evidence of carcinogenicity in robust occupational epidemiological studies. We evaluated and integrated all lines of evidence for chloroprene carcinogenicity to assess whether the 2010 EPA IUR could be scientifically substantiated. Due to clear interspecies differences in carcinogenic response to chloroprene, we applied a physiologically based pharmacokinetic model for chloroprene to calculate a species-specific internal dose (amount metabolized/gram of lung tissue) and derived an IUR that is over 100-fold lower than the 2010 EPA IUR. Therefore, we recommend that EPA's IUR be updated.     

Correlation Between Carcinogenic Potency of Chemicals in Animals and Humans

Twenty-three chemicals were selected for comparison of the carcinogenic potencies estimated from epidemiological data to those estimated from animal carcinogenesis bioassays. The chemicals were all those for which reasonably strong evidence of carcinogenicity could be found in humans or animals and for which suitable data could be obtained for quantifying carcinogenic potencies in both humans and animals. Many alternative methods of analyzing the bioassay data were investigated. Almost all of the methods yielded potency estimates that were highly correlated with potencies estimated from epidemiological data; correlations were highly statistically significant (p less than 0.001), with the corresponding correlation coefficients ranging as high as 0.9. These findings provide support for the general use of animal data to evaluate carcinogenic potential in humans and also for the use of animal data to quantify human risk.     

Assessing Cancer Risks from Short-Term Exposures in Children

For the vast majority of chemicals that have cancer potency estimates on IRIS, the underlying database is deficient with respect to early-life exposures. This data gap has prevented derivation of cancer potency factors that are relevant to this time period, and so assessments may not fully address children's risks. This article provides a review of juvenile animal bioassay data in comparison to adult animal data for a broad array of carcinogens. This comparison indicates that short-term exposures in early life are likely to yield a greater tumor response than short-term exposures in adults, but similar tumor response when compared to long-term exposures in adults. This evidence is brought into a risk assessment context by proposing an approach that: (1) does not prorate children's exposures over the entire life span or mix them with exposures that occur at other ages; (2) applies the cancer slope factor from adult animal or human epidemiology studies to the children's exposure dose to calculate the cancer risk associated with the early-life period; and (3) adds the cancer risk for young children to that for older children/adults to yield a total lifetime cancer risk. The proposed approach allows for the unique exposure and pharmacokinetic factors associated with young children to be fully weighted in the cancer risk assessment. It is very similar to the approach currently used by U.S. EPA for vinyl chloride. The current analysis finds that the database of early life and adult cancer bioassays supports extension of this approach from vinyl chloride to other carcinogens of diverse mode of action. This approach should be enhanced by early-life data specific to the particular carcinogen under analysis whenever possible.     

Characterizing Dose-Response Relationships in Multiple Cancer Bioassays

In the evaluation of chemical compounds for carcinogenic risk, regulatory agencies such as the U.S. Environmental Protection Agency and National Toxicology Program (NTP) have traditionally fit a dose-response model to data from rodent bioassays, and then used the fitted model to estimate a Virtually Safe Dose or the dose corresponding to a very small increase (usually 10(-6)) in risk over background. Much recent interest has been directed at incorporating additional scientific information regarding the properties of the specific chemical under investigation into the risk assessment process, including biological mechanisms of cancer induction, metabolic pathways, and chemical structure and activity. Despite the fact that regulatory agencies are currently poised to allow use of nonlinear dose-response models based on the concept of an underlying threshold for nongenotoxic chemicals, there have been few attempts to investigate the overall relationship between the shape of dose-response curves and mutagenicity. Using data from an historical database of NTP cancer bioassays, the authors conducted a repeated-measures Analysis of the estimated shape from fitting extended Weibull dose-response curves. It was concluded that genotoxic chemicals have dose-response curves that are closer to linear than those for nongenotoxic chemicals, though on average, both types of compounds have dose-response curves that are convex and the effect of genotoxicity is small.     

The Social Benefits of Expedited Risk Assessments

The present regulation of carcinogens is quite slow; hundreds of substances that have tested positive for carcinogenicity in animal bioassays have not been addressed by the U.S. regulatory system. This carries with it unappreciated social, economic, and public health costs. However, there are readily available expedited approximation procedures for assessing the potency of carcinogens whose use has substantial benefits that outweigh any costs from less science-intensive and less extensively documented assessments. These benefits can be seen by using a model to suggest the magnitude of social costs in regulating carcinogens by current conventional methods compared with expedited procedures for assessing the potency of known carcinogens. Two scenarios, one in accordance with current agency presumptions and one which assumes extreme unreliability in animal data and in the accuracy of potency assessments, compare conventional science-intensive and expedited procedures. On both, the total social costs of expedited procedures are lower than conventional procedures across a wide range of values assigned for individual mistakes of under regulation and over regulation. It appears better to evaluate a larger universe of known carcinogens somewhat less intensively for each substance than to evaluate a small proportion of that same universe very carefully and delay considering the rest.     

The Role of Cancer Risk in the Regulation of Industrial Pollution

The extent of carcinogen regulation under existing U.S. environmental statutes is assessed by developing measures of the scope and stringency of regulation. While concern about cancer risk has played an important political role in obtaining support for pollution control programs, it has not provided the predominant rationale for most regulatory actions taken to date. Less than 20% of all standards established to limit concentrations of chemicals in various media address carcinogens. Restrictions on chemical use are more frequently based on concerns about noncancer human health or ecological effects. Of the chemicals in commercial use which have been identified as potential human carcinogens on the basis of rodent bioassays, only a small proportion are regulated. There is an inverse relationship between the scope of regulatory coverage and the stringency of regulatory requirements: the largest percentages of identified carcinogens are affected by the least stringent requirements, such as information disclosure. Standards based on de minimis cancer risk levels have been established for only 10% of identified carcinogens and are restricted to one medium: water. Complete bans on use have affected very few chemicals. The general role that carcinogenicity now plays in the regulatory process is not dramatically different from that of other adverse human health effects: if a substance is identified as a hazard, it may eventually be subject to economically achievable and technically feasible restrictions.     

Evaluating the Risk of Liver Cancer in Humans Exposed to Trichloroethylene Using Physiological Models

Trichloroethylene (TCE) is a widespread environmental pollutant. TCE is classified as a rodent carcinogen by the U.S. Environmental Protection Agency (EPA). Using the rodent cancer bioassay findings and estimates of metabolized dose, the EPA has estimated lifetime exposure cancer risks for humans that ingest TCE in drinking water or inhale TCE. In this study, a physiologically based pharmacokinetic (PB-PK) model for mice was used to simulate selected gavage and inhalation bioassays with TCE. Plausible dose-metrics thought to be linked with the mechanism of action for TCE carcinogenesis were selected. These dose-metrics, adjusted to reflect an average amount per day for a lifetime, were metabolism of TCE (AMET, mg/kg/day) and systemic concentration of TCA (AUCTCA, mg/L/day). These dose-metrics were then used in a linearized multistage model to estimate AMET and AUCTCA values that correspond to liver cancer risks of 1 in 1 million in mice. A human PB-PK model for TCE was then used to predict TCE concentrations in drinking water and air that would provide AMET and AUCTCA values equal to the predicted mice AMET and AUCTCA values that correspond to liver cancer risks of 1 in 1 million. For the dose-metrics, AMET and AUCTCA, the TCE concentrations in air were 10.0 and 0.1 ppb TCE (continuous exposure), respectively, and in water, 7 and 4 μg TCE/L, respectively.     

An Evaluation of Risk Estimation Procedures for Mixtures of Carcinogens

The estimation of health risks from exposure to a mixture of chemical carcinogens is generally based on the combination of information from several available single compound studies. The current practice of directly summing the upper bound risk estimates of individual carcinogenic components as an upper bound on the total risk of a mixture is known to be generally too conservative. Gaylor and Chen (1996, Risk Analysis) proposed a simple procedure to compute an upper bound on the total risk using only the upper confidence limits and central risk estimates of individual carcinogens. The Gaylor-Chen procedure was derived based on an underlying assumption of the normality for the distributions of individual risk estimates. In this paper we evaluated the Gaylor-Chen approach in terms of the coverage probability. The performance of the Gaylor-Chen approach in terms the coverages of the upper confidence limits on the true risks of individual carcinogens. In general, if the coverage probabilities for the individual carcinogens are all approximately equal to the nominal level, then the Gaylor-Chen approach should perform well. However, the Gaylor-Chen approach can be conservative or anti-conservative if some or all individual upper confidence limit estimates are conservative or anti-conservative.     

Ambiguous carcinogens and their regulation

Examination of five animal and one human studies suggest that certain agents increase the incidence of some cancers but simultaneously reduce the incidence of other cancers. Yellow die #3, for example, sharply increases the incidence of liver tumors but practically eliminates naturally occurring leukemia/lymphoma in F-344 male rates. Such ambiguity in the action of presumed carcinogens suggests that caution must be used by regulatory bodies in proscribing suspected carcinogens, or even in recommending changes in lifestyle or dietary habits as a means of reducing incidence of cancer.     

One-Hit Models of Carcinogenesis: Conservative or Not?

One-hit formulas are widely believed to be "conservative" when used to analyze carcinogenesis bioassays, in the sense that they will rarely underestimate risks of cancer at low exposures. Such formulas are generally applied to the lifetime incidence of cancer at a specific site, with risks estimated from animal data at zero dose (control), and two or more additional doses that are appreciable fractions of a maximum tolerated dose. No empirical study has demonstrated that the one-hit formula is conservative in the sense described. The Carcinogenesis Bioassay Database System contains data on 1212 separate bioassays of 308 chemical substances tested at exactly three evaluable doses. These provided sufficient data to examine 8432 specific combinations of cancer site with sex, species, and chemical. For each of these we fitted a one-hit formula to the zero and maximum dose data points, then examined the relation of the fitted curve to the incidence rate observed at the mid-dose, with and without adjustment for intercurrent mortality. Both underestimates and overestimates of risk at mid-dose occurred substantially more often than expected by chance. We cannot tell whether such underestimates would occur at lower doses, but offer six biological reasons why underestimates might be expected. In a high percentage of animal bioassays, the one-hit formula is not conservative when applied in the usual way to animal data. It remains possible that the one-hit formula may indeed be conservative at sufficiently low doses (below the observational range), but the usual procedure, applied to the usual dose range, can be nonconservative in estimating the slope of the formula at such low doses. Risk assessments for regulation of carcinogens should incorporate some measure of additional uncertainty.     

Uncertainties in the CIIT Model for Formaldehyde-Induced Carcinogenicity in the Rat: A Limited Sensitivity Analysis–I

Scientists at the CIIT Centers for Health Research (Conolly et al., 2000, 2003; Kimbell et al., 2001a, 2001b) developed a two-stage clonal expansion model of formaldehyde-induced nasal cancers in the F344 rat that made extensive use of mechanistic information. An inference of their modeling approach was that formaldehyde-induced tumorigenicity could be optimally explained without the role of formaldehyde's mutagenic action. In this article, we examine the strength of this result and modify select features to examine the sensitivity of the predicted dose response to select assumptions. We implement solutions to the two-stage cancer model that are valid for nonhomogeneous models (i.e., models with time-dependent parameters), thus accounting for time dependence in variables. In this reimplementation, we examine the sensitivity of model predictions to pooling historical and concurrent control data, and to lumping sacrificed animals in which tumors were discovered incidentally with those in which death was caused by the tumors. We found the CIIT model results were not significantly altered with the nonhomogeneous solutions. Dose-response predictions below the range of exposures where tumors occurred in the bioassays were highly sensitive to the choice of control data. In the range of exposures where tumors were observed, the model attributed up to 74% of the added tumor probability to formaldehyde's mutagenic action when our reanalysis restricted the use of the National Toxicology Program (NTP) historical control data to only those obtained from inhalation exposures. Model results were insensitive to hourly or daily temporal variations in DNA protein cross-link (DPX) concentration, a surrogate for the dose-metric linked to formaldehyde-induced mutations, prompting us to utilize weekly averages for this quantity. Various other biological and mathematical uncertainties in the model have been retained unmodified in this analysis. These include model specification of initiated cell division and death rates, and uncertainty and variability in the dose response for cell replication rates, issues that will be considered in a future paper.     

Evaluation of False Positive and False Negative Outcomes in NTP Long-Term Rodent Carcinogenicity Studies

The decision-making process used by the National Toxicology Program (NTP) in its evaluation of long-term rodent carcinogenicity studies was investigated to determine whether or not this procedure resulted in an excessive number of false positive or false negative outcomes. All site-specific tumor incidences that were found to be significantly ( p < 0.05) increased either by a trend test or by pairwise comparisons of each dosed group against the controls in 218 NTP 2-year studies with Fischer 344 rats and/or B6C3F1 mice were tabulated and compared to the number of statistically significant tumor increases expected to occur by chance. Our evaluation suggests that false positive rates are fairly low in NTP long-term studies. Assessing false negative rates is more difficult because of the limited sensitivity of the bioassay for detecting subtle carcinogenic effects. Moreover, reduced body weights frequently occur in dosed animals, and the positive correlation between the incidences of certain site-specific tumors and body weight may mask the detection of carcinogenic effects. Despite these difficulties, our analysis did identify one tumor showing evidence of false negative outcomes: interstitial cell tumors of the testis in male Fischer 344 (F344) rats. This tumor showed considerably more significant ( p > 0.05) increased incidences than expected by chance, yet none were considered to be chemically-related. However, the biological significance of interstitial cell tumor increases in F344 rats is uncertain because of the high background rate of neoplasia (>90%) for this target site.