Nonlinear Cancer Response at Ultralow Dose: A 40800-Animal ED₀₀₁ Tumor and Biomarker Study

Abstract

Assessment of human cancer risk from animal carcinogen studies is severely limited by inadequate experimental data at environmentally relevant exposures, and procedures requiring modeled extrapolations many orders of magnitude below observable data. We used rainbow trout, an animal model well suited to ultra low-dose carcinogenesis research, to explore dose-response down to a targeted 10 excess liver tumors per 10,000 animals (ED001). A total of 40,800 trout were fed 0–225 ppm dibenzo[a,l]pyrene (DBP) for four weeks, sampled for biomarker analyses, and returned to control diet for nine months prior to gross and histologic examination. Suspect tumors were confirmed by pathology, and resulting incidences were modeled and compared to the default EPA LED10 linear extrapolation method. The study provided observed incidence data down to two above-background liver tumors per 10,000 animals at lowest dose (that is, an un-modeled ED0002 measurement). Among nine statistical models explored, three were determined to fit the liver data well - linear probit, quadratic logit, and Ryzin-Rai. None of these fitted models is compatible with the LED10 default assumption, and all fell increasingly below the default extrapolation with decreasing DBP dose. Low-dose tumor response was also not predictable from hepatic DBP-DNA adduct biomarkers, which accumulated as a power function of dose (adducts = 100 * DBP1.31). Two-order extrapolations below the modeled tumor data predicted DBP doses producing one excess cancer per million individuals (ED10−6) that were 500–1500-fold higher than that predicted by the five-order LED10 extrapolation. These results are considered specific to the animal model, carcinogen, and protocol used. They provide the first experimental estimation in any model of the degree of conservatism that may exist for the EPA default linear assumption for a genotoxic carcinogen.

The vanishing zero revisited: Thresholds in the age of genomics

“…One could surmise that low adduct levels can be efficiently repaired and hence are not highly mutagenic [15, 16]. In support of such argument, recent studies with large numbers of animals and mass spectrometry approaches that can detect DNA adducts as low as 1 per 10 9 to 10 10 have revealed that adduct formation at low doses is not linear [17]. Moreover, for adducts that can be formed by endogenous processes, the number of adducts induced by the exposure may be small compared to existing background levels, and therefore both endogenous and exogenous DNA adducts must be taken into account [13].…”

“…Even though there are examples of mutagens/genotoxic carcinogens for which the existence of a threshold was convincingly demonstrated, the expert consensus for regulatory purposes is that DNA adduct data should always be trumped by mutational and carcinogenicity data [22]. Nonetheless, the availability of highly sensitive methods for detection of DNA adducts [12, 17] continues to fuel the vanishing zero debate.…”

Mechanistic Models Fit to Ed001 Data on >40,000 Trout Exposed to Dibenzo[A,L]Pyrene Indicate Mutations Do Not Drive Increased Tumor Risk

“…was not reported by Bailey et al (2009) as noted above, tumors were all assumed to be malignant. In the absence of time-to-tumor data, this assumption did not clearly constrain modeling results to predict responses that differ substantially from those that could be obtained by modeling separate benign and malignant DAH pathways.…”

Developing mechanism-based and exposure biomarkers for mycotoxins in animals

“…However, one should not assume that such a relationship will hold for all hepatocarcinogens. In a recent ED 001 study, almost 40 000 trout were administered dibenzo(def,p)chrysene (DBC) (formally called dibenzo[a,l]pyrene) in the diet to determine the dose that statistically would give one additional cancer in 1 000 animals (ED 001 ) (Bailey et al, 2009). Although DBC-DNA adducts in liver were linear at the lowest dose administered, hepatocarcinogenesis statistically became sub-linear at low dose.…”