Long-term Rodent Carcinogenicity Studies Research Articles

A rat subchronic study transcriptional point of departure estimates a carcinogenicity study apical point of departure

Considerations of human relevance and animal use are driving research to identify new approaches to inform risk assessment of chemicals and replace guideline-based rodent carcinogenicity tests. Here, the hypothesis was tested across four agrochemicals that 1) a rat 90-day transcriptome-based BEPOD is protective of a rat carcinogenicity study and 2) a subchronic liver or kidney BEPOD would approximate a cancer bioassay apical POD derived from other organs and a rat subchronic BEPOD would approximate a mouse cancer bioassay apical POD. Using RNA sequencing and BMDExpress software, liver and/or kidney BEPOD values were generated in male rats exposed for 90 days to either Triclopyr Acid, Pronamide, Sulfoxaflor, or Fenpicoxamid. BEPOD values were compared to benchmark dose-derived apical POD values generated from rat 90-day and rodent carcinogenicity studies. Across all four agrochemicals, findings showed that a rat 90-day study BEPOD approximated the most sensitive apical POD (within 10-fold) generated from the 90-day rat study and long-term rodent carcinogenicity studies. This study supports the conclusion that a subchronic transcriptome-based BEPOD could be utilized to estimate an apical POD within a risk-based approach of chronic toxicity and carcinogenicity agrochemical assessment, abrogating the need for time- and resource-intensive rodent carcinogenicity studies and minimizing animal testing.

Identification of specific mRNA signatures as fingerprints for carcinogenesis in mice induced by genotoxic and nongenotoxic hepatocarcinogens.

Long-term rodent carcinogenicity studies for evaluation of chemicals and pharmaceuticals concerning their carcinogenic potential to humans are currently receiving critical revision. Additional data from mechanistic studies can support cancer risk assessment by clarifying the underlying mode of action. In the course of the IMI MARCAR project, a European consortium of EFPIA partners and academics, which aims to identify biomarkers for nongenotoxic carcinogenesis, a toxicogenomic mouse liver database was generated. CD-1 mice were orally treated for 3 and 14 days with 3 known genotoxic hepatocarcinogens: C.I. Direct Black 38, Dimethylnitrosamine and 4,4'-Methylenedianiline; 3 nongenotoxic hepatocarcinogens: 1,4-Dichlorobenzene, Phenobarbital sodium and Piperonyl butoxide; 4 nonhepatocarcinogens: Cefuroxime sodium, Nifedipine, Prazosin hydrochloride and Propranolol hydrochloride; and 3 compounds that show ambiguous results in genotoxicity testing: Cyproterone acetate, Thioacetamide and Wy-14643. By liver mRNA expression analysis using individual animal data, we identified 64 specific biomarker candidates for genotoxic carcinogens and 69 for nongenotoxic carcinogens for male mice at day 15. The majority of genotoxic carcinogen biomarker candidates possess functions in DNA damage response (eg, apoptosis, cell cycle progression, DNA repair). Most of the identified nongenotoxic carcinogen biomarker candidates are involved in regulation of cell cycle progression and apoptosis. The derived biomarker lists were characterized with respect to their dependency on study duration and gender and were successfully used to characterize carcinogens with ambiguous genotoxicity test results, such as Wy-14643. The identified biomarker candidates improve the mechanistic understanding of drug-induced effects on the mouse liver that result in hepatocellular adenomas and/or carcinomas in 2-year mouse carcinogenicity studies.

A Linear Prediction Rule Based on Ensemble Classifiers for Non-Genotoxic Carcinogenicity

This article presents a novel approach for developing a linear prediction rule to predict the non-genotoxic carcinogenicity potential of a new compound in the drug development pipeline. We construct the approach using data from 24-hour microarray experiments on rats treated with the compound. This method was developed to address an actual problem that we were presented with by scientists in mechanistic toxicology. Short-term, preclinical assays for non-genotoxic carcinogenicity, a toxicity commonly observed in long-term rodent carcinogenicity studies, have proven difficult to develop. A quick and early preclinical assay, such as this, is of particular interest and urgency. The linear prediction rule is derived using an Ensemble Linear Discriminant classifier. This is a hybrid approach which leverages the advantages of ensemble approaches for addressing over-fitting (a problem endemic to microarray data), and that of LDA for interpretability. In a limited comparison with some other classifiers, including random forest, we show that our approach has good predictive performance in addition to being interpretable.

Evaluation of carcinogenicity studies of medicinal products for human use authorised via the European centralised procedure (1995–2009)

Carcinogenicity data of medicinal products for human use that have been authorised via the European centralised procedure (CP) between 1995 and 2009 were evaluated. Carcinogenicity data, either from long-term rodent carcinogenicity studies, transgenic mouse studies or repeat-dose toxicity studies were available for 144 active substances contained in 159 medicinal products. Out of these compounds, 94 (65%) were positive in at least one long-term carcinogenicity study or in repeat-dose toxicity studies. Fifty compounds (35%) showed no evidence of a carcinogenic potential. Out of the 94 compounds with positive findings in either carcinogenicity or repeat-dose toxicity studies, 33 were positive in both mice and rats, 40 were positive in rats only, and 21 were positive exclusively in mice. Long-term carcinogenicity studies in two rodent species were available for 116 compounds. Data from one long-term carcinogenicity study in rats and a transgenic mouse model were available for eight compounds. For 13 compounds, carcinogenicity data were generated in only one rodent species. One compound was exclusively tested in a transgenic mouse model. Six compounds were tumourigenic in repeat-dose toxicity studies in rats. The majority of tumour findings observed in rodent carcinogenicity studies were considered not to be relevant for humans, either due to a rodent-specific mechanism of carcinogenicity, a high safety margin between exposures at the NOAEL (No Observed Adverse Effect Level) in rodents and recommended therapeutic doses in humans, or based on historical control data, a small effect size and lack of dose–response relationship and tumours typically observed in rodent strains used, or were considered not to be relevant for humans based on literature and clinical data or likely differences in metabolism/local concentrations between rodents and humans. Due to the high number of rodent tumour findings with unlikely relevance for humans, the value of the currently used testing strategy for carcinogenicity appears questionable. A revision of the carcinogenicity testing paradigm is warranted.

Poly-k-Trend Tests for Survival Adjusted Analysis of Tumor Rates Formulated as Approximate Multiple Contrast Test

In long-term rodent carcinogenicity studies without cause of death information, poly-k-adjusted tumor rates are commonly used to assess the carcinogenic potential of a compound. Testing trend on proportions, global tests such as the Cochran–Armitage test and Williams-type test have been proposed. Here are introduced simultaneous confidence intervals and adjusted p-values for multiple contrasts on poly-k-adjusted tumor rates, based on approximation with the multivariate normal distribution. Williams-type contrasts and Dunnett-type comparisons to control are special cases of this approach. In simulation studies, the acceptable performance for finite sample sizes is demonstrated. The methods are applied to a real data example and are implemented in a package for R.

The potential use of mutation spectra in cancer related genes in genetic toxicology: a statement of a GUM working group

In recent years, there has been widespread interest in the relationship between carcinogenic exposure and mutation spectra in cancer-related genes. To evaluate potential benefits and/or limitations in the use of mutation spectra in genetic toxicology, a GUM working group has been established to discuss this subject. Based on methodological possibilities and limitations, the impact of mutation spectra in the interpretation of animal experiments and in the identification of etiological agents in human cancer has been considered. With respect to experimental animals, the analyses of mutation spectra within long-term rodent carcinogenicity studies may provide some additional information on the mode of action of the respective carcinogen, however, the interpretation of results should be done carefully and only in context with other toxicological data available. Regarding human exposure, the analysis of mutation spectra in p53 or ras genes supplies information on the genotoxic properties of the respective agent. Nevertheless, on the individual level, the presence or absence of defined mutations in cancer-related genes in human tumors does not permit a definite conclusion about the causative agent.

Correlations among tumor types in mouse cancer bioassays: liver adenomas, liver carcinomas, leukemias and lymphomas.

In an examination of rodent bioassays Young and Gries [Young S.S., and Gries C.L. Exploration of the negative correlation between proliferative hepatocellular lesions and lymphoma in rats and mice--establishment and implications. Fundam. Appl. Toxicol. 1984: 4: 632-6401 and Haseman et al. [Haseman J.K., et al. Body weight-tumor incidence correlations in long-term rodent carcinogenicity studies. Toxicol. Pathol. 1997: 25: 256-263] noticed that there is a negative correlation (anticorrelation) between development of liver tumors and leukemia or lymphomas. If an animal has a lymphoma or leukemia it is less likely to develop liver tumors. These studies noted that this applies to several strains of animals. The anticorrelation appeared in control animals. In this paper we study this anticorrelation in the quarter of a million rodents exposed in the Carcinogenesis Bioassay Database System (CBDS) database of the National Toxicology program in both control and dosed animals. We failed to completely replicate Young and Gries or Haseman et al. However, when benign liver tumors (adenomas or nodules) and malignant liver tumors (carcinomas) are considered separately AND different leukemia and lymphoma types are considered, a strong anticorrelation appears. We identify survival and the time period (in years) during which the bioassay was completed as an important factor in interpreting correlations and anticorrelations. Differences between liver adenomas and carcinomas and lymphoma types contribute to a more general question of grouping of the individual tumor types. In our classification scheme (originally developed about 1986 by Dr. Bailar) an effort is made to distinguish benign and malignant neoplasms, while other investigators group all tumors at a specific site. For many analyses liver adenomas and carcinomas have been lumped together. This is because it is suspected that the diagnoses by pathologists may not distinguish, or ignore the (benign) adenoma when a (malignant) carcinoma is present. Possible biological differences in tumor mechanisms may require separate evaluation of these tumors with all the dangers of "pathologist bias" that this introduces.

An update of the National Toxicology Program database on nasal carcinogens

Nearly 500 long-term rodent carcinogenicity studies carried out by the National Cancer Institute and the National Toxicology Program were examined, and 12 chemicals were identified that produced nasal tumors: allyl glycidol ether, p-cresidine, 1,2-dibromo-3-chloropropane, 1,2-dibromoethane, 2,3-dibromo-1-propanol, dimethylvinyl chloride, 1,4-dioxane, 1,2-epoxybutane, iodinated glycerol, procarbazine, propylene oxide, and 2,6-xylidine. All 12 of these chemicals produced nasal tumors in rats, and 5 also produced nasal tumors in mice. Most of the nasal carcinogens (1) produced tumor increases in both sexes, (2) produced tumors at other sites as well, (3) had significantly reduced survival at doses that were carcinogenic, and (4) were genotoxic. Only 5 of the 12 nasal carcinogens were administered by inhalation. A variety of different types of nasal cavity tumors were produced, and specific tumor rates are given for those chemicals causing multiple tumor types. Increased incidences of nasal neoplasms were often accompanied by suppurative/acute inflammation, epithelial/focal hyperplasia and squamous metaplasia. However, high incidences of these nonneoplastic nasal lesions were also frequently seen in inhalation studies showing no evidence of nasal carcinogenicity, suggesting that in general nasal carcinogenesis is not associated with the magnitude of chronic toxicity observed at this site.

Body weight-tumor incidence correlations in long-term rodent carcinogenicity studies.

Associations between body weight and tumor incidence and among the incidences of selected site-specific tumors were examined for more than 4,000 male and female Fischer-344 rats and B6C3F1 mice in the National Toxicology Program historical control database. Incidences of certain site-specific tumors, most notably mammary gland and pituitary gland tumors in rats and liver tumors in mice, were shown to have a strong positive correlation with 52-wk body weight. Using individual animal data, logistic regression models were derived for predicting site-specific tumor incidence as a function of 52-wk body weight, age, and other factors. This association between body weight and tumor incidence can explain many of the decreased tumor incidences observed in National Toxicology Program carcinogenicity studies. Body weight differences between dosed and control groups can also mask carcinogenic effects for those sites sensitive to body weight changes. Thus, when designing long-term rodent carcinogenicity studies, measures should be taken to minimize potential body weight differences between dosed and control groups. There were a number of small but significant negative correlations among tumor incidences, reflecting primarily the lethality of the tumors in question. None of these correlations (nor the 2 small positive correlations found) are likely to have any impact on the interpretation of experimental results. However, the high negative correlation between pituitary gland and testis tumors in male Fischer-344 rats cannot be dismissed so easily, does not reflect tumor lethality, and is currently being studied further.

Data Analysis: Statistical Analysis and Use of Historical Control Data

Survival-adjusted methods for the statistical analysis of tumor data from long-term rodent carcinogenicity studies are described. Although most of these methods require knowledge of whether individual tumors are "fatal" or "incidental," such determinations may be difficult. Several methods for dealing with this and with other data analysis issues are discussed. Historical control tumor data may be useful in the interpretation of rodent carcinogenicity studies, particularly for rare tumors and for borderline effects. Although statistical methods are available for using historical control data in a formal testing framework, the primary difficulty is establishing a database that is truly comparable to the study under evaluation with respect to those factors known to influence tumor occurrence. Major sources of variability in tumor incidence include the animal room environment, dietary factors/body weight, gross necropsy and slide preparation procedures, and histopathology diagnosis. The National Toxicology Program′s use of historical control data is briefly described and illustrated.

Use of statistical decision rules for evaluating laboratory animal carcinogenicity studies

In the evaluation of long-term rodent carcinogenicity studies, many different tumor sites and types are evaluated, which may increase the likelihood of a statistical false positive. To deal with this issue, a number of statistical decision rules have been proposed that take into account multiple comparisons. This paper discusses the various types of decision rules and evaluates the factors that may lead to different interpretations of experimental results. These concepts are illustrated by examining the statistical decision procedures used by three analysts to evaluate the results of 25 long-term rodent carcinogenicity studies carried out by the National Cancer Institute. Agreement among these decision rules is shown to be greater than originally reported. It is also concluded that while the application of statistical decision rules may be of value in some instances to guard against statistical false positives, the final interpretation of the data should be based on biological as well as statistical considerations. Thus, statistical decision rules should not be employed as a substitute for sound scientific judgment in the overall evaluation of these experiments.

Use of Statistical Decision Rules for Evaluating Laboratory Animal Carcinogenicity Studies

Use of Statistical Decision Rules for Evaluating Laboratory Animal Carcinogenicity Studies. HASEMAN, J. K. (1990). Fundam. Appl. Toxicol. 14, 637–648. In the evaluation of long-term rodent carcinogenicity studies, many different tumor sites and types are evaluated, which may increase the likelihood of a statistical false positive. To deal with this issue, a number of statistical decision rules have been proposed that take into account multiple comparisons. This paper discusses the various types of decision rules and evaluates the factors that may lead to different interpretations of experimental results. These concepts are illustrated by examining the statistical decision procedures used by three analysts to evaluate the results of 25 long-term rodent carcinogenicity studies carried out by the National Cancer Institute. Agreement among these decision rules is shown to be greater than originally reported. It is also concluded that while the application of statistical decision rules may be of value in some instances to guard against statistical false positives, the final interpretation of the data should be based on biological as well as statistical considerations. Thus, statistical decision rules should not be employed as a substitute for sound scientific judgment in the overall evaluation of these experiments.

Chemicals showing no evidence of carcinogenicity in long-term, two-species rodent studies: the need for short-term test data.

Studies to determine the performance of short-term tests (STT) in predicting chemical carcinogenicity are frequently characterized by a poor representation of noncarcinogens among the test chemicals. As a result, the performance of several STT is well characterized with carcinogenic chemicals, but the ability of the tests to discriminate between carcinogens and noncarcinogens is largely unknown because of the shortage of test results for noncarcinogens. A list of 70 chemicals that have been tested by the National Cancer Institute or the National Toxicology Program in long-term rodent carcinogenicity studies and that have yielded no evidence of cancer induction in male and female rats and mice is presented. The list helps document the shortage of STT results of noncarcinogens and provides a potential source of chemicals for consideration in future studies designed to assess the ability of STT to predict chemical carcinogenicity.