ABOUT AUTHOR:
Jitendra kumar
M.S. Pharmacology & Toxicology
NIPER raebareli
jkumar.kumar382@gmail.com
INTRODUCTION
This chapter presents an historical overview of cancer, carcinogenicity testing, and human cancer causes. Cancer has been known for a very long time, but the awareness of human carcinogenicity caused by chemicals is a phenomenon of the 20th century. This in turn has produced legislation that prohibits the use of carcinogens in the food chain and has provided guidelines for carcinogenicity testing in animals. Lifetime studies (18–24 months) in two main rodent species (rat and mouse), also known as the “Standard Chronic Bioassay,” have been conducted since the 1960s
Meanwhile, various deficiencies have been detected in the Chronic Bioassay; over-sensitivity is the major one. Hundreds of compounds have been tested with the Chronic Bioassay method, and about 50% have yielded (false) positive results. Lack of relevance to man has often been demonstrated by additional mechanistic studies. Additionally, more mechanistic and molecular knowledge has been gained in regards to the human carcinogenicity concept, including genotoxic versus epigenetic carcinogens, the multi-stage cancer theory, and human life style factors involved in carcinogenesis.
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The above evolutions have opened up new opportunities for carcinogenicity testing, including short-term alternative carcinogenicity models. In addition, carcinogenicity testing is evolving from a standard chronic bioassay to a weight-of-evidence approach, where the mechanisms involved in rodent and human carcinogenesis are considered, and where communication between industry and regulatory authorities is encourage Carcinogenicity Testing Guidelines
In the 1970s and 1980s, the US, European, and Japanese Registration Authorities established guidelines for carcinogenicity testing in animals for the various chemicals characterized by possible long-term intake by man. These chemicals included food and color additives, agrochemicals, industrial chemicals, solvents, human pharmaceuticals, and veterinary products. The guidelines were based upon the Chronic Bioassay of the NTP and gave indications for route and frequency of dosing, dose levels, group sizes, duration of the study, and observations during the study.
Table 1 Establishment of guidelines for carcinogenicity testing in animals
EPA: Environmental Protection Agency; FDA: Food and Drug Administration; FIFRA: Federal Insecticide Fungicide and Rodenticide Act;MAFF: Ministry Agricultural Forestry and Fisheries; MHW: Ministry for Health and Welfare; OECD: Organisation for Economic Co-Operation and Development; TCSA: Toxic Substances Control Act.
CURRENT CARCINOGENICITY TESTING
A description of the standard approaches in carcinogenicity testing for the safety of chemicals is provided.
Standard Rodent Chronic Bioassay
Species and strains
According to the previously described guidelines, carcinogenicity studies have to be performed in two rodent species, usually the rat and the mouse. Ideally, the strains should have a low spontaneous incidence of cancer, but they should also be sensitive to induction of cancer by human carcinogens. Commonly used species are the Sprague Dawley, Fisher F344, or Wistar strains in rats, and the CD-1 or C57BL-based strains in mice. At least 50 animals are included per sex in each dose group. Today, at least three dose groups are used as well as at least one negative control group, which results in minimally 200 males and 200 females for one study.
Doses and route of administration
The animals are exposed daily to the test compound from the age of 6 weeks onward. The administration procedure should simulate human exposure as closely as possible; oral intake is the most common route.In the past, the test item was often mixed in the drinking water or the feed, either in a fixed concentration in the feed during the entire study or with regular adaptations to maintain a steady ratio of mg/kg of body weight intake during the entire life expectancy. Nowadays, oral gavage administration in the stomach is used, except for agrochemicals and food additives, where feed administration is still applicable. Oral gavage administration provides more certainty of test item intake, but also leads to another pattern of test item exposure in the body (peak concentrations after dosing). Dose-selection for the various dose groups has been based mainly upon a MTD, which is defined to elicit slight target organ toxicity but will not shorten the treated animals’ survivability from any toxic effects other than the induction of neoplasms. For the most part, a body weight gain loss of 10% is considered acceptable as evidence of minimal toxicity. The medium dose may elicit minimal toxicity; however, the low dose should be free of any toxicity.
Duration
The studies are designed to last for at least 24 months and survival should be at least 25 animals per sex in the control and low dose groups, both in males and females. In the past, the studies were often extended beyond 24 months because survival (especially in the control and low dose group) was still above 25 animals/group/sex, and because of concern that the carcinogenic effect might become visible only at a later end point. Currently, most of the studies are not extended since geriatric pathology increases, which can complicate and obscure the assessment of carcinogenicity.
Experimental condition
Experimental conditions are of utmost importance because they can influence the results of the study. Factors such as hygiene, temperature, relative humidity, number of air replacements, and light have to be maintained and monitored consistently during the study Today, carcinogenicity studies are performed under Specific Pathogen Free (SPF) conditions. This means that SPF animals are obtained from commercial breeders and are placed in SPF rooms after arrival in the experimental unit. Other factors taken into account are quarantine, health monitoring, and hygienic measures during handling, such as sterile gloves and mouth masks. Good Laboratory Practice (GLP) has also contributed to improved test conditions. GLP not only applies to the way animals are handled, but also to appropriate documentation and recording of all actions during a study. This leads to better traceability, reconstruction, and interpretation of study data and results. All the improvements in experimental conditions have led to increased survivability in the animals. In the past, various deaths occurred due to respiratory or other infectious diseases; these are almost totally excluded within the current improved health condition of the animals.
Parameters examined in the study
During the 24-month study, various study parameters are examined.The daily follow-up is of utmost importance in order to pick up unexpected findings. If problems arise, the study director’s, veterinarian’s, or pathologist’s attention is drawn, and immediate and appropriate actions are requested. After necropsy during or at the end of the study, a mean list of 30 tissues is sampled and examined macroscopically. This may lead to a total number of 12,000 or more tissue samples for a single carcinogenicity study. All tissues are fixed and processed for further microscopic examination for neoplastic and nonneoplastic changes. These examinations are done by pathologists specialized in rodent pathology. The final aim is to detect the number of animals with tumors, but also multiplicity of tumors and whether the tumors caused death of the animal.
Histopathological evaluation
Histopathological examination is performed on all animals to detect “nonneoplastic” and especially “neoplastic” changes induced by the test compound. Nonneoplastic changes may include inflammatory, degenerative, or other changes in various tissues, either caused by the test item or by geriatric pathology. Neoplastic changes, or tumors, can be divided by “benign” and “malignant” neoplasms. Benign neoplasms are well defined, often encapsulated, noninvasive, and well differentiated. They grow relative slowly, display relative few mitoses, and are not metastatic. Malignant tumors are less well defined and usually not well encapsulated. They are invasive and relatively undifferentiated; they grow rapidly, display abundant mitosis, and finally undergo metastasis).
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Historical control data
Historical control data can be used to interpret the changes seen in carcinogenicity data. These control data can apply to the various parameters studied, such as hematology, biochemistry, and the incidences of tumors. They may be used when differences are seen between the incidences of tumors in the dosed groups and spontaneous incidences in concurrent control animals, where coincidence is suspected, or for tumors with very sporadic incidences. Spontaneous incidences in tumors are commonly seen in untreated rats and mice, and vary from strain to strain. Examples include pituitary and mammary tumors in rats, and liver and lung tumors in mice. The incidences of tumors can vary, and even today, there is no clear understanding of their etiology, except for ad-libitum feeding. On the other hand, caloric restriction could retard aging (associated with a reduction in the rate of cell, replication), and reduce the incidence of degenerative diseases and tumor incidences.
Statistical analysis
Statistical analysis is performed on all parameters in the study. Its most fundamental objective is to determine whether administration of the test agent results in an increase in tumor incidence rates as compared to those in unexposed controls. Various statistical methods can be used. Tests for increased tumor occurrence rates between dosages may be based on “pair-wise comparisons,” such as the Chi-square test, the Fisher’s exact test, or the Cochan–Armitage test. These tests are most appropriate when survival rates do not differ appreciably in the various dose groups
If the treatment results in reduced survival, early mortality in the high-dose groups may preclude the development of tumors and other statistical methods are required. Peto proposed a test for differences in tumor occurrence rates due to treatment, taking into account differences in survival and the times at which tumors were observed. This procedure requires information on the cause of death of each animal, and is based on a time-stratified contingency table analysis of the prevalence of incidental tumors that did not kill their host and a similar analysis of fatal tumors that resulted in death prior to the study. These two analyses are then combined to arrive at an overall test for increase in trend in tumor occurrence rates allowing for differential survival rates among the treatment group.
Cancer Risk Assessment
Once carcinogenicity testing has been performed, carcinogenicity risk assessment must be performed. Regulatory agencies have the responsibility to identify and assess compounds that are administered in food, provided as pharmaceuticals, or have the potential to be released in the environment at levels that warrant concern. Various topics have to be addressed when characterizing the carcinogenic risk. These include hazard identification (i.e., the likelihood to be a human carcinogen), doseresponse, and extent of human exposure. Each of these assessments involve the use of many assumptions and estimations, the magnitude of which may be decreased by the incorporation of more information (e.g., mechanistic studies, pharmacokinetic data, and improved low dose extrapolation models).In addition, the International Agency for Cancer Research (IARC) has evaluated and published carcinogenic risk to humans for hundreds of chemicals In both systems, chemicals, including pharmaceuticals, are assigned to five groups: 1) carcinogenic to humans; 2) probably carcinogenic to humans; 3) possibly carcinogenic to humans; 4) not classifiable for human carcinogenicity; and 5) probably not carcinogenic to humans. Assignment to one of these groups is based on scientific judgement of data derived from studies in humans and animals as well as supporting data. Data are estimated a providing sufficient, limited, or inadequate evidence for carcinogenicity in humans and rodents.
Other Future Opportunities
Data from short to medium-term toxicity studies that precede carcinogenicity studies reveal that most of the nongenotoxic agents which induce tumors in rodents also produce other pathological changes in the tissues in which the tumors develop and at dose levels at which tumors are observed. These early changes range from altered hormone levels, impaired ion balance, and organ enlargement to specific and marked histopathological changes. These findings may be used for early detection of nongenotoxic carcinogens, and may also be extremely valuable for designing protocols for long-term bioassays. Furthermore, a thorough understanding of such early indicators will lead to the elucidation of specific mechanisms involved in carcinogenesis. Together with examination of possible thresholds for underlying toxic events, this confirms the basis for assessment of carcinogenic risk and for the regulation of human exposureBased upon the above rationale, a “tier approach in carcinogenicity testing and assessment” of pharmaceuticals can be followed, possibly with refinement, reduction or replacement of test methodologies in carcinogenicity testing A first approach, according to the ICH S1A guideline scenario on the need for carcinogenicity testing, prescribes long-term carcinogenicity testing (in one or two species) for compounds with continuous or intermittent exposure to humans and compounds with cause for concern. If there is no long-term exposure to the compound, and if there is no cause for concern, no further action is recommended, whereas short- or long-term studies may be warranted for suspicious findings.A second approach postulates that much of the information necessary to assess the carcinogenic potential of a new drug without a bioassay is usually available by the end of the first clinical studies in patients. (Suspicious findings from in vivo genotoxicity studies and 3-6 month toxicology studies aimed at assessing risk factors associated with carcinogenicity in humans include: genotoxicity, immune suppression, hormonal activity, and chronic irritation/mitogenic activity.) Evaluation of this package will, therefore, identify the presence or absence of the known causes of cancer from pharmaceuticals in humans, under conditions relevant to the use of the drug in question. If cause for concern remains at this stage, useful information on long-term adverse effects that might represent a carcinogenic hazard to humans may be obtained (e.g., from a 12-month study, usually in rats, conducted at clinically relevant dose levels)Finally, a third approach has been proposed with five stages that focus on the chemical structure, DNAreactivity, epigenetic effects, limited bioassays, and finally, the application of “accelerated bioassays.” These accelerated bioassays require 40 weeks and apply to the use of sensitive markers for induction of neoplasia in comparison to positive control compounds for important organs in human carcinogenesis. It enables data acquisition of the entire carcinogenesis process directed toward developing mechanistic information. This system would have the potential to replace the chronic bioassay in rodents in some circumstances and could serve an alternative to a chronic bioassay in a second species.
CONCLUSION
In the 20th century, the concept of carcinogenesis and carcinogenicity testing has evolved enormously, although the standard Chronic Bioassay still contains many of deficiencies. New carcinogenicity testing strategies, however, are to be expected. Also, validation results with regards to the alternative carcinogenicity models will become available and lead into new insights in the most appropriate short-term carcinogenicity studies.
REFERENCES
(1)Ciminera, J.L.; Allen, H.L. Carcinogenicity Testing. Encyclopedia of Pharmaceutical Technology; Marcel Dekker, Inc.: New York, 1998; 285–317
(2) Williams, G.M.; Whysner, J. Epigenetic Carcinogens: Evaluation and Risk Assessment. Exp. Toxic. Pathol 1996,48, 189-195
(3) Robinson, D. The International Life Sciences Institute’s Role in the Evaluation of Alternative Methodologies for the Assessment of Carcinogenic Risk. Toxicol. Pathol. 1998 26(4), 474–475 .
(4) Inveresk Research International. Rodent Carcinogenicity and Chronic Toxicity. A Review of Test Protocols for Pharmaceuticals, Agrochemicals, Food Additives and Industrial Chemicals According to European, American and Japanese Guidelines. Regulatory Guidelines 1990, 1, 1–13.
(5)Feron, V.J.; Schwarz, M.; Hemminki, K.; Krewski, D. Long- and Medium-Term Carcinogenicity Studies in Animals and Short-Term Genotoxicity Tests, IARC Scientific Publications, No. 131; International Agency for Research on Cancer: Lyon, 1999; 103–129
(6)Statistical Methods in Cancer Research. The Design and Analysis of Long-Term Animal Experiments; Gart, J.J., Krewsky, D., Lee, P.N., Tarone, R.E., Wahrendorf, J., Eds.; No. 79 IARC Scientific Publications; 1986; 3.
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