Info

(genotoxic)

(genotoxic)

Nongenotoxic

Test model (duration)

initiator

carcinogen

carcinogen

TG.AC mice (6 months)

Negative

Positive

Positive

Tg rasH2 mice (6 months)

Negative (?)

Positive

Positive

p53/- mice (6 months)

Positive (?)

Positive

Negative

XPA-- mice (6 months)

Positive (?)

Positive

Negative

Newborn mice (1 year)

Positive

Positive

Negative

mutagenesis research and in the regulatory decision-making process, has been reviewed previously (67). The possibility of their use in carcinogenic risk assessment has also been explored (68).

The ICH guidance "Testing for carcinogenicity of pharmaceuticals" (S1B), in its Note 3, has introduced the potential for regulatory use of transgenic animal models, such as those mentioned above for carcinogenicity assessment of pharmaceuticals. The reliability of the different transgenic tumor models is under investigation in several studies, including an international collaborative study coordinated by the International Life Science Institute (ILSI), Health and Environmental Science Institute (HESI), Alternatives to Carcinogenicity Testing Committee. Contrera and DeGeorge (64) have pointed out that, "Based on available information there is sufficient experience with some in vivo transgenic rodent carcinogenicity models to support their application as complementary second species studies in conjunction with a single 2-year rodent carcinogenicity study." They go on to state, "The use of the in vivo transgenic mouse model in place of a second 2-year mouse study will improve the assessment of carcinogenic risk by contributing insights into the mechanisms of tumorigenesis and potential human relevance not available from a standard 2-year bioassay."

Certain of the regulatory authorities in the ICH regions have established a system by which to provide scientific advice to the private sector regarding the use of alternative toxicological methods. Such guidance is also available to pharmaceutical companies that intend to use alternative models for carcinogenicity testing.

4. Other Alternative In Vivo Models

In addition to transgenic tumor models, the ICH guidance S1B mentions, in its Note 3, two specific tumor models that may be used to replace a traditional rodent bioassay, that is, initiation-promotion test methodlogies and the neonatal rodent tumorigenicity model (newborn mouse model). Neither approach to test for tumorigenicity is new, and their suitability has been assessed in ICH negotiations on the background of a large database (69, 70). The newborn mouse model seems to be suitable primarily for detection of clearly genotoxic compounds, that is, "pure initiators" and "complete carcinogens" (Table 5), because the limitation of treatment to two to three doses over the first 2 weeks of life would generally not facilitate detection of nongenotoxic mechanisms of tumorigenesis.

5. In Vitro Models to Detect Cell Transformation

Several in vitro assays for malignant cell transformation have been developed in the past, the majority of which use fibroblastic target cells. Among them, the Syrian hamster embryo cell transformation assay (SHE assay) makes use of early passage (most often primary, secondary, or tertiary) cultures of embryo cells, which, upon treatment with known carcinogens, are "transformed" into morpho logically anomalous cells, recognized by their atypical clonal growth patterns into morphologically aberrant colonies (71-73). Other assays such as the BALB/3T3 (74) and the C3H10T'/2 (75) assays use established mouse cell lines. Aberrant cellular growth patterns atop confluent, contact-inhibited, monolayers of these cell lines are manifest as foci of transformed, misoriented, non-contact-inhibited cells.

Each of these in vitro transformation assay systems offers advantages and disadvantages as well as differences in their inherent capacity to metabolize (activate/detoxify) compounds to bioreactive metabolites or respond to metabolites generated by an exogenous metabolic supplement (76, 77). Transformed cell populations derived by expansion of transformed colonies or foci are tumorigenic in vivo, which is the basis for the use of such assays to predict carcinogenic potential of chemicals. These and other properties of transformation assay systems methodologies as well as surveys of their respective carcinogenic predicitivity have been reported (78-83).

Irrespective of the transformation system, however, the mechanism(s) of the transformation event has not been fully elucidated (83, 84). Furthermore, because the overwhelming majority of rodent and human tumors is of epithelial origin, it would appear that the predictivity of in vitro transformation systems (the preponderance of which rely on fibroblastic cell types) is independent of in vivo tumor type or tumor etiology. A successful malignant transformation of human primary cells of epithelial (embryonic kidney) and fibroblastic (foreskin) origin has been reported (85). In addition to loss of tumor suppressor gene function (P53 and RB) and gain of oncogenic activity (ras), expression of telomerase activity was found to be essential for creating tumorigenic human cells from normal ones (85).

Although in vitro cell transformation assays seem to be good predictors of animal carcinogenicity (80, 82, 86, 87), their added contribution in assessing risk remains obscure. One possible advantage of cell transformation assays vis-à-vis genetic toxicology assays as screening systems for carcinogens may be related to the apparent ability of the former to detect nongenotoxic carcinogens (80). However, it is not clear whether the supposed mechanism(s) of nongenotoxic carcino-genesis in rodents and in cell transformation assays in vitro are the same. The ICH carcinogenicity guidance S1C makes reference to the use of in vitro cell transformation assays for prediction of carcinogenicity "at the compound selection stage," and thereby encourages the use of such assays along with the routinely used standard genetic toxicology screening tests.

C. The Future of Regulatory Carcinogenicity Testing

1. Integrated Approaches to Evaluate Genotoxicity and Carcinogenicity

The preponderance of available scientific information suggests that cancer is a process in which genetic changes are inevitably involved (88). The testing of compounds for their genotoxic potential mainly addresses safety concerns regarding a carcinogenic potential by induction of irreversible changes in the genetic material of cells as the initial step in the carcinogenesis process. The new ICH guidances on genotoxicity and carcinogenicity represent a more integrated approach to these fields than has previously existed. For instance, it is acknowledged in the ICH guidance that "unequivocally genotoxic compounds, in the absence of other data, are presumed to be trans-species carcinogens, implying a hazard to humans. Such compounds need not be subjected to long-term carcinogenicity studies." To allow for this type of situation, appropriate product labeling has been put into practice to reflect the presumed carcinogenicity on the basis of positive genotoxicity, when other (e.g., carcinogenicity) results are lacking. Both the ICH genotoxicity and carcinogenicity guidances seek additional genotoxicity data "for compounds that were negative in the standard test battery (for geno-toxicity) but which have shown effects in a carcinogenicity test with no clear evidence for an epigenetic mechanism." Such additional data may come from investigations into DNA adduct formation, mutation induction in transgenes, or changes in tumor-related genes. In addition, new transgenic tumor models may provide other options for evaluating potential carcinogenicity of compounds demonstrated to be genotoxic, such as the p53+/- knockout model (64).

2. Carcinogenicity Testing of Nongenotoxic Compounds

Mechanistic understanding of the action of human carcinogens, that is, IARC Category 1 carcinogens, suggests that such agents may be subdivided roughly into five carcinogenic groups: (1) genotoxic compounds, (2) fibers, (3) compounds resulting in hormonal imbalances, (4) immunosuppressants, (5) mito-gens. Representatives of these groups, with the exception of carcinogenic fibers, are also found among pharmaceuticals (89). The question may be posed whether future experience and a more detailed epidemiological analysis of human car-cinogenesis will add to the list of carcinogens others that may produce tumors by completely different modes of action. For the present, however, it seems reasonable to concentrate mainly on the detection of possible human pharmaceutical carcinogens that operate through the above-named mechanisms. It is understood that the majority of genotoxic carcinogens are identifiable by the genotoxicity test battery plus short-term tumor models such as the p53+/- knockout mouse (see previous sections). Conversely, the identification of relevant nongenotoxic carcinogens seems to necessitate the exploitation of alternative approaches.

The current database for the rodent bioassay includes results for each of the above-specified mechanisms. Yet, the database includes compounds whose tum-origenicity in rodents may not be relevant for human exposure situations. Induction of liver tumors in rats through peroxisome proliferation, induction of mammary tumors in rodents by enhanced secretion of prolactin, induction of liver tumors in rodents by effects on the cytochrome p450 system (barbiturates)

are examples of tumor responses in rodents from exposure to pharmaceuticals that are believed not to be relevant for the patient situation. Information on all of these probable mechanisms can be derived from toxicological studies other than the rodent bioassay. Further investigations into existing and evolving alternative predictive models may ultimately provide the desired knowledge to supplement an abridged version of the rodent bioassay. It is expected that such an integrated approach should, in most instances, accurately predict tumor outcome. When that situation is achieved, the conduct of rodent bioassays in two species on new compounds in well-defined pharmacological classes of action may, at times, be considered superfluous. Monroe's conclusion that "if a pharmaceutical is not (i) genotoxic, (ii) an immunosuppressant, (iii) hormonal, or (iv) a chronic irritant at human exposure levels, it is unlikely to pose a carcinogenic hazard to humans" may be seen as an extreme position (90).

The ICH guidances emphasize the importance of using all corroborative information when designing the rodent bioassay experiment for carcinogenicity assessment. The future, however, could witness the replacement of the rodent bioassay completely by an assessment of the "likelihood of tumorigenesis" using "alternative" information from pharmacological and toxicological studies plus various short-term tumor models without any forfeiture of safety. In this regard, transgenic short-term tumor models capable of detecting both genotoxic effects and relevant nongenotoxic mechanisms would be most advantageous. Current evidence suggests that the human c-Ha-ras transgenic mouse model fulfills some of these conditions, because it may not respond to nongenotoxic mouse-only carcinogens (65). Further pursuit of such model systems may, hopefully, abbreviate the effort, time, and expense, as well as limit the use of animals, associated with current approaches to assess carcinogenic potential. In fact, recent developments already indicate that short-term transgenic tumor models could ultimately replace the traditional rodent bioassays for assessing both genotoxic and non-genotoxic carcinogenesis (64, 65, 91, 92). As explained by Contrera and DeGeorge (64), there are several potential and promising applications of alternative assay systems for assessing carcinogenicity. Some of these are to serve as (a) surrogates for the 2-year rodent carcinogenicity study performed in a second species; (b) complementary and confirmatory tests for equivocal carcinogenicity results derived from the rodent bioassay; (c) prescreens for carcinogenic potential before conducting comprehensive carcinogenicity studies; (d) an option to repeating an inadequate, unacceptable, or uninformative lifetime rodent bioassay; (e) methods for determining the carcinogenic activity of newly introduced genotoxic impurities or degradants present in the final marketed product that may not have been present at the time that the rodent carcinogenicity bioassay was performed. With these encouraging prospects, it is not unreasonable to predict that the future of regulatory decision-making for pharmaceuticals will rely heavily on alternative methodologies and mechanistic investigations on issues of of genotoxic and non-

genotoxic carcinogenesis, and that this reliance could supplant the current dependence on guidance-conforming performance of rodent carcinogenicity bioassays.

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