The role of transgenic mouse models in carcinogen identification. (Research).

In this article, we examine existing data on the use of transgenic mouse models for identification of human carcinogens. We focus on the three most extensively studied of these mice, Trp53+/-, Tg/AC, and RasH2, and compare their performance with the traditional 2-year rodent bioassay. Data on 99 chemicals were evaluated. Using the International Agency for Research on Cancer/Report on Carcinogens determinations for the carcinogenicity of these chemicals to humans as the standard for comparison, we evaluated a variety of potential testing strategies ranging from individual transgenic models to combinations of these three models with each other and with traditional rodent assays. The individual transgenic models made the "correct" determinations (positive for carcinogens; negative for noncarcinogens) for 74-81% of the chemicals, with an increase to as much as 83% using combined strategies (e.g., Trp53+/- for genotoxic chemicals and RasH2 for all chemicals). For comparison, identical analysis of chemicals in this data set that were tested in the 2-year, two-species rodent bioassay yielded correct determinations for 69% of the chemicals. However, although the transgenic models had a high percentage of correct determinations, they did miss a number of known or probable human carcinogens, whereas the bioassay missed none of these chemicals. Therefore, we also evaluated mixed strategies using transgenic models and the rat bioassay. These strategies yielded approximately 85% correct determinations, missed no carcinogens, and cut the number of positive determinations for human noncarcinogens in half. Overall, the transgenic models performed well, but important issues of validation and standardization need further attention to permit their regulatory acceptance and use in human risk assessment. Key words: carcinogens, hazard identification, mouse model, mutagenesis screening, transgenic models.

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The National Toxicology Program (NTP) is responsible for evaluating the toxicity and carcinogenicity of environmental agents, developing and validating improved testing methods, and strengthening the science base of toxicology. A variety of end points are used to assess the systemic toxicity of environmental chemicals, but the mainstay of chemical carcinogenicity testing has been the 2-year rodent bioassay. This highly standardized method has been widely adopted throughout the world. However, like any other approach, the rodent bioassay has its strengths and weaknesses. In particular, the 2-year bioassay is expensive, both in resources and time required and in the numbers of animals needed. Thus, the advent of transgenic and gene knockout technology in the early 1980s and increasing knowledge of the mechanisms involved in carcinogenesis led a number of investigators to examine whether faster, less costly, and more predictive models might be developed. The National Institute of Environmental Health Sciences (NIEHS) has been actively involved in this effort for more than a decade, and several model systems using transgenic and knockout models have been investigated (Bucher 1998; Eastin et al. 1998; Tennant 1993; Tennant et al. 1995).

Transgenic models have a number of potential advantages for use in carcinogen identification programs. For example, because tumors arise more quickly in the genetically engineered models, the assays can be more rapid. For the studies reviewed here, the assay length was 24-26 weeks, significantly shorter than the standard 2-year rodent bioassay. Transgenic models may also provide the opportunity to reduce the number of animals used in testing. Shorter assays using fewer animals could also reduce the overall cost of testing programs. However, proprietary issues and the limited availability of some models may impact cost savings. Furthermore, with appropriate model selection, it may become possible to more accurately predict the human response, contributing directly to the ease and effectiveness of risk assessment and regulatory decisions. Finally, by virtue of the specific genetic modification(s) in transgenic models, it should be possible to gain additional insights into the mechanisms involved in tumor induction and development. Such insights would facilitate identification of important mechanisms of the tumor response and chemical features associated with carcinogenesis.

Although they have great promise, transgenic models also have actual or potential limitations for use in a carcinogen identification effort. For example, many current transgenic models (including those evaluated here) have mutations in only one pathway that may or may not be relevant to human cancer processes for a given chemical. In addition, the specific gene defect may influence tumor development and type, increasing the difficulty of modeling the human response. Likewise, the strain (genetic) background can influence tumor type, incidence, and location. Thus, short-term, gene-specific transgenic assays may lose biological information obtained in longer term bioassays (e.g., multiple target organ effects and/or interactions of time and age that are important in chemical carcinogenicity). These issues do not preclude the use of transgenic models, but they must certainly be considered in their development and selection and in interpretation of data obtained using transgenic models.

Given the potential and the limitations of the transgenic models, the goals of the current assessment were to a) review progress in this field of research, b) determine if the models reviewed show sufficient merit for use in a carcinogen identification program, and c) identify research needs and knowledge gaps that should be addressed to increase the effectiveness of transgenic models.

Review of Research Progress

Many transgenic models are available for various investigational uses, but three transgenic models have been most widely used for carcinogen identification: Trp53+/-, Tg.AC, and RasH2. We selected these three models for this assessment because they have the extensive data set needed for this analysis. Their selection does not indicate that they are deemed superior a priori to other transgenic models.

Extensive recent reviews of these three models have been published, and only their main features are briefly reviewed here. They were developed based on dysregulation of either the Trp53 tumor-suppressor gene or the rasproto-oncogene, both of which are critical to cancer development and which represent the two main classes of human cancer genes. The p53 protein suppresses cancer in humans and rodents and is mutated or dysfunctional in more than 50% of all cancers (Donehower et al. 1992; Hollstein et al. 1991; Weinberg 1991a). As a transcription factor, p53 regulates the activity of a variety of genes involved in cell cycle arrest, apoptosis, anti-angiogenesis, differentiation, DNA repair, and genomic stability (el-Deiry 1998; Prives and Hall 1999). The ras proto-oncogene protein (H-, K-, and N-ras isoforms) is integral to cell proliferation through signaling by growth factors and noxious agents (chemicals, UV radiation, etc.) that act via the mitogen-activated protein kinase kinase (MAPKK) pathway (Campbell et al. 1998; Gupta et al. 2000; Pruitt and Der 2001). Activation and dysregulation of ras through mutations at specific sites within the gene are often observed in both human and rodent cancers (Bos 1989; Hruban et al. 1993; Vogelstein et al. 1990; Yunis et al. 1989). In addition, increased expression of oncogenic ras protein is often seen during tumorigenesis by aneuploidy of the ras-bearing chromosomes, which may be analogous to overexpression of induced transgenic ras protein. Overall, ras is overexpressed in more than 50% of all cancers.

The Trp53 heterozygous null allele (+/-) mouse. This model uses B6129 N5 mice heterozygous for a wild-type Trp53 tumor-suppressor gene and a null allele that is not transcribed or translated (Donehower et al. 1992; Harvey et al. 1993). These Trp53 heterozygotes (+/-) have a low spontaneous tumor incidence up to 9 months of age but have increased spontaneous tumor rates thereafter with approximately 50% survival at 18 months. Therefore, short-term (26 week) exposure to test and positive control agents during the period between 7 and 33 weeks of age makes it possible to distinguish between treatment-induced and spontaneous tumors that may arise independently and confound a cancer bioassay (Haseman and Elwell 1996; Karstadt and Haseman 1997). The Trp53 model appears to be particularly useful as an in vivo test for mutagenic carcinogens (Donehower et al. 1992; Eastin et al. 1998; Harvey et al. 1993; Kemp et al. 1993, 1994; Tennant et al. 1995). In human cancers, where mutations have been found in up to 50% of all tumors (Greenblatt et al. 1994; Hollstein et al. 1991), point mutations or deletions in one allele of the Trp53 gene that create a heterozygous allelic state are usually accompanied by loss of the normal allele (loss of heterozygous or LOH) (Weinberg 1991b). Because Trp53 +/- mice only carry one copy (germline) of the gene, these mice were expected, according to the two-hit hypothesis (Knudson 1996; Knudson et al. 1975), to show a shorter latency period for tumors induced by genotoxic agents. However, there is evidence that the acceleration of tumorigenesis in Trp53 +/- mice may be due to a gene dosage effect and a haplo-insufficient phenotype such that a second (p53 LOH) event is not required (French et al. 2001; Venkatachalam et al. 1998).

The Tg.AC (v-Ha-ras) mouse. The Tg.AC transgenic mouse model provides a reporter phenotype (skin papillomas) in response to either genotoxic or nongenotoxic carcinogens, including tumor promoters (Spalding et al. 1993, 1999; Tennant et al. 1999). Tg.AC mice are hemizygous for a mutant v-Ha-ras transgene. The model was developed by Leder et al. (1990), with an inducible [xi]-globin promoter driving the expression of a mutated v-Ha-ras oncogene and is regarded as a genetically initiated model. With the exception of the bone marrow, constitutive expression of the transgene cannot be detected in adult tissues. The transgene is transcriptionally silent until activated by full-thickness wounding, UV irradiation, or specific chemical exposure (Cannon et al. 1997; Trempus et al. 1998). Topical application of carcinogens to the shaved dorsal surface of Tg.AC mice induces epidermal squamous cell papillomas or carcinomas, a reporter phenotype that defines the activity of the chemical. The oral route of administration can also generate tumor responses in the skin of Tg.AC mice and in addition lead to squamous cell papillomas and/or carcinomas of the forestomach. To date, the appearance of either spontaneous or induced tumors has been shown to require activation of transgene expression. However, the mechanism of response by the Tg.AC model to chemical carcinogens is not yet understood.

The rasH2 mouse. The rasH2 mouse is hemizygous for the human c-Ha-ras transgene under control of its endogenous promoter and enhancer sequences. It was developed by Saitoh et al. (1990) in CB6F1 mice to evaluate the association of chemically induced transgene expression and tumor induction (Katsuki et al. 1991; Yamamoto et al. 1996, 1998a). The transgene encodes a prototype c-H-ras gene product, p21, that does not induce transformation in NIH3T3 cells. Approximately three copies of the human transgene were integrated into the mouse genome in a tandem array through pronuclear injection (Suemizu et al. 2002). Expression of the transgenic protein is observed in normal tissues and increased approximately 2-fold in chemically induced tumors (Maruyama et al. 2001). Mutation of the endogenous mouse ras genes or of the transgene is infrequent and unpredictable (Katsuki et al. 1991), suggesting that a 2- to 3-fold increase in ras protein expression is sufficient to cooperate with other carcinogen-induced changes (genetic and/or epigenetic) to predispose this mouse to development of neoplasia.

Merits of the Models

Data collection. To assess the potential merit of the three transgenic models in a research and testing program, we assembled available information on responses to chemical treatment in each model (Tables 1-3). The primary sources of these data were the recent publications of the International Life Sciences Institute (ILSI) Assay Working Groups for the Trp53+/-, Tg.AC, and RasH2 Mouse Alternative Models (Popp 2001; Robinson and MacDonald 2001), NTP evaluations, and published independent laboratory research using alternative or conventional rodent models for carcinogen identification (for specific references, see Tables 1-3). The resulting data set consists of 99 chemicals that were tested at the maximum tolerated dose (MTD) or proportional fractions of MTD as determined by toxicokinetic and range-finding studies in the test strain using positive and negative controls groups and nongenetically altered co-isogenic reference controls. Dosing routes, study duration, number of animals per group, and extent of histopathologic evaluation varied between studies and chemicals. Despite these limitations, for the purposes of this analysis, peer-reviewed published findings were accepted as reported.

Criteria for analysis. Because the goal of NTP carcinogenicity testing is prediction of human carcinogenicity of chemicals, the merit of each transgenic model was evaluated by determining the ability of the model to identify human carcinogens. Classification of human carcinogens was based on evaluations by the NTP Report on Carcinogens (ROC) (NTP 2002) and the International Agency for Research on Cancer (IARC 2002) chemical evaluations/classifications. Both the ROC and IARC assessments are based on comprehensive evaluations of all relevant human and animal data from the published literature. The designation of an agent as a "known human carcinogen" by IARC (group 1) or the ROC requires definitive data from human epidemiologic studies or strong mechanistic data from human systems in conjunction with similar mechanistic and cancer data from experimental animals. Less convincing evidence (e.g., limited human data and/or sufficient animal data) will generally lead to the designation of the agent as a "probable" (group 2A) or "possible" (group 2B) human carcinogen by IARC or a "reasonably anticipated" human carcinogen in the ROC. A chemical that shows inadequate evidence of carcinogenicity in humans and animals will generally result in an IARC designation of "not classifiable" (group 3). The ROC has no equivalent to IARC group 3 and does not list such chemicals. Rodent carcinogenicity was not used as the primary targeted response in our analysis. Nevertheless, for completeness we did consider the correlation of each transgenic model with the outcomes of National Cancer Institute (NCI)/NTP long-term rodent tests. We also examined whether these transgenic assays were more or less accurate in predicting human carcinogenicity of genotoxic versus nongenotoxic chemicals, as defined by either a positive result in the Salmonella (Ames) test and/or in vivo rodent micronucleus assay.

A total of 99 chemicals have been studied in one or more of these three transgenic models. For this analysis, we divided these chemicals into three groups: a) known human carcinogens (IARC group 1 and/or ROC known; 14 chemicals, Table 1); b) probable/possible human carcinogens (IARC groups 2A and 2B or ROC reasonably anticipated; 32 chemicals, Table 2); and c) chemicals with inadequate evidence of carcinogenicity (IARC group 3, NTP bioassay negative, and/or not listed in the ROC or by IARC; 53 chemicals, Table 3).

Tables 1-3 identify each chemical by Chemical Abstracts Service (CAS) number and give the IARC and/or the ROC evaluations. For those chemicals evaluated in the NTP rodent bioassay, carcinogenicity results are given for each sex-species group (male rats, female rats, male mice, female mice). Genotoxicity outcomes from the Salmonella (Ames) assay and the in vivo micronuclei assays are also given. Finally, the results of carcinogenicity testing in each of the three transgenic models are given. The route of administration is noted, as well as the published reference source. For chemicals tested more than once in the transgenic models, each result is given separately.

For each of the transgenic models and for the rodent bioassay, a chemical is designated as a carcinogen if positive (carcinogenic) effects were found in one or more of the sex--species groups. Similarly, a chemical found to be positive in either the Salmonella assay or the in vivo micronudei assay is considered to be genotoxic.

Analysis of the models. Based on the 99-chemical database from Tables 1-3, 10 possible strategies were considered for using transgenic models to identify chemicals as known or suspected human carcinogens or as noncarcinogens. For comparison, the standard 2-year, two-species rodent bioassay and a modified strategy using the rat bioassay in conjunction with genotoxicity were also analyzed in an identical fashion. Thus, 12 strategies in all were considered:

* Strategy 1: Trp53+/- model

* Strategy 2: Trp53+/- model, but only for genotoxic chemicals

* Strategy 3: Tg.AC model

* Strategy 4: RasH2 model

* Strategy 5: Trp53+/- model for genotoxic chemicals; RasH2 model for nongenotoxic chemicals

* Strategy 6: Trp53+/- model for genotoxic chemicals; RasH2 model for all chemicals

* Strategy 7: Trp53+/- model for genotoxic chemicals; Tg.AC model for nongenotoxic chemicals

* Strategy 8: Trp53+/- model for genotoxic chemicals; Tg.AC model for all chemicals

* Strategy 9: NTP bioassay

* Strategy 10: NTP rat bioassay plus the Tg.AC model for nongenotoxic chemicals or the Trp53+/- model for genotoxic chemicals

* Strategy 11: NTP rat bioassay plus the RasH2 model for nongenotoxic chemicals or the Trp53+/- model for genotoxic chemicals

* Strategy 12: NTP rat bioassay plus genotoxicity.

When evaluating strategies that were conditional on genotoxicity (strategies 5-8, 10-11), the following conventions were established: a) a chemical was considered genotoxic if either the Salmonella or in vivo micronuclei assays were positive; b) a chemical was considered nongenotoxic only if both assays were negative; and c) when a chemical's genotoxicity could not be determined definitively (i.e., negative in one assay and not tested in the other), the chemical was excluded from the analysis, unless the genotoxicity status of the chemical had no impact on the transgenic mouse result (i.e., both transgenic models were positive or both were negative).

A valid transgenic rodent model should successfully identify (test positive) the IARC/ROC known or suspected human carcinogens listed in Tables 1 and 2. Likewise, such a model should identify as noncagcinogens (test negative) those chemicals in Table 3 that were shown in NTP long-term bioassays to be negative. Although many of the remaining chemicals in Table 3 were positive in a long-term rodent bioassay, these results were not considered by the IARC and/or ROC to be sufficiently convincing to merit the categorization of the chemical as a known, possible, probable, or reasonably anticipated human carcinogen. For these chemicals, it is uncertain if the response of the transgenic models should be positive or negative as carcinogens. Thus, our initial analysis (Table 4) included only those group 3 chemicals with negative results in the NTP rodent bioassay. Table 5 examines the same data set as Table 4 but considers each IARC/ROC classification separately to ensure that pooling carcinogen groups in these analyses did not lose important distinctions between assay responses to strong or weak carcinogens.

In addition, as summarized in Table 6, we have conducted a second analysis in which all chemicals in Table 3 are regarded as human noncarcinogens (i.e., we have assumed, for the sake of direct comparison between transgenic and traditional NTP bioassays, that more extensive testing of these chemicals would confirm their lack of human carcinogenicity). This assumption permits exactly the same criteria to be applied to all strategies, transgenic and traditional alike. Finally, although human carcinogenicity was used as the targeted response in our analysis, a similar analysis was conducted in which the transgenic assay responses were compared with the results of the NTP bioassay (Table 7).

Results and Discussion

Scope of analysis. Before discussing the analysis, it is critical to reiterate the precise limitations and assumptions implicit in our analysis. First, this evaluation was limited to those chemicals with definitive published transgenic results available at the time of our analysis. We recognize that this is a dynamic field of research. Thus, additional transgenic studies will become available over time, and it is possible that some chemicals listed in Tables 1-3 could be reclassified after consideration of such new data. However, we suggest that the analyses for these 99 chemicals are sufficiently robust that the addition, subtraction, and/or reassignment of chemicals will not alter the conclusions, provided that uniform criteria are applied.

Second, optimal protocol designs for specific transgenic animal cancer bioassays have not been identified and validated. Thus, the study designs that form the basis of this evaluation may differ from each other with regard to study duration, sample sizes, dose selection strategy, number of doses, tissues examined, methods of statistical analysis, historical controls, and the use of positive and negative controls.

Third, we made no interpretative decisions ourselves in regard to study results. For assessments of possible human cancer risk, we relied on the authoritative judgments of IARC and the ROC. Likewise, we accepted the authors' interpretations of the data. However, there was uniformity of study design and interpretation for a sizable number of the studies involved in the International Life Sciences Institute (ILSI) Alternatives to Carcinogenicity Testing consortium. It was beyond the scope of our analysis to reevaluate and reinterpret each individual study.

Fourth, we acknowledge that a positive transgenic study may reflect a wide range of carcinogenic responses, with some positive results being limited to a marginal increase in a single tumor type in a single sex-species group, while others reflect striking multisite, multisex, carcinogenic effects. Although future refinements in statistical evaluation may permit sub-classification and rank order documentation for the various positive transgenic responses, we have not attempted to do so at this stage in the development of transgenic rodent bioassays.

Finally, we recognize that certain chemicals listed in Table 3 may ultimately be shown to be known or suspected human carcinogens, especially those with positive rodent bioassay results. However, our current state of knowledge does not permit a higher classification of these chemicals. As noted below, the frequency of positive transgenic results for Table 3 chemicals was essentially the same for those chemicals that were evaluated by the IARC (and assigned to category 3) and those that were not yet evaluated and are thus unclassified. This suggests that there are few, if any, important human carcinogens among the unclassified chemicals in Table 3.

Performance of strategies. The overall performance of each transgenic strategy is summarized in Table 4. With the caveat that data on all chemicals were not available for each model and thus, that the subset of chemicals actually tested in each model may influence the specific outcomes reported, each of the three transgenic mouse models predicted human carcinogenesis for 77-81% of the chemicals studied in that model, ranging from 77% for the Trp53+/-, 77% for the Tg.AC, and 81% for the RasH2. Use of the Trp53+/- for only genotoxic chemicals increased its predictiveness to 84%. The combined strategies that use more than one transgenic model (strategies 5-8; as defined above) were somewhat more predictive, ranging from 82 to 90%. The best strategy (Trp53+/- for genotoxic chemicals and RasH2 for all chemicals; strategy 6) correctly predicted the human outcome for 90% of the agents (Table 4). Strategy 8 (Trp53+/- for genotoxic chemicals and Tg.AC for all chemicals) was only slightly less predictive (84%).

Our initial analysis (Table 4) defined the targeted population of human carcinogens as the pool of chemicals from Tables 1 and 2, in which IARC classifications ranged from 1 to 2B. A further breakdown of these chemicals is shown in Table 5. Note that a) the transgenic models (considered collectively) are more apt to be positive for the more certain human carcinogens (IARC groups 1 and 2A) than for the less certain human carcinogens (group 2B); b) there is a striking difference in the proportion of positive transgenic responses between the 1/2A/2B chemicals and the group 3 chemicals or those not evaluated; and c) the IARC group 3 chemicals and those not evaluated show a similar rate of overall transgenic responses, indicating that most of the unclassified chemicals listed in Table 3 may be human noncarcinogens.

Our initial analysis (Table 4) was somewhat restrictive, in that it defined human noncarcinogens as being only those chemicals from Table 3 with negative NCI/NTP rodent bioassay results. However, Table 5 suggests that it is reasonable to expand this classification and regard all Table 3 chemicals as human noncarcinogens. This analysis is summarized in Table 6, which allows more direct comparison of the performance of the transgenic models with the traditional NTP two-species bioassay. Transgenic and traditional testing strategies each show strengths and weakness. These strengths and weaknesses differ. For the transgenic models, particularly the RasH2 and the Trp53+/-, there are relatively few positive findings for noncarcinogens (i.e., group 3 chemicals, either known negatives or chemicals unlisted by IARC/ROC, that gave evidence of carcinogenicity in the assay). In fact, as shown in Table 4, RasH2 and Trp53+/- have no positive results for noncarcinogens if those group 3 chemicals that lack a negative rat and mouse bioassay are eliminated from the analysis (in effect, eliminating those chemicals with greater uncertainty as to their carcinogenic potential). The Tg.AC model was more prone to this type of error than the other two transgenic models reviewed (Tables 4 and 6). The combined transgenic strategies (strategies 5-8) did not improve predictability.

A more frequent shortcoming of the transgenic models (including those strategies using multiple transgenic models) was the number of negative tests for known or suspected human carcinogens (i.e., those listed in Tables 1 and 2) (Tables 4 and 6). For example, even the most predictive combination (the combined results of Trp53+/- for genotoxic chemicals plus Tg.AC for nongenotoxic chemicals; strategy 7) still had 6 negative results for IARC/ROC known carcinogens among the total of 53 chemicals tested in both (Table 6).

In contrast, the NTP two-species bioassay identified all IARC/NTP known/probable human carcinogens (Tables 1 and 2). Thus, as shown in Table 6 (strategy 9), among the 58 chemicals evaluated in the NTP bioassay, there were no negative results for known human carcinogens. However, this is not without a downside in the form of numerous positive findings for chemicals that are considered to be noncarcinogens in humans (Table 3). In this data set, there were 18 positive assay results for IARC/ROC noncarcinogens among a total of 58 chemicals tested, or 31% (Table 6). Certainly, there is a cost of this type of error as well, specifically unneeded regulation and/or additional testing. It is this propensity for positive findings for chemicals considered to be human noncarcinogens that yielded the surprisingly low 69% concordance between the standard NTP bioassay and human cancer--surprising because many of the ROC and IARC determinations are based in large part on animal data and the NTP bioassay in particular. In fact, all three transgenic models had a modestly higher concordance with human carcinogens (Tables 1 and 2) than the rodent 2-year bioassay (Trp53+/- 81%, RasH2 76%, and Tg.AC 74%; Table 6). Of course, this difference is also reflected in the modest success (54-75%) of the transgenic models as predictors of the bioassay response (Table 7).

It should be emphasized that it is possible that many of the 18 NTP rodent carcinogens labeled in our analysis as "positive for noncarcinogens" (Table 6, strategy 9) may ultimately prove to be actual human carcinogens as additional data become available. However, at this time the positive rodent data are not sufficiently compelling for the IARC or the NTP ROC to consider these chemicals to be known, probable, possible, or reasonably anticipated human carcinogens. In those rare cases where the IARC and ROC disagreed (e.g., diethylhexyl phthalate) we used the most recent determination. Moreover, these 18 chemicals collectively were positive in only 23% (7/30) of the three transgenic assays evaluated, as compared with 66% (29/44) positive transgenic assays conducted on the 24 known/probable carcinogens. This difference strongly suggests that the transgenic assays are selectively identifying the trans-species carcinogens.

Because both transgenic models and the bioassay have strengths and weakness in correctly identifying carcinogenic chemicals, we examined the performance of composite strategies using both transgenic and conventional rodent models to determine if such a strategy might capitalize on the strengths of both types of models. Strategies 10 and 11 address this possibility (Table 6). Strategy 10 (rat bioassay for all chemicals plus the Trp53+/- model for genotoxic agents or the Tg.AC for nongenotoxic chemicals) provided an improvement in performance. Overall concordance increased to 84% versus the 69% of the bioassay itself. More important, negative results for known carcinogens were completely eliminated, and positive findings for noncarcinogens were reduced to 16% (9/57) versus the 31% (18/58) for the bioassay. A similar strategy (strategy 11) substituting RasH2 for Tg.AC gave similar results, with an overall concordance of 85% (45/53), or just 15% (8/53) with positive results for noncarcinogens.

For those chemicals evaluated in both the NTP bioassay and the transgenic models, the substitution of the transgenic models (strategy 10: Trp53+/- for genotoxic chemicals; the Tg.AC for nongenotoxic chemicals) for the B6C3[F.sub.1] mouse used in the standard bioassay resulted in a net reduction of four positive findings. Four chemicals (coconut oil diethanolamine, diethanolamine, N-methyloacrylamide, and methylphenidate) were negative in the transgenic models and the NTP rat bioassay. In the B6C3[F.sub.1] mouse, the first two of these chemicals produced liver tumors (both sexes) and kidney adenomas (males only). N-Methyloacrylamide produced tumors of the Harderian gland, liver, lung, and ovary. Methylphenidate produced liver tumors only. None of these chemicals has been classified as a known/probable human carcinogen by the IARC or the NTP ROC (Tables 1-3).

Historically, genotoxicity has proven to be an important clue as to the likely carcinogenesis of chemicals (Ashby and Tennant 1991; Shelby 1988). In addition, as shown in Table 4, it increases the predictiveness of the Trp53+/- model. Thus, to provide a more complete assessment of possible testing strategies, we compared an additional strategy (strategy 12, Table 6) that consists of substitution of genotoxicity data for the transgenic models to be used in concert with the rat bioassay (strategies 10 and 11, Table 6). Strategy 12 does, like the bioassay itself, avoid negative results for known carcinogens. It also has modest concordance with human carcinogenesis 65% (43 of 66), but it has 23 positive results for noncarcinogens out of 66 chemicals (35%). A number of the other strategies do better.

Conclusions. Given the complementary strengths demonstrated by the transgenic models and the 2-year rodent bioassay as presented above and summarized in Table 6, it appears that a strategy employing both types of models would have advantages over either alone. Thus, strategies 10 and 11 that use the standard rat bioassay in conjunction with Trp53+/- for genotoxic chemicals and Tg.AC or RasH2 for nongenotoxic chemicals are promising, based on their performance with these 99 chemicals.

Research Needs

This analysis demonstrates that transgenic models have the potential to play an important role in identification of potential human carcinogens. However, several research needs and data gaps remain to be addressed to ensure that the use of transgenic models has been adequately evaluated and that protocols have been optimized or standardized for such use, critical requirements for the regulatory acceptance of transgenic model data and its use in human risk assessment.

Validation of study design. The study design for each transgenic model must be rigorously evaluated and optimized for the testing paradigm used (e.g., toxicity, mutagenicity, or carcinogenicity). Therefore, additional research will be required for each model evaluated and used in the NTP testing program. As mentioned previously, the testing strategies, animal numbers, duration of dosing, extent of pathology, and interpretation of results varied among the studies evaluated. In particular, an optimal design for transgenic models has not yet been identified that clearly eliminates the potential for false negatives in carcinogen identification. Two possible strategies for increasing the power of the study (thereby reducing the negative results for known human carcinogens) are to increase the sample size beyond the 15 animals per group commonly used and/or to increase the duration of the study to allow more time for tumors to develop. The performance of the transgenics under these different conditions should be thoroughly investigated and standardized. A perhaps less obvious possibility would be to compile a rigorous historical control database for the various transgenic models and to make use of this information in weight-of-evidence decisions. Many of the tissues in the transgenic mouse models have a low spontaneous tumor incidence. Thus, the occurrence of two or three of these tumors in a dosed group in a given study, although perhaps not statistically significant when tested against the concurrent controls, may nevertheless be significant when the low historical control incidence is taken into account. For example, three of the seven negative results for known/suspected carcinogens associated with the RasH2 model (cyclosporin A, melphalan, and 1,4-dioxane) produced tumor effects that were considered equivocal. Had it been possible to consider these tumor responses in the context of a large historical control database, certain borderline cases might have been regarded as biologically significant, thereby reducing the number of incorrect findings.

Improve understanding of chemical outcomes. One problem in our analysis was in identifying a rational basis to explain discordant results. For example, the most significant shortcoming of a combined (transgenic plus rat bioassay) strategy was not the negative results for known carcinogens, but rather the apparent number of positive chemicals in the rat bioassay that are not listed as known or reasonably anticipated to be human carcinogens (e.g., the 8 of the 53 chemicals for strategy 11; Table 6). How might this be improved? First, it might be possible to design additional studies to investigate whether or not these are truly noncarcinogenic chemicals. As discussed above, the targeted response in our investigation is imperfect, as it represents a scientific judgment by IARC and/or the ROC regarding potential carcinogenicity based on available data. In many cases, the existing data are insufficient for a definitive decision to be made. Additional research could reduce the number of positive results for supposed noncarcinogens simply by revealing that certain of these chemicals are in fact carcinogens. Other options that might be considered to reduce this type of error include a rat transgenic model (if done in a manner that did not yield negative results for known carcinogens) or improvements in the design of the rat bioassay itself.

Development of a chemical database to validate transgenics. The data set summarized in Tables 1-3 may provide an important resource if appropriate statistical considerations could be developed to allow selection of an informative subset of chemicals to evaluate new models and/or modify current protocols. Such a set of chemicals that represents a spectrum of mechanisms or modes of action consistent with human carcinogenesis would not only be valuable in the context of the models discussed above but would lend itself to the evaluation and validation of any new model, transgenic or otherwise.

Development of models. In the current analysis we examined the Trp53+/-, Tg.AC, and RasH2 transgenic models because these models had the most complete data sets available. Other models are also under evaluation at the NIEHS/NTP (p16Ink4a and p19Arf deficient mice) or elsewhere (XPA-Trp53 deficient mice). A new generation of transgenic models is also currently being developed (Berns 2001), such as one incorporating a point mutation in k-Ras (Johnson et al. 2001), or models subject to premature aging or having telomere dysfunction (Artandi and DePinho 2000; Rudolph et al. 2001). If the NTP incorporates transgenic models into routine testing, it must necessarily include a strong research program aimed at developing the transgenic models appropriate for chemical carcinogenesis investigation and identification of carcinogens of the greatest risk to humans. As our analysis shows, the best strategy for testing may be combining different transgenic models depending on their particular attributes and utility. Thus, the NTP should develop an arsenal of models. Likewise, site-specific or mechanism-specific models could be developed and used in both basic research and carcinogen identification. The NTP could also develop or support research to evaluate transgenic rats or in assessment of possible refinements in the 2-year rat bioassay.

Correction

After the manuscript form was published online in EHP-In-Press, the authors discovered errors in several values, most of them in Tables 4-7. These errors have been corrected. None of the errors affected the interpretation of the data.

Table 1. Comparison of results from 14 known human carcinogens tested
in rodent NCI/NTP cancer bioassays, Salmonella (Sal) and/or in vivo
micronuclei (Mn) genotoxicity assays, and/or three transgenic mouse
cancer bioassays.

                           CAS      IARC      NTP
Agent                      no.      group     ROC

Benzene                  71-43-2      1      Known
Cyclophosphamide        6055-19-2     1      Known
Melphalan                148-82-3     1      Known
Cyclosporin A           79217-60-0    1      Known
Diethylstilbestrol       56-53-1      1      Known
17[beta]-Estradiol (a)   50-28-2      1    Reasonable
TCDD (b)                1746-01-6     1      Known
UVR (312-450 nM)            NA        1      Known
Asbestos fibers         1332-21-4     1      Known
Beryllium               7440-41-7     1      Known
Plutonium-239               NA        1      Known
Cobalt-60 (LET)             NA        1      Known
Sodium arsenate         7784-46-5     1      Known
Thio-TEPA                52-24-4      1      Known

                          NCI/NTP     Genotoxicity
Agent                    bioassays     (Sal; Mn)        p53 +/-

Benzene                 +; +; +; +g       -; +      +g; +g (French et
                        (NTP 1986a)                 al. 2001)
                                                    -g; -g (Storer et
                                                    al. 2001)
Cyclophosphamide        +; +; +; +ip      +; +      +g (Storer et al.
                        (Weisburger                 2001)
                           1977)
Melphalan               +; +; +; +ip      +; +      +ip (Eastin et al.
                        (Weisburger                 1998; Storer et
                           1977)                    al. 2001)
Cyclosporin A                NT           -; -      -g; +f; +f (Eastin
                                                    et al. 1998;
                                                    Storer et al. 2001)
Diethylstilbestrol           NT          -; NT      -sc; +f (Eastin et
                                                    al. 1998; Storer et
                                                    al. 2001)
17[beta]-Estradiol (a)       NT           -; -      [+ or -] g; -g
                                                    (Storer et al.
                                                    2001)
TCDD (b)                +; +; +; +f      -; NT      -g (Eastin et al.
                          (NCI/NTP                  1998)
                           1982a)
UVR (312-450 nM)             NT           +; +      +d (Jiang et al.
                                                    1999)
Asbestos fibers          -; -; NT;       NT; -      +ip (Marsella et
                            NT f                    al. 1997)
                        (NTP 1988a)
Beryllium                    NT           -; -      +l (Finch et al.
                                                    1998)
Plutonium-239                NT           +; +      +l (Finch et al.
                                                    1998)
Cobalt-60 (LET)              NT           -; +      +wb (Kemp et al.
                                                    1994)
Sodium arsenate              NT          NT; NT     NT
Thio-TEPA               +; +; +; +g      +; NT      NT
                          (NCI/NTP
                           1978a)

Agent                          Tg.AC                  RasH2

Benzene                 +d; +g (Blanchard      +g (Yamamoto et al.
                        et al. 1998;           1998b)
                        Spalding et al.
                        1999)
Cyclophosphamide        [+ or -] d; +g         [+ or -] g; +g;
                        (Eastin et al. 2001)   +g (Usui et al. 2001;
                                               Yamamoto et al. 1998b)
Melphalan               [+ or -] d; +g         [+ or -] ip (Yamamoto
                        (Eastin et al. 1998;   et al. 1998b)
                        2001)
Cyclosporin A           +d; [+ or -] f         [+ or -] g (Maronpot
                        (Eastin et al. 1998;   et al. 2000; Usui et
                        2001)                  al. 2001; Yamamoto et
                                               al. 1998a)
Diethylstilbestrol      +d; -g (Eastin et      +f (Usui et al. 2001)
                        al. 1998; 2001)
17[beta]-Estradiol (a)  +d; -g (Eastin et      -g (Usui et al. 2001)
                        al. 2001)
TCDD (b)                +d (Fastin et al.      NT
                        1998)
UVR (312-450 nM)        +d (Trempus et al.     NT
                        1998)
Asbestos fibers         NT                     NT

Beryllium               NT                     NT
Plutonium-239           NT                     NT
Cobalt-60 (LET)         NT                     NT
Sodium arsenate         -d (Germolic et al.    NT
                        1997)
Thio-TEPA               NT                     +ip (Yamamoto et al.
                                               1998b)

Abbreviations: -, negative; +, positive; [+ or -], equivocal; d,
dermal; f, food; g, gavage; l, inhalation; ip, intraperitoneal; LET,
linear energy transfer; NT, not tested or no published record; sc,
subcutaneous; wb, whole body. Individual results were found in the
cited references or in the IARC (2002) or the NTP databases (NTP
2002). NCI/NTP peer-reviewed conclusions are reported for male rat,
female rat, male mouse, and female mouse, respectively. Results from
transgenic models are presented as the summary conclusion for each
route of exposure using one or both sexes of the strain used.

(a) Both dermal and gavage studies in the Tg.AC mice employed ethinyl
estradiol (CAS no. 57-63-6), a synthetic form of 17[beta]-estradiol.
(b) 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

Table 2. Comparison of results from 32 suspected human carcinogens
tested in rodent NCI/NTP cancer bioassays, Salmonella (Sal) and/or
in vivo micronuclei (MN) genotoxicity assays, and/or three transgenic
mouse bioassays.

                                             IARC
Agent                            CAS no.    group      ROC

p-Cresidine                     120-71-8      2B    Reasonable

Glycidol                        556-52-5      2A    Reasonable

Phenolphthalein                  77-09-8      2B    Reasonable

4-Vinyl-1-cyclohexene           106-87-6      2B    Reasonable
 diepoxide

2,4-Diaminotolulene              95-80-7      2B    Reasonable

Chloroprene                     126-99-8      2B    Reasonable

Pentachlorophenol                87-86-5      2B    Not listed

Phenacetin                       62-44-2      2A    Reasonable

Phenobarbital                    50-06-6      2B    Not listed

Chloroform                       67-66-3      2B    Reasonable

Benzo[a]pyrene                   50-32-8      2A    Reasonable

Dimethylnitrosamine              62-75-9      2A    Not listed

7,12-                            57-97-6      NE    Not listed
 Dimethylbenzanthracene (b)

N-Ethyl-N-nitrosourea           759-73-9      2A    Not listed

2-Amino-3-methyl                6180-96-6     2A    Not listed
 imidazo[4,5-f]quinoline

N-Butyl-N-(4-hydroxybutyl)     64091-91-4     2B    Not listed
 nitrosamine (BBN)

N-Methyl-N-nitrosourea          684-93-5      2A    Not listed

Urethane                         51-79-6      2B    Reasonable

Oxymetholone                    434-07-1      2A    Reasonable

1, 2-Dimethylhydrazine          540-73-8      2A    Not listed

1,4-Dioxane                     123-91-1      2B    Reasonable

Ethylene thiourea                96-45-7      2B    Reasonable

Methylazoxymethanol acetate     592-62-1      2B    Not listed

Procarbazine                    366-70-1      2A    Reasonable

4,4'-Thiodianiline              139-65-1      2B    Not listed

MNNG                             70-25-7      2A    Reasonable

Cupferron                       135-20-6      2A    Reasonable

N-Nitrosodiethylamine            55-18-5      2A    Reasonable

Dimethylvinylchloride           513-37-1      2B    Not listed

4-Nitroquinoline N-oxide (d)     56-57-5      NE    Not listed

4-Hydroxyaminoquinoline-        4637-56-3     NE    Not listed
 1-oxide (d)

Mirex                           2385-85-5     2B    Reasonable

                                    NCI/NTP        Genotoxicity
Agent                              bioassays        (Sal; Mn)

p-Cresidine                       +; +; +; +f          +; -
                                (NCI/NTP 1979a)

Glycidol                          +; +; +; +g          +; +
                                  (NTP 1990a)

Phenolphthalein                   +; +; +; +f          -; +
                                  (NTP 1996a)

4-Vinyl-1-cyclohexene             +; +; +; +d          +; +
 diepoxide                        (NTP 1989a)

2,4-Diaminotolulene               +; +; -; +f          +; -
                                (NCI/NTP 1979b)

Chloroprene                       +; +; +; +1          -;-
                                  (NTP 1998a)

Pentachlorophenol               + (a); -; +; +f        -;-
                              (NTP 1989b, 1999a)

Phenacetin                            NT              -; NT

Phenobarbital                         NT             wk+; NT

Chloroform                        +; -; +; +w          -; +
                                (Griesemer and
                                  Cueto 1980)

Benzo[a]pyrene                        NT              +; NT

Dimethylnitrosamine                   NT              +; NT

7,12-                           NT; NT; +; +d,         +; +
 Dimethylbenzanthracene (b)     i-p (NTP 1996b)

N-Ethyl-N-nitrosourea                 NT               +; +

2-Amino-3-methyl                      NT               +; +
 imidazo[4,5-f]quinoline

N-Butyl-N-(4-hydroxybutyl)            NT              NT; -
 nitrosamine (BBN)

N-Methyl-N-nitrosourea                NT              NT; +

Urethane                              NT               +; +

Oxymetholone                  [+ or -]; +; NT; NT      -; -
                                  (NTP 1999b)

1, 2-Dimethylhydrazine                NT            - (c); NT

1,4-Dioxane                       +; +; +; +w          -; +
                                (NCI/NTP 1978b)

Ethylene thiourea                 +; +; +; +f         -; NT
                                  (NTP 1992a)

Methylazoxymethanol acetate           NT              -; NT

Procarbazine                     +; +; +; +ip          +; +
                                (NCI/NTP 1979c)

4,4'-Thiodianiline                +; +; +; +f         +; NT
                                (NCI/NTP 1978c)

MNNG                              +; +; +d ip         +; NT
                                  (NTP 1996b)

Cupferron                         +; +; +; +f         +; NT
                                (NCI/NTP 1978d)

N-Nitrosodiethylamine                 NT              +; NT

Dimethylvinylchloride             +; +; +; +g          +; +
                                  (NTP 1986b)

4-Nitroquinoline N-oxide (d)          NT              +; NT

4-Hydroxyaminoquinoline-              NT              +; NT
 1-oxide (d)

Mirex                           +; +; NT; NT f        -; NT
                                  (NTP 1990b)

Agent                               p53+/-

p-Cresidine                   +f; +g (Storer et al. 2001;
                              Tennant et al. 1995)

Glycidol                      -g (Tennant et al. 1999)

Phenolphthalein               +f
                              (Dunnick et al. 1997)

4-Vinyl-1-cyclohexene         +d (Tennant et al. 1995)
 diepoxide

2,4-Diaminotolulene           [+ or -] f (Eastin et al. 1998)

Chloroprene                   -i (French. Personal
                              communication)

Pentachlorophenol             -f (Spalding et al. 2000)

Phenacetin                    -f; -g (Storer et al. 2001)

Phenobarbital                 -f; -f (Sagartz et al. 1998;
                              Storer et al. 2001)

Chloroform                    [+ or -] g (Storer et al. 2001)

Benzo[a]pyrene                +d, g (Martin et al. 2001)

Dimethylnitrosamine           +w (Harvey et al. 1993)

7,12-                         +d (Kemp et al. 1993)
 Dimethylbenzanthracene (b)

N-Ethyl-N-nitrosourea         +ip (Mitsumori et al. 2000)

2-Amino-3-methyl              +g (Morimura 1999)
 imidazo[4,5-f]quinoline

N-Butyl-N-(4-hydroxybutyl)    +w (Ozaki et al. 1998)
 nitrosamine (BBN)

N-Methyl-N-nitrosourea        +ip (Yamamoto et al. 2000)

Urethane                      +ip (Carmichael et al. 2000)

Oxymetholone                  -g (Stoll et al. 1999)

1, 2-Dimethylhydrazine        NT

1,4-Dioxane                   NT

Ethylene thiourea             NT

Methylazoxymethanol acetate   NT

Procarbazine                  NT

4,4'-Thiodianiline            NT

MNNG                          NT

Cupferron                     NT

N-Nitrosodiethylamine         NT

Dimethylvinylchloride         NT

4-Nitroquinoline N-oxide (d)  NT

4-Hydroxyaminoquinoline-      NT
 1-oxide (d)

Mirex                         NT

Agent                                Tg.AC

p-Cresidine                   +d (Tennant et al. 1999)

Glycidol                      -d; -g (Tennant et al. 1999)

Phenolphthalein               NT

4-Vinyl-1-cyclohexene         -d (Tennant et al. 1999)
 diepoxide

2,4-Diaminotolulene           +d (Eastin et al. 1998)

Chloroprene                   -i (French. Personal
                              communication)

Pentachlorophenol             +d (Spalding et al. 2000)

Phenacetin                    -d; -f (Eastin et al. 2001)

Phenobarbital                 ia d; ia g; ia f
                              (Eastin et al. 2001)

Chloroform                    -g (Delker et al. 1999)

Benzo[a]pyrene                +d (Martin et al. 2001)

Dimethylnitrosamine           NT

7,12-                         +d (Spalding et al. 1993)
 Dimethylbenzanthracene (b)

N-Ethyl-N-nitrosourea         NT

2-Amino-3-methyl              NT
 imidazo[4,5-f]quinoline

N-Butyl-N-(4-hydroxybutyl)    NT
 nitrosamine (BBN)

N-Methyl-N-nitrosourea        NT

Urethane                      +d (Spalding et al. 1993)

Oxymetholone                  +d (Stoll et al. 1999)

1, 2-Dimethylhydrazine        NT

1,4-Dioxane                   NT

Ethylene thiourea             NT

Methylazoxymethanol acetate   NT

Procarbazine                  NT

4,4'-Thiodianiline            NT

MNNG                          NT

Cupferron                     NT

N-Nitrosodiethylamine         NT

Dimethylvinylchloride         +d (Stoll et al. 1999)

4-Nitroquinoline N-oxide (d)  NT

4-Hydroxyaminoquinoline-      NT
 1-oxide (d)

Mirex                         +d (Stoll et al. 1999)

Agent                                RasH2

p-Cresidine                   +f (Yamamoto et al. 1998b)

Glycidol                      +g (Usui et al. 2001)

Phenolphthalein               -f (Koujitani et al. 2000)

4-Vinyl-1-cyclohexene         +d (Yamamoto et al. 1998b)
 diepoxide

2,4-Diaminotolulene           NT

Chloroprene                   NT

Pentachlorophenol             NT

Phenacetin                    +f (Yamamoto et al. 1998b)

Phenobarbital                 -g (Usui et al. 2001)

Chloroform                    -g (Usui et al. 2001)

Benzo[a]pyrene                NT

Dimethylnitrosamine           NT

7,12-                         NT
 Dimethylbenzanthracene (b)

N-Ethyl-N-nitrosourea         +ip (Yamamoto et al. 1998b)

2-Amino-3-methyl              NT
 imidazo[4,5-f]quinoline

N-Butyl-N-(4-hydroxybutyl)    NT
 nitrosamine (BBN)

N-Methyl-N-nitrosourea        +ip (Yamamoto et al. 1998b)

Urethane                      +ip (Mori et al. 2000;
                              Umemura et al. 1999)

Oxymetholone                  NT

1, 2-Dimethylhydrazine        +d (Yamamoto et al. 1998b)

1,4-Dioxane                   [+ or -] w (Yamamoto et al. 1998b)

Ethylene thiourea             +f (Yamamoto et at. 1998b)

Methylazoxymethanol acetate   +sc (Yamamoto et al. 1998b)

Procarbazine                  +ip (Yamamoto et al. 1998b)

4,4'-Thiodianiline            +f (Yamamoto et al. 1998b)

MNNG                          +g (Yamamoto et al. 1998b)

Cupferron                     +f (Yamamoto et al. 1998b)

N-Nitrosodiethylamine         +ip (Yamamoto et al. 1998b)

Dimethylvinylchloride         NT

4-Nitroquinoline N-oxide (d)  +sc (Yamamoto et al. 1998b)

4-Hydroxyaminoquinoline-      +ip (Yamamoto et al. 1998b)
 1-oxide (d)

Mirex                         NT

Abbreviations: -, negative; +, positive; [+ or -], equivocal;
d, dermal; f, food; g, gavage; l, inhalation; ia, inadequately
evaluated; ip, intraperitoneal; i-p, initiation--promotion; MNNG,
N-methyl-N'-nitro-N-nitrosoguanidine; NE, not evaluated; NT, not
tested or no published record; sc, subcutaneous; w, drinking water;
wk, weakly. Individual results are found in the cited references or
in the IARC (2002) or the NTP databases (NTP 2002). NCI/NTP
peer-reviewed conclusions are reported for male F344 rat, female
F344 rat, male B6C3[F.sub.1] mouse, and female B6C3[F.sub.1] mouse,
respectively. Results from transgenic models are presented as the
summary conclusion for each route of exposure using one or both sexes.

(a) Positive in 1,000-ppm 1-year exposure stop study but not with
2-year exposure to purified pentachlorophenol at lower levels
(NTP 1989b, 1999a).

(b) Reasonably anticipated to be a human carcinogen based on its use
as a prototypical mutagenic carcinogen in initiation-promotion and
complete carcinogenicity studies.

(c) 1,2-Dimethylhydrazine dihydrochloride (CAS no. 306-37-6) tested in
Salmonella mutagenicity assay. (d) Reasonably expected to be a human
carcinogen based upon its use as a prototypical mutagenic carcinogen
for mechanistic investigation of chemical carcinogenesis.

Table 3. Comparison of results from 53 chemicals with insufficient
evidence to be considered potential human carcinogens tested in rodent
NCI/NTP cancer bioassays, Salmonella (Sal) and/or in vivo micronuclei
genotoxicity assays, and/or three transgenic mouse bioassays.

                                              IARC
Agent                            CAS no.      group     ROC

p-Anisidine                      90-04-0        3    Not listed

1-Chloro-2-propanol             127-00-4       NE    Not listed

2,6-Diaminotoluene              820-40-5       NE    Not listed

8-Hydroxyquinoline              148-24-3        3    Not listed

Coconut oil diethanolamine     68603-42-9      NE    Not listed

Diethanolamine                  111-42-2        3    Not listed

Ethyl acrylate                  140-88-5       2B     Delisted

Furfuryl alcohol                 98-00-0       NE    Not listed

Lauric acid diethanolamine      120-40-1       NE    Not listed

N-Methyloacrylamide             924-42-5        3    Not listed

Methylphenidate                 298-59-9       NE    Not listed

Pyridine                        110-86-1        3    Not listed

Reserpine                        50-55-5        3    Reasonable

Rotenone                         83-79-4       NE    Not listed

Resorcinol                      108-46-3        3    Not listed

Oleic acid diethanolamide        93-83-4       NE    Not listed

Clolfibrate                     637-07-0        3    Not listed

Dieldrin                         60-57-1        3    Not listed

Methapyrilene HCl               135-23-9       NE    Not listed

Haloperidol                      52-86-8       NE    Not listed

Chlorpromazine HCl               69-09-0       NE    Not listed

Metaproterenol                  586-06-1       NE    Not listed

WY-14643                       50892-23-4      NE    Not listed

Di(2-ethylhexyl)phthalate       117-81-7        3    Reasonable

Sulfamethoxazole                723-46-6        3    Not listed

Sulfisoxazole                   127-69-5        3    Not listed

Ampicillin                      7177-48-2       3    Not listed

D-Mannitol                       69-65-8       NE    Not listed

1,1,2-Trichloroethane            79-00-5        3    Not listed

Xylenes (mixed)                 1330-20-7       3    Not listed

Furfural                         98-01-1        3    Not listed

5-Nitro-o-toluidine              99-55-8        3    Not listed

Benzethonium chloride           121-54-0       NE    Not listed

o-Benzyl-p-chlorophenol         120-32-1       NE    Not listed

2-Chloroethanol                 107-07-3       NE    Not listed

Phenol                          108-95-2        3    Not listed

Triethanolamine                 102-71-6        3    Not listed

Acetic anhydride                108-24-7       NE    Not listed

2,4-Dinitro-1-fluorobenzene      70-34-8       NE    Not listed

Diisopropylcarbodiimide         693-13-0       NE    Not listed

Dicyclohexylcarbodiimide        538-75-0       NE    Not listed

Fluocinolone acetonide           67-73-2       NE    Not listed

Tripropylene glycol
diacrylate                     42978-66-5      NE    Not listed

D-Limonene                      5989-27-5       3    Not listed

Foreign body (transponder)         NA          NE    Not listed

Acetone                          67-64-1       NE    Not listed

Benzoyl peroxide                 94-36-0        3    Not listed

Ethanol                          64-17-5       NE    Not listed

Methyl ethyl ketone peroxide    1338-23-4      NE    Not listed

4-Nitro-o-phenylenediamine       99-56-9        3    Not listed

6-Nitrobenzimidazole             94-52-0       NE    Not listed

Cholestyramine                 11041-12-6      NE    Not listed

Magnetic fields (60 mhz)           NA          NE    Not listed

                                            NCI/NTP
Agent                                      bioassays

p-Anisidine                              +; -; -: -f,
                                        (NCI/NTP 1978e)

1-Chloro-2-propanol                       -; ; -; -w
                                          (NTP 1998b)

2,6-Diaminotoluene                        -; -; -; f
                                        Battershill and
                                         Fielder 1998)

8-Hydroxyquinoline                        -; -; -; f
                                          (NTP 1985b)

Coconut oil diethanolamine            -; [+ or -]; +; +d
                                          (NTP 2001)

Diethanolamine                            -; -; +; +d
                                          (NTP 1999c)

Ethyl acrylate                            +; +; +; +g
                                          (NTP 1986c)

Furfuryl alcohol                          +; +; +; -i
                                          (NTP 1999d)

Lauric acid diethanolamine                -; -; -; +d
                                          (NTP 1999e)

N-Methyloacrylamide                       -; -; +; +g
                                          (NTP 1989c)

Methylphenidate                           -; -; +; +f
                                          (NTP 1995a)

Pyridine                                  +; [+ or -]; +w
                                          (NTP 2000)

Reserpine                                 +; -: +; +f
                                        (NCI/NTP 1982b)

Rotenone                              [+ or -]; -; -; -f
                                          (NTP 1988b)

Resorcinol                                -; -; -; -g
                                          (NTP 1992b)

Oleic acid diethanolamide                 -; -; -; -d
                                          (NTP 1999f)

Clolfibrate                                   NT

Dieldrin                              -; -; [+ or -]; -f
                                        (NCI/NTP 1978f)

Methapyrilene HCI                       +; +; NT; NT f
                                        (Lijinsky 1980)

Haloperidol                                   NT

Chlorpromazine HCI                            NT

Metaproterenol                                NT

WY-14643                                      NT

Di(2-ethylhexyl)phthalate                 +; +; +; +f
                                          (NTP 1982c)

Sulfamethoxazole                              NT

Sulfisoxazole                            -; -; -l; -f
                                        (NCI/NTP 1979d)

Ampicillin                            [+ or -]; -; -; -f
                                          (NTP 1987)

D-Mannitol                                -; -; -; -f
                                        (NCI/NTP 1982d)

1,1,2-Trichloroethane                     -; -; +; +g
                                        (NCI/NTP 1978g)

Xylenes (mixed)                         -; -; -; -; -g
                                          (NTP 1986d)

Furfural                                  +; -; +; +g
                                          (NTP 1990c)

5-Nitro-o-toluidine                       -; -; +; +f
                                        (NCI/NTP 1978h)

Benzethonium chloride                     -; -; -; -d
                                          (NTP 1995b)

o-Benzyl-p-chlorophenol               -; [+ or -]; +; -g
                                          (NTP 1994)

2-Chloroethanol                           -; -; -; -d
                                          (NTP 1985a)

Phenol                                   -; -; -; -dw
                                        (NCI/NTP 1980)

Triethanolamine                      [+ or -]; -; ia; ia d
                                          (NTP 1999g)

Acetic anhydride                              NT

2,4-Dinitro-1-fluorobenzene                   NT

Diisopropylcarbodiimide                   In progress

Dicyclohexylcarbodiimide                  In progress

Fluocinolone acetonide                        NT

Tripropylene glycol diacrylate                NT

D-Limonene                                +; -; -; -f
                                          (NTP 1990d)

Foreign body (transponder)                    NT

Acetone                                       NT

Benzoyl peroxide                             +i-p
                                          (NTP 1996b)

Ethanol                                   In progress

Methyl ethyl ketone peroxide              In progress

4-Nitro-o-phenylenediamine                -; -; -; -f
                                        (NCI/NTP 1979e)

6-Nitrobenzimidazole                      -; -; +; +f
                                        (NCI/NTP 1979f)

Cholestyramine                                NT

Magnetic fields (60 mhz)                 -; -; -; -wb
                                          (NTP 1999h)

                                         Genotoxicity
Agent                                      (Sal; Mn)

p-Anisidine                                  +; -

1-Chloro-2-propanol                          +; NT

2,6-Diaminotoluene                           +; -

8-Hydroxyquinoline                           +; -

Coconut oil diethanolamine                   -; +

Diethanolamine                               -; -

Ethyl acrylate                               -; -

Furfuryl alcohol                             -; -

Lauric acid diethanolamine                   -; -

N-Methyloacrylamide                          -; -

Methylphenidate                              -; NT

Pyridine                                     -; -

Reserpine                                    -; -

Rotenone                                     -; NT

Resorcinol                                   -; +

Oleic acid diethanolamide                    -; NT

Clolfibrate                                  -; -

Dieldrin                                     -; NT

Methapyrilene HCI                            -; -

Haloperidol                                 NT; NT

Chlorpromazine HCI                           -; NT

Metaproterenol                              NT; NT

WY-14643                                    NT; NT

Di(2-ethylhexyl)phthalate                    -; -

Sulfamethoxazole                             -; NT

Sulfisoxazole                                -; NT

Ampicillin                                   -; NT

D-Mannitol                                   -; -

1,1,2-Trichloroethane                        -; -

Xylenes (mixed)                              -; NT

Furfural                                     -; NT

5-Nitro-o-toluidine                          +; NT

Benzethonium chloride                        -; NT

o-Benzyl-p-chlorophenol                      -; NT

2-Chloroethanol                              +; -

Phenol                                       -; +

Triethanolamine                              -; -

Acetic anhydride                             -; NT

2,4-Dinitro-1-fluorobenzene                  +; NT

Diisopropylcarbodiimide                      -; +

Dicyclohexylcarbodiimide                     +; +

Fluocinolone acetonide                      NT; NT

Tripropylene glycol diacrylate               -; -

D-Limonene                                   -; NT

Foreign body (transponder)                   -; -

Acetone                                      -; -

Benzoyl peroxide                             -; NT

Ethanol                                      -; NT

Methyl ethyl ketone peroxide                 +; -

4-Nitro-o-phenylenediamine                +; [+ or -]

6-Nitrobenzimidazole                         +; NT

Cholestyramine                              NT; NT

Magnetic fields (60 mhz)                     -; -

Agent                                       p53+/-

p-Anisidine                     -f (Tennant et al. 1995)

1-Chloro-2-propanol             -g (Tennant et al. 1999)

2,6-Diaminotoluene              -f (Eastin et al. 1998)

8-Hydroxyquinoline              -f (Eastin et al. 1998)

Coconut oil diethanolamine      -d (Spalding et al. 2000)

Diethanolamine                  NT

Ethyl acrylate                  NT

Furfuryl alcohol                NT

Lauric acid diethanolamine      -f (Spalding et al. 2000)

N-Methyloacrylamide             -g (Tennant et al. 1995)

Methylphenidate                 -f (Tennant et al. 1999)

Pyridine                        -g (Spalding et al. 2000)

Reserpine                       -f (Tennant et al. 1995)

Rotenone                        -f (Eastin et al. 1998)

Resorcinol                      -g (Eastin et al. 1998)

Oleic acid diethanolamide       -d (Spalding et al. 2000)

Clolfibrate                     -g; -g (Storer et al. 2001)

Dieldrin                        -f (Storer et al. 2001)

Methapyrilene HCI               -g; -f (Storer et al. 2001)

Haloperidol                     -g (Storer et al. 2001)

Chlorpromazine HCI              -g; -g (Storer et al. 2001)

Metaproterenol                  -f; -f (Storer et al. 2001)

WY-14643                        -f (Storer et al. 2001)

Di(2-ethylhexyl)phthalate       [+ or -] f (Storer et al. 2001)

Sulfamethoxazole                -f (Storer et al. 2001)

Sulfisoxazole                   -f (Storer et al. 2001)

Ampicillin                      -g (Storer et al. 2001)

D-Mannitol                      -f (Storer er al. 2001)

1,1,2-Trichloroethane           NT

Xylenes (mixed)                 NT

Furfural                        NT

5-Nitro-o-toluidine             NT

Benzethonium chloride           NT

o-Benzyl-p-chlorophenol         NT

2-Chloroethanol                 NT

Phenol                          NT

Triethanolamine                 NT

Acetic anhydride                NT

2,4-Dinitro-1-fluorobenzene     NT

Diisopropylcarbodiimide         In progress

Dicyclohexylcarbodiimide        NT

Fluocinolone acetonide          NT

Tripropylene glycol diacrylate  NT

D-Limonene                      -g (Carmichael et al. 2000)

Foreign body (transponder)      +sc (Blanchard et al. 1999)

Acetone                         NT

Benzoyl peroxide                NT

Ethanol                         NT

Methyl ethyl ketone peroxide    NT

4-Nitro-o-phenylenediamine      NT

6-Nitrobenzimidazole            NT

Cholestyramine                  NT

Magnetic fields (60 mhz)        -wb (McCormick et al. 1998)

Agent                                        Tg.AC

p-Anisidine                     -d (Tennant et al. 1995)

1-Chloro-2-propanol             -d (Tennant et al. 1999)

2,6-Diaminotoluene              -d (Eastin et al. 1998)

8-Hydroxyquinoline              -d (Eastin et al. 1998)

Coconut oil diethanolamine      -d (Spalding et al. 2000)

Diethanolamine                  -d (Spalding et al. 2000)

Ethyl acrylate                  -d (Nylander-French and
                                French 1998; Tice et al. 1997)

Furfuryl alcohol                -d (Spalding et al. 2000)

Lauric acid diethanolamine      +d (Spalding et al. 2000)

N-Methyloacrylamide             -d; -g (Eastin et al. 1998)

Methylphenidate                 -d (Tennant et al. 1999)

Pyridine                        -d (Spalding et al. 2000)

Reserpine                       -d; -g (Tennant et al. 1995)

Rotenone                        +d; -g (Eastin et al. 1998)

Resorcinol                      +d (Fastin et al. 1998)

Oleic acid diethanolamide       -d (Spalding et al. 2000)

Clolfibrate                     +d (Eastin et al. 2001)

Dieldrin                        NT

Methapyrilene HCI               -d (Eastin et al. 2001)

Haloperidol                     NT

Chlorpromazine HCI              NT

Metaproterenol                  NT

WY-14643                        -d; [+ or -] f (Eastin et al. 2001)

Di(2-ethylhexyl)phthalate       -d; -f (Eastin et al. 2001)

Sulfamethoxazole                -d; -g (Eastin et al. 2001)

Sulfisoxazole                   -d; -g (Eastin et al. 2001)

Ampicillin                      NT

D-Mannitol                      NT

1,1,2-Trichloroethane           NT

Xylenes (mixed)                 NT

Furfural                        NT

5-Nitro-o-toluidine             NT

Benzethonium chloride           -d (Spalding et al. 1999)

o-Benzyl-p-chlorophenol         +d (Spalding et al. 1999)

2-Chloroethanol                 -d (Spalding et al. 1999)

Phenol                          -d (Spalding et al. 1999)

Triethanolamine                 -d (Spalding et al. 1999)

Acetic anhydride                -d (Spalding et al. 1999)

2,4-Dinitro-1-fluorobenzene     +d (Albert et al. 1996)

Diisopropylcarbodiimide         +d (Spalding et al. 1999)

Dicyclohexylcarbodiimide        -d (Spalding et al. 1999)

Fluocinolone acetonide          -d (Albert et al. 1996)

Tripropylene glycol diacrylate  +d (Albert et al. 1996)

D-Limonene                      NT

Foreign body (transponder)      -sc (French J. Personal
                                communication)

Acetone                         -d (Spalding et al. 1999;
                                Spalding et al. 1993)

Benzoyl peroxide                +d (Spalding et al. 1993)

Ethanol                         -d (Spalding et al. 1999)

Methyl ethyl ketone peroxide    +d (Spalding et al. 1993)

4-Nitro-o-phenylenediamine      NT

6-Nitrobenzimidazole            NT

Cholestyramine                  NT

Magnetic fields (60 mhz)        -wb (McCormick et al. 1998)

Agent                                        RasH2

p-Anisidine                     -g (Maronpot et al, 2000)

1-Chloro-2-propanol             NT

2,6-Diaminotoluene              NT

8-Hydroxyquinoline              NT

Coconut oil diethanolamine      NT

Diethanolamine                  NT

Ethyl acrylate                  +g (Yamamoto et al. 1998b)

Furfuryl alcohol                NT

Lauric acid diethanolamine      NT

N-Methyloacrylamide             NT

Methylphenidate                 NT

Pyridine                        NT

Reserpine                       -f (Yamamoto et al. 1998b)

Rotenone                        -g (Yamamoto et al. 1998b)

Resorcinol                      -g (Maronpot et al. 2000)

Oleic acid diethanolamide       NT

Clolfibrate                     [+ or -] g; +g (Usui et al. 2001)

Dieldrin                        -f (Usui et al. 2001)

Methapyrilene HCI               -g (Yamamoto et al. 1996)

Haloperidol                     -g (Usui et al. 2001)

Chlorpromazine HCI              -g (Usui et al. 2001)

Metaproterenol                  -f (Yamamoto et al. 1998b)

WY-14643                        NT

Di(2-ethylhexyl)phthalate       +(Usui et al, 2001)

Sulfamethoxazole                -f (Usui et al. 2001)

Sulfisoxazole                   -f (Usui et al. 2001)

Ampicillin                      -g (Usui et al. 2001)

D-Mannitol                      -f (Yamamoto et al. 1998b)

1,1,2-Trichloroethane           -g (Yamamoto et al. 1998b)

Xylenes (mixed)                 -g (Yamamoto et al. 1998b)

Furfural                        +g (Yamamoto et al, 1998b)

5-Nitro-o-toluidine             +f (Yamamoto et al. 1998b)

Benzethonium chloride           NT

o-Benzyl-p-chlorophenol         NT

2-Chloroethanol                 NT

Phenol                          NT

Triethanolamine                 NT

Acetic anhydride                NT

2,4-Dinitro-1-fluorobenzene     NT

Diisopropylcarbodiimide         NT

Dicyclohexylcarbodiimide        NT

Fluocinolone acetonide          NT

Tripropylene glycol diacrylate  NT

D-Limonene                      NT

Foreign body (transponder)      NT

Acetone                         NT

Benzoyl peroxide                NT

Ethanol                         NT

Methyl ethyl ketone peroxide    NT

4-Nitro-o-phenylenediamine      [+ or -] f (Yamamoto et al. 1998b)

6-Nitrobenzimidazole            -f (Yamamoto et al. 1998b)

Cholestyramine                  -f (Yamamoto et al. 1998b)

Magnetic fields (60 mhz)        NT

Abbreviations: -, negative; +, positive; [+ or -], equivocal;
d, dermal; f, food; g, gavage; i, inhalation; i-p,
initiation-promotion; NE, not evaluated; NT, not tested; sc,
subcutaneous; w, drinking water; wb, whole body. Individual results
are found in the cited references or in the IARC (2002) or the NTP
databases (NTP 2002). NCI/NTP peer-reviewed conclusions are reported
for male F344 rat, female F344 rat, male B6C3[F.sub.1] mouse, and
female B6C3[F.sub.1] mouse, respectively. Results from transgenic
models are presented as the summary conclusion for each route of
exposure using one or both sexes.

Table 4. Summary performance of each strategy versus likely human
cancer.

                        Positive for    Negative for
Strategy                carcinogens    noncarcinogens

Trp53+/-                     21              12
Trp53+/- (genotoxic)         16               5
Tg.AC                        17              10
RasH2                        21               9
Trp53+/- (genotoxic);        17              10
 RasH2 (nongenotoxic)
Trp53+/- (genotoxic);        30               7
 RasH2 (all)
Trp53+/-(genotoxic);         21               8
 Tg.AC (nongenotoxic)
Trp53+/- (genotoxic);        25               7
 Tg.AC for all

                        Positive for    Negative for    Overall
Strategy               noncarcinogens   carcinogens     accuracy

Trp53+/-                     0              10         77% (33/43)
Trp53+/- (genotoxic)         0               4         84% (21/25)
Tg.AC                        2               6         77% (27/35)
RasH2                        0               7         81% (30/37)
Trp53+/- (genotoxic);        0               6         82% (27/33)
 RasH2 (nongenotoxic)
Trp53+/- (genotoxic);        0               4         90% (37/41)
 RasH2 (all)
Trp53+/-(genotoxic);         0               6         83% (29/35)
 Tg.AC (nongenotoxic)
Trp53+/- (genotoxic);        2               4         84% (32/38)
 Tg.AC for all

All chemicals in Tables 1 and 2 are included as human carcinogens, but
only those chemicals in Table 3 with negative NCI/NTP bioessay results
are regarded as true human noncarcinogens. Definitions: positive for
carcinogens, positive assay results for IARC/ROC carcinogens; negative
for noncarcinogens, negative assay results for IARC/ROC noncarcinogens;
positive for noncarcinogens, positive assay results for IARC/ROC
noncarcinogens; negative for carcinogens, negative assay results for
IARC/ROC carcinogens.

Table 5. Proportion of positive responses in the three transgenic
models as a function of the IARC classification of these 99 chemicals.

IARC classification      Trp53+/-          Tg.AC

Group 1                 83% (10/12)      89% (8/9)
Group 2A                62% (5/8)        50% (2/4)
Group 2B (b)            55% (6/11)       64% (7/11)
Group 3                  0% (0/13)       21% (3/14)
Not evaluated            7% (1/15)       29% (7/24)

IARC classification        RasH2          Overall

Group 1                 57% (4/7) (a)    79% (22/28)
Group 2A               100% (9/9)        76% (16/21)
Group 2B (b)            69% (9/13)       63% (22/35)
Group 3                 29% (4/14)       17% (7/41)
Not evaluated            0% (0/8)        17% (8/47)

(a) Two of the three that were not positive were equivocal.

(b) Includes 7,12-dimethylbenzanthracene, 4-nitroquinoline
N-oxide, and 4-hydroxyaminoquinoline-1-oxide.

Table 6. Summary of performance of each strategy versus likely
human cancer when all chemicals in Table 3 are regarded as true
human noncarcinogens.

                          Positive for    Negative for    Positive for
Strategy                  carcinogens    noncarcinogens  noncarcinogens

Trp53+/-                       21              27               1
Trp53+/- (genotoxic)           16               6               0
Tg.AC                          17              29              10
RasH2                          21              18               5
Trp53+/-(genotoxic);           17              18               3
 RasH2 (nongenotoxic)
Trp53+/-(genotoxic);           30              14               5
 RasH2 (all)
Trp53+/- (genotoxic);          21              23               3
 Tg.AC (nongenotoxic)
Trp53+/- (genotoxic);          25              22              10
 Tg.AC for all
NTP rodent bioassay            23              17              18
NTP rat bioassay; Tg.AC        35              13               9
 (nongenotoxic);
 Trp53+/- (enotoxic)
NTP rat bioassay; RasH2        33              12               8
 (nongenotoxic);
 Trp53+/- (genotoxic)
NTP rat bioassay;              36               7              23
 genotoxicity

                          Negative for      Overall
Strategy                  carcinogens       accuracy

Trp53+/-                       10         81% (48/59)
Trp53+/- (genotoxic)            4         85% (22/26)
Tg.AC                           6         74% (44/62)
RasH2                           7         76% (39/51)
Trp53+/-(genotoxic);            6         80% (35/44)
 RasH2 (nongenotoxic)
Trp53+/-(genotoxic);            4         83% (44/53)
 RasH2 (all)
Trp53+/- (genotoxic);           6         83% (44/53)
 Tg.AC (nongenotoxic)
Trp53+/- (genotoxic);           4         77% (47/61)
 Tg.AC for all
NTP rodent bioassay             0         69% (40/58)
NTP rat bioassay; Tg.AC         0         84% (48/57)
 (nongenotoxic);
 Trp53+/- (genotoxic)
NTP rat bioassay; RasH2         0         85% (45/53)
 (nongenotoxic);
 Trp53+/- (genotoxic)
NTP rat bioassay;               0         65% (43/66)
 genotoxicity

Definitions: positive for carcinogens, positive assay results
for IARC/ROC carcinogens; negative for noncarcinogens, negative
assay results for IARC/ROC noncarcinogens; positive for noncarcinogens,
positive assay results for IARC/ROC non-carcinogens; negative for
carcinogens, negative assay results for IARC/ROC carcinogens.

Table 7. Summary performance of each strategy
(vs. NTP rodent cancer results).

                          Positive for    Negative for    Positive for
Strategy                  carcinogens    noncarcinogens  noncarcinogens

Trp53+/-                        7              12              0
Trp53+/- (genotoxic)            7               5              0
Tg.AC                          14              10              2
RasH2                          15               9              0
Trp53+/- (genotoxic);           9              10              0
 RasH2 (nongenotoxic)
Trp53+/- (genotoxic);          17               7              0
 RasH2 (all)
Trp53+/-(genotoxic);           10               8              0
 Tg.AC (nongenotoxic)
Trp53+/- (genotoxic);          16               7              2
 Tg.AC for all

                          Negative for      Overall
Strategy                  carcinogens       accuracy

Trp53+/-                       16         54% (19/35)
Trp53+/- (genotoxic)            4         75% (12/16)
Tg.AC                          14         60% (24/40)
RasH2                           8         75% (24/32)
Trp53+/- (genotoxic);           8         70% (19/27)
 RasH2 (nongenotoxic)
Trp53+/- (genotoxic);           3         89% (24/27)
 RasH2 (all)
Trp53+/- (genotoxic);          14         56% (18/32)
 Tg.AC (nongenotoxic)
Trp53+/- (genotoxic);          13         61% (23/38)
 Tg.AC for all

Definitions: positive for carcinogens, positive assay results for NTP
rodent carcinogens; negative for noncarcinogens, negative assay results
for NTP rodent noncarcinogens; positive for noncarcinogens, positive
assay results for NTP rodent noncarcinogens; negative for carcinogens,
negative assay results for NTP rodent carcinogens.

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--. 1978e. Bioassay of p-Anisidine Hydrochloride for Possible Carcinogenicity (CAS No. 20265-97-8). Technical Report 116. Bethesda, MD:National Cancer Institute.

--. 1978f. Bioassay of Dieldrin for Possible Carcinogenicity (CAS No. 60-57-1). Technical Report 22. Bethesda, MD:National Cancer Institute.

--. 1978g. Bioassay of 1,1,2-Trichloroethane for Possible Carcinogenicity (CAS No. 79-00-5). Technical Report 74. Bethesda, MD:National Cancer Institute.

--. 1978h. Bioassay of 5-Nitro-o-toluidine for Possible Carcinogenicity (CAS No. 99-55-8) Technical Report 107. Bethesda, MD:National Cancer Institute.

--. 1979a. Bioassay of p-Cresidine for Possible Carcinogenicity (CAS No. 120-71-8). Technical Report 142. Bethesda, MD:National Cancer Institute.

--. 1979b. Bioassay of 2,4-Diaminotoluene for Possible Carcinogenicity (CAS No. 95-60-7). Technical Report 162. Bethesda, MD:National Cancer Institute.

--. 1979c. Bioassay of Procarbazine for Possible Carcinogenicity (CAS No. 366-70-1). Technical Report 19. Bethesda, MD:National Cancer Institute.

--. 1979d. Bioassay of Sulfisoxazole for Possible Carcinogenicity (CAS No. 127-69-5). Technical Report 138. Bethesda, MD:National Cancer Institute.

--.1979e. Bioassay of 4-Nitro-o-phenylenediamine for Possible Carcinogenicity (CAS No. 99-56-9). Technical Report 180. Bethesda, MD:National Cancer Institute.

--. 1979f. Bioassay of 6-Nitrobenzimidazole for Possible Carcinogenicity (CAS No. 94-52-0). Technical Report 117. Bethesda, MD:National Cancer Institute.

--. 1980. Bioassay of Phenol for Possible Carcinogenicity (CAS No. 108-95-2). Technical Report 203. Bethesda, MD: National Cancer Institute.

--. 1982a. Carcinogenesis Bioassay of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (CAS No. 1746-01-6)in Osborne-Mendel Rats and B6C3[F.sub.1] Mice (Gavage Study). Technical Report 209. Research Triangle Park, NC:National Cancer Institute/National Toxicology Program.

--. 1982b. Bioassay of Reserpine for Possible Carcinogenicity (CAS No. 50-55-5). Technical Report 193. Bethesda, MD:National Cancer Institute.

--. 1982c. Carcinogenesis Bioassay of Di(2-ethylhexyl)phthalate (CAS No. 117-81-7)in F344 Rats and B6C3[F.sub.1] Mice (Feed Studies). Technical Report 217. Research Triangle Park, NC:National Cancer Institute/National Toxicology Program.

--. 1982d. Carcinogenesis Bioassay of D-Mannitol (CAS No. 69-65-8)in F344/N Rats and B6C3[F.sub.1] Mice (Feed Study). Technical Report 236. Research Triangle Park, NC:National Cancer Institute/National Toxicology Program.

NTP. 1985a. Toxicology and Carcinogenesis Studies of 2-Chloroethanol (Ethylene Chlorohydrin) (CAS No. 107-073) in F344/N Rats and Swiss CD-1 Mice (Dermal Studies). Technical Report 275. Research Triangle Park, NC:National Toxicology Program.

--. 1985b. Toxicology and Carcinogenesis Studies of 8-Hydroxyquinoline (CAS No. 148-24-3) in F344/N Rats and B6C3[F.sub.1] Mice (Feed Studies). Technical Report 276. Research Triangle Park, NC:National Toxicology Program.

--. 1986a. Toxicology and Carcinogenesis Studies of Benzene in F344/N rats and B6C3[F.sub.1] Mice. Technical Report 289. Research Triangle Park, NC:National Toxicology Program.

--. 1986b. Toxicology and Carcinogenesis Studies of Dimethylvinyl Chloride (1-Chloro-2-Methylpropene) (CAS No. 513-37-1)in F344/N Rats and B6C3[F.sub.1] Mice (Gavage Studies). Technical Report 316. Research Triangle Park, NC:National Toxicology Program.

--. 1986c. Carcinogenesis Studies of Ethyl Acrylate (CAS No. 140-88-5) in F344/N Rats and B6C3[F.sub.1] Mice (Gavage Studies). Technical Report 259. Research Triangle Park, NC:National Toxicology Program.

--. 1986d. Toxicology and Carcinogenesis Studies of Xylenes (Mixed) (60% m-Xylene, 14% p-Xylene, 9% o-Xylene, and 17% Ethylbenzene) (CAS No. 1330-20-7) in F344/N Rats and B6C3[F.sub.1] Mice (Gavage Studies). Technical Report 327. Research Triangle Park, NC:National Toxicology Program.

--. 1987. Toxicology and Carcinogenesis Studies of Ampicillin Trihydrate (CAS No. 7177-48-2) in F344/N Rats and B6C3[F.sub.1] Mice (Gavage Studies). Technical Report 318. Research Triangle Park, NC:National Cancer Institute.

--. 1988a. Toxicology and Carcinogenesis Studies of Crocidolite Asbestos (CAS No. 12001-28-4) in F344/N Rats (Feed Studies). Technical Report 280. Research Triangle Park, NC:National Toxicology Program.

--. 1988b. Toxicology and Carcinogenesis Studies of Rotenone (CAS No. 83-79-4) in F344/N Rats and B6C3[F.sub.1] Mice (Feed Studies). Technical Report 320. Research Triangle Park, NC:National Toxicology Program.

--. 1989a. Toxicology and Carcinogenesis Studies of 4-Vinyl-1-cyclohexene Diepoxide (CAS No. 106-87-6) in F344/N Rats and B6C3[F.sub.1] Mice (Dermal Studies). Technical Report 362. Research Triangle Park, NC:National Toxicology Program.

--. 1989b. Toxicology and Carcinogenesis Studies of Two Pentachlorophenol Technical-Grade Mixtures (CAS No. 87-86-5) in B6C3[F.sub.1] Mice (Feed Studies). Technical Report 349. Research Triangle Park, NC:National Toxicology Program.

--. 1989c. Toxicology and Carcinogenesis Studies of N-Methylolacrylamide (CAS No. 924-42-5)in F344/N Rats and B6C3[F.sub.1] Mice (Gavage Studies). Technical Report 352. Research Triangle Park, NC:National Toxicology Program.

--. 1990a. Toxicology and Carcinogenesis Studies of Glycidol (CAS No. 556-52-5) in F344/N Rats and B6C3[F.sub.1] Mice (Gavage Studies). Technical Report 374. Research Triangle Park, NC:National Toxicology Program.

--. 1990b. Toxicology and Carcinogenesis Studies of Mirex (1,1a,2,2,3,3a,4,5,5,5a,5b,6-Dodecachlorooctahydro-1,3, 4metheno-1H-cyclouta[cd]pentalene) (CAS No. 2385-85-5) in F344/N Rats (Feed Studies). Technical Report 313. Research Triangle Park, NC:National Toxicology Program.

--. 1990c. Toxicology and Carcinogenesis Studies of Furfural (CAS No. 98-01-1)in F344/N Rats and B6C3[F.sub.1] Mice (Gavage Studies). Technical Report 382. Research Triangle Park, NC:National Toxicology Program.

--. 1990d. Toxicology and Carcinogenesis Studies of d-Limonene (CAS No. 5989-27-5) in F344/N Rats and B6C3[F.sub.1] Mice (Gavage Studies). Technical Report 347. Research Triangle Park, NC:National Toxicology Program.

--. 1992a. Toxicology and Carcinogenesis Studies of Ethylene Thiourea (CAS: 96-45-7) in F344 Rats and B6C3[F.sub.1] Mice (Feed Studies). Technical Report 388. Research Triangle Park, NC:National Toxicology Program.

--. 1992b. Toxicology and Carcinogenesis Studies of Resorcinol (CAS No. 108-46-3) in F344 Rats and B6C3[F.sub.1] Mice (Gavage Studies). Technical Report 403. Research Triangle Park, NC:National Toxicology Program.

--. 1994. Toxicology and Carcinogenesis Studies of o-Benzyl-p-Chlorophenol (CAS No. 120-32-1) in F344/N Rats and 86C3[F.sub.1] Mice (Garage Studies). Technical Report 424. Research Triangle Park, NC:National Toxicology Program.

--. 1995a. Toxicology and Carcinogenesis Studies of Methylphenidate Hydrochloride (CAS No. 298-59-9) in F344/N Rats and B6C3[F.sub.1] Mice (Feed Studies). Technical Report 439. Research Triangle Park, NC:National Toxicology Program.

--. 1995b. Toxicology and Carcinogenesis Studies of Benzethonium Chloride (CAS No. 121-54-0) in F344/N Rats and B6C3[F.sub.1] Mice (Dermal Studies). Technical Report 438. Research Triangle Park, NC:National Toxicology Program.

--. 1996a. Toxicology and Carcinogenesis Studies of Phenolphthalein in F344/N rats and B6C3[F.sub.1] mice. Technical Report 465. Research Triangle Park, NC:National Toxicology Program.

--. 1996b. Comparative Initiation/Promotion Skin Paint Studies of B6C3[F.sub.1] Mice, Swiss (CD-1) Mice, and SENCAR Mice. Technical Report 441. Research Triangle Park, NC:National Toxicology Program.

--. 1998a. Toxicology and Carcinogenesis Studies of Chloroprene (CAS No. 126-99-8) in F344/N Rats and B6C3[F.sub.1] Mice (Inhalation Studies). Technical Report 467. Research Triangle Park, NC:National Toxicology Program.

--. 1998b. Toxicology and Carcinogenesis Studies of 1-Chloro-2-propanol (Technical Grade)(CAS NO. 127-00-4) in F344/N Rats and B6C3[F.sub.1] Mice (Drinking Water Studies). Technical Report 477. Research Triangle Park, NC:National Toxicology Program.

--. 1999a. Toxicology and Carcinogenesis Studies of Pentachlorophenol (CAS No. 87-86-5) in F344/N Rats (Feed Studies). Technical Report 483. Research Triangle Park, NC:National Toxicology Program.

--. 1999b. Toxicology and Carcinogenesis Studies of Oxymetholone (CAS No. 434-07-1) in F344/N Rats and Toxicology Studies of Oxymetholone in B6C3[F.sub.1] Mice (Garage Studies). Technical Report 485. Research Triangle Park, NC:National Toxicology Program.

--. 1999c. Toxicology and Carcinogenesis Studies of Diethanolamine (CAS No. 111-42-2) in F344/N Rats and B6C3[F.sub.1] Mice (Dermal Studies). Technical Report 478. Research Triangle Park, NC:National Toxicology Program.

--. 1999d. Toxicology and Carcinogenesis Studies of Furfuryl Alcohol (CAS No. 98-00-0) in F344/N Rats and B6C3[F.sub.1] Mice (Inhalation Studies). Technical Report 482. Research Triangle Park, NC:National Toxicology Program.

--. 1999e. Toxicology and Carcinogenesis Studies of Lauric Acid Diethanolamine Condensate (CAS No. 120-401) in F344/N Rats and B6C3[F.sub.1] Mice (Dermal Studies). Technical Report 480. Research Triangle Park, NC:National Toxicology Program.

--. 1999f. Toxicology and Carcinogenesis Studies of Oleic Acid Diethanolamine Condensate (CAS No. 93-83-4)in F344/N Rats and B6C3[F.sub.1] Mice (Dermal Studies). Technical Report 481. Research Triangle Park, NC:National Toxicology Program.

--. 1999g. Toxicology and Carcinogenesis Studies of Triethanolamine (CAS No. 102-71-6) in F344 Rats and B6C3[F.sub.1] Mice (Dermal Studies). Technical Report 449. Research Triangle Park, NC:National Toxicology Program.

--. 1999h. Toxicology and Carcinogenesis Studies of Magnetic Field Promotion (DMBA Initiation) in Female Sprague-Dawley Rats (Whole-body Exposure/Gavage Studies). Technical Report 489. Research Triangle Park, NC:National Toxicology Program.

--. 2000. Toxicology and Carcinogenesis Studies of Pyridine (CAS No. 110-86-1) in F344/N Rats, Wistar Rats, and B6C3[F.sub.1] Mice (Drinking Water Studies). Technical Report 470. Research Triangle Park, NC:National Toxicology Program.

--. 2001. Toxicology and Carcinogenesis Studies of Coconut Oil Acid Diethanolamine Condensate (CAS No. 68603-42-9) in F344/N Rats And B6C3[F.sub.1] Mice (Dermal Studies). Technical Report 479. Research Triangle Park, NC:National Toxicology Program.

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John B. Pritchard, (1) John E. French, (2) Barbara J. Davis, (3) and Joseph K. Haseman (4)

(1) Laboratory of Pharmacology and Chemistry and (2) Laboratory of Experimental Carcinogenesis and Mutagenesis, Environmental Toxicology Program; and (3) Laboratory of Women's Health, and (4) Biostatistics Branch, Environmental Biology and Medicine Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA

Address correspondence to J.B. Pritchard, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709 USA. Telephone: [phone]. Fax: [phone]. E-mail: [e-mail]

Received 15 May 2002; accepted 5 September 2002.