Genetically altered mouse models carry activated oncogenes or inactivated tumor suppressor genes known to be involved in neoplastic processes in both humans and rodents. For some chemicals, this trait allows these mice to respond or show the effects of carcinogens in less than the usual 2 years of conventional rodent carcinogenicity tests and within a time frame in which few, if any, spontaneous tumors would arise. Target or reporter genes also allow direct molecular and cellular analysis of a chemical’s effects and can provide additional mechanistic information about its mode of action. The National Toxicology Program (NTP) has been using a number of transgenic rodent models for studies of carcinogenesis [p16^Ink4a, p53^+/−, Tg.AC (v-Ha-ras), and Tg.NK (MMTV/c-neu)] and mutagenesis [C57BL/6JTgN (phiX174am3, cs70) 54Hvm] for the last few years. The p53 haploinsufficent model has demonstrated preferential identification of genotoxic/mutagenic carcinogens, and the Tg.AC hemizygous model has responded to both genotoxic and nongenotoxic carcinogens. The goal of the NTP is to select transgenic animal models that best mimic human tissue processes, providing a firmer foundation for applying hazard data from animals to humans (Chhabra et al., 2003).

Studies on diisopropylcarbodiimide and dicyclohexylcarbodiimide are part of the database the NTP is establishing to help in developing more rapid and economical assays to reduce dependence on the 2-year bioassay. Dicyclohexylcarbodiimide and diisopropylcarbodiimide are representatives of the carbodiimide chemical class, are widely used reagents in the chemical and pharmaceutical industries, and are used increasingly in the field of bioenergetics. Dicyclohexylcarbodiimide and diisopropylcarbodiimide are widely used as coupling and condensation agents, especially in peptide synthesis, and as stabilizing agents. Of the two chemicals, dicyclohexylcarbodiimide currently has the greater usage. However, this use pattern might change because the reaction side products of diisopropylcarbodiimide are easier to remove than those of dicyclohexylcarbodiimide, and the use of diisopropylcarbodiimide is increasing. Dicyclohexylcarbodiimide and diisopropylcarbodi imide are listed in the Toxic Substances Control Act (TSCA) Inventory and on the European Inventory of existing commercial chemical substances, but information on specific production volumes is not available. Research scientists in the bioenergetics field are particularly at risk. Occupational exposure to these chemicals occurs primarily by dermal contact or inhalation.

The NTP has completed traditional dermal toxicity and carcinogenicity studies on diisopropylcarbodiimide and reported the results in a Technical Report (NTP, 2006). There were no diisopropylcarbodiimide treatment-related increases in the incidences of neoplastic lesions in those studies. In the current studies, two genetically altered mouse models with either a loss of heterozygosity in a critical cancer suppressor gene (p53) or a gain of oncogene function (Tg.AC) were used to determine how these animals would respond to diisopropylcarbodiimide treatment. The dose concentrations used in the studies were derived from the 3-month toxicity study performed in the B6C3F₁ mice that showed skin as the primary target site for diisopropylcarbodiimide toxicity (NTP, 2006). The dose concentrations selected for the 2-year bioassay were 0, 10, 20, and 40 mg/kg. Because of different strain backgrounds in the transgenic mouse models and shorter durations of treatment, the doses selected for these studies were 0, 4.38, 8.75, 17.5, 35, and 70 mg/kg. This broader dosing regimen consisting of five dose groups was expected to achieve maximum tolerated doses at the upper end of the dosing range.

The findings from the Tg.AC hemizygous mouse study show that with diisopropylcarbodiimide treatment there were no neoplastic or nonneoplastic lesions. Similarly, there were no diisopropylcarbodiimide treatment-related neoplastic or nonneoplastic lesions in the p53 haploinsufficient mouse model with the exception of epidermal hyperplasia in the 70 mg/kg group at the site of application. These findings agree with the lack of diisopropylcarbodiimide carcinogenic activity in the traditional 2-year rodent bioassay (NTP, 2006). However, the diispropylcarbodiimide studies performed in the Tg.AC hemizygous model were some of the earliest NTP studies exploring the value of the model as an alternate to the traditional bioassay. The protocol used does not meet the current study design standards with respect to the number of animals per group. Also, the animals could have tolerated higher doses than 70 mg/kg since no chemical-related effects were seen at this concentration.

Pritchard et al. (2003), in a review of the role of transgenic mouse models in carcinogen identification, reported that if a testing strategy of evaluating chemicals in both the p53 haploinsufficient and Tg.AC hemizygous mouse models were adopted, predictions of the carcinogenic potential of chemicals in humans could be made with approximately 83% accuracy. If the 2-year bioassay were added to this combination, approximately the same percentage of predictability could be expected. In comparison, the 2-year bioassay alone yielded a correct determination for 69% of chemicals tested. This lower predictability could be due to a lack of sufficient information in humans for classification of the chemicals that have been identified as carcinogens in the 2-year bioassay. Although transgenic models had a high percentage of correct determinations, they did fail to respond to a number of known or probable human carcinogens, whereas the traditional bioassay identified all of these chemicals. The studies reported by Pritchard et al. (2003) are based on a limited database of approximately 100 chemicals from human and animal studies with varying study designs and approaches. Therefore, acceptance of transgenic mouse models individually or in combination as a substitute for the 2-year bioassay remain controversial. Additional studies such as those reported here, as well as basic research in the suitability of these models for identification of chemical carcinogens, is needed. In the meantime, studies from genetically modified models may contribute to the weight of evidence in the risk assessment of chemicals causing adverse health effects in humans.

Diisopropylcarbodiimide is not mutagenic in the Salmonella assay (NTP, 2006) and therefore, the chemical might be expected to be negative for carcinogenic activity in the transgenic mouse strains used in this study. However, results of in vivo mutagenicity tests showed clear evidence of increased micronucleus frequencies in blood of male and female mice following three or more months of exposure via skin painting (Witt et al., 1999). The results of the peripheral blood micronucleus tests are somewhat surprising because a strong correlation has been reported between positive results in subchronic peripheral blood micronucleus tests and rodent carcinogenicity (Witt et al., 2000). The number of positive studies from which this correlation derives is small, although additional support for the relationship between positive rodent micronucleus test data and carcinogenicity was provided by Morita et al. (1997), who reported a 90.5% correlation between carcinogenic activity in humans and positive results in the rodent micronucleus test when data were corrected for known structure-activity considerations with regard to micronucleus assay sensitivity. In addition, Zeiger (1998) reported a 70% correlation between rodent carcinogenicity and positive results in the mouse bone marrow micronucleus test in an unadjusted dataset of 83 NTP chemicals. Thus, the pattern of activity shown by diisopropylcarbodiimide is unusual.

Footnotes

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Explanation of Levels of Evidence of Carcinogenic Activity is on page 8. A summary of the Technical Reports Review Subcommittee comments and the public discussion on this Report appears on page 10.