Role of chronic toxicology studies in revealing new toxicities.

Chronic (>3 months) preclinical toxicology studies are conducted to support the safe conduct of clinical trials exceeding 3 months in duration. We have conducted a review of 32 chronic toxicology studies in non-rodents (22 studies in dogs and 10 in non-human primates) and 27 chronic toxicology studies in rats dosed with Merck compounds to determine the frequency at which additional target organ toxicities are observed in chronic toxicology studies as compared to subchronic studies of 3 months in duration. Our review shows that majority of the findings are observed in the subchronic studies since additional target organs were not observed in 24 chronic non rodent studies and in 21 chronic rodent studies. However, 6 studies in non rodents and 6 studies in rodents yielded new findings that were not seen in studies of 3-month or shorter duration. For 3 compounds the new safety findings did contribute to termination of clinical development plans. Although the incidence of compound termination associated with chronic toxicology study observations is low (∼10%), the observations made in these studies can be important for evaluating human safety risk.

A low volume, high-throughput forward mutation assay in Salmonella typhimurium based on fluorouracil resistance.

A forward mutation assay based on 5-fluorouracil (FU) resistance has been developed using a strain of Salmonella typhimurium derived from the Ames strain TA100. The sensitivity of the assay benefits from the genetic characteristics present in the standard Ames strain that enhances the response to genotoxic agents. A mutation conferring resistance to 5-fluorouridine was also introduced into the test strain to avoid unwanted toxicity resulting from cross-feeding of 5-fluorouridine between wild-type and FU-resistant (FU(R)) cells during selection with FU. In the mutation assay 1 ml aliquots of exponentially growing bacteria are exposed to the test agent for 2 h in the presence and absence of a rat-liver S-9 metabolizing system. The aliquots are then diluted, allowed to grow for 3 h, and assessed for treatment-related toxicity/inhibition by optical density. The cultures are diluted a second time and grown overnight to permit full recovery from toxicity and to allow expression of the FU(R) phenotype. Samples of the cultures are then plated in 384-well microtiter dishes in the presence of 2 microg/ml FU and the pH-sensitive indicator bromcresol purple. Three days later positive wells containing FU(R) colonies are detected by their yellow color and enumerated. Results were obtained using a variety of 45 compounds to validate the assay. Of the 25 mutagens and 20 non-mutagens tested, the results correlated 100% with those collected using the battery of standard Ames reversion strains, further supporting the use of the FU Assay as an alternative screen to the Ames assay. The use of a single strain exposed in liquid suspension permits assessment of high concentrations of test compound but with a low overall compound requirement (30 mg total). The highly parallel nature of culture handling/dilution and use of standard microtiter plates also offers the possibility of assay automation.

5-fluorouracil forward mutation assay in Salmonella: determination of mutational target and spontaneous mutational spectra.

A forward mutation assay in Salmonella typhimurium that selects for 5-fluoruracil (FU) resistance has been developed. The two genes possibly involved in FU resistance, the uracil phosphoribosyl transferase gene (upp) and the uracil transport protein (uraA), have been cloned from S. typhimurium and sequenced. One hundred percent of FU-resistant clones display sequence changes in the upp gene, indicating that its loss is the major mechanism involved in FU resistance. The spontaneous mutational spectra at the upp locus were then determined in two S. typhimurium strains, FU100 and FU1535, that differ only in the presence of pKM101 plasmid. The pKM101 plasmid provides error-prone replicative bypass of DNA lesions and renders FU100 more susceptible to induced mutagenesis. Fluctuation analysis of FU-resistant clones demonstrated a 10-fold higher spontaneous mutation rate at the upp locus in FU100 relative to FU1535. Over 300 independent FU-resistant clones were then used to generate the spectra at the upp locus in both the strains. Approximately 40% of all the mutations were base substitutions, present at the same relative percentage in both the strains. Frameshift mutations also accounted for approximately 40% of the total; however, their incidence was slightly elevated in FU100. The remaining mutations were larger insertions and deletions, which were both slightly elevated in FU1535. pKM101 significantly elevated the rate of all classes of mutations at the upp locus, with profound effects on A:T to T:A transversions and -2-base frameshift mutations. These initial mutational spectra at the upp locus reveal 147 mutable sites, or 23% of the total 627-base coding sequence and suggest that the target can detect a diverse spectrum of mutagenic events.