Practical and Science-Based Strategy for Establishing Acceptable Intakes for Drug Product -Nitrosamine Impurities.

The potential for -nitrosamine impurities in pharmaceutical products presents a challenge for the quality management of medicinal products. -Nitrosamines are considered cohort-of-concern compounds due to the potent carcinogenicity of many of the structurally simple chemicals within this structural class. In the past 2 years, a number of drug products containing certain active pharmaceutical ingredients have been withdrawn or recalled from the market due to the presence of carcinogenic low-molecular-weight ,-dialkylnitrosamine impurities.

Lack of response to multiple genotoxic agents at the hprt locus in peripheral blood T-lymphocytes of cynomolgus monkeys.

We tested the ability of a series of known genotoxic agents to cause mutations at the hprt locus in peripheral blood T-lymphocytes of cynomolgus monkeys as measured by the ability to form clones in the presence of 6-thioguanine. Ethylmethane sulfonate (EMS, 300 mg/kg i.p.

DNA sequence analysis of hprt mutants persisting in peripheral blood of cynomolgus monkeys more than two years after ENU treatment.

We have been studying in vivo mutagenesis at the hypoxanthine phosphoribosyl transferase (hprt) locus in cynomolgus monkey T-lymphocytes. This primate model allows us to study mutations and their kinetics under well-controlled conditions. Previously, we reported mutations detected at various times after intraperitoneal treatment with ethylnitrosourea (ENU, 77 mg/kg).

DNA sequence analysis of spontaneous hprt mutations arising in vivo in cynomolgus monkey T-lymphocytes.

To study the mechanisms of mutagenesis in vivo, we analyzed mutations at the hypoxanthine phosphoribosyl transferase (hprt) locus using cDNA from cynomolgus monkey T-lymphocytes. In the present study, the spectrum of spontaneous hprt mutations arising in vivo in wild-caught cynomolgus monkey peripheral T-lymphocytes is described. Cells were isolated from peripheral blood, and mutant clones were selected in 6-thioguanine, propagated, and stored frozen.

Southern blot analysis of T-cell receptor gene rearrangements in cynomolgus monkeys, and identification of a progenitor cell HPRT mutation.

Increases in peripheral blood T-lymphocyte HPRT mutant frequency may reflect either a number of independent HPRT gene mutational events or clonal proliferation of a single HPRT mutant. Sequence analysis of HPRT mutations in conjunction with T-cell receptor (TCR) gene rearrangement pattern analysis can distinguish these possibilities. Our laboratory previously characterized a nonhuman primate model for in vivo mutation studies using the clonal HPRT mutation assay.