Considerations when deriving compound-specific limits for extractables and leachables from pharmaceutical products: Four case studies.

Leachables from pharmaceutical container closure systems are a subset of impurities that present in drug products and may pose a risk to patients or compromise product quality. Extractable studies can identify potential leachables, and extractables and leachables (E&Ls) should be evaluated during development of the impurity control strategy. Currently, there is a lack of specific regulatory guidance on how to risk assess E&Ls; this may lead to inconsistency across the industry. This manuscript is a cross-industry Extractables and Leachables Safety Information Exchange (ELSIE) consortium collaboration and follow-up to Broschard et al. (2016), which aims to provide further clarity and detail on the conduct of E&L risk assessments. Where sufficient data are available, a health-based exposure limit termed Permitted Daily Exposure (PDE) may be calculated and to exemplify this, case studies of four common E&Ls are described herein, namely bisphenol-A, butylated hydroxytoluene, Irgafos® 168, and Irganox® 1010. Relevant discussion points are further explored, including the value of extractable data, how to perform route-to-route extrapolations and considerations around degradation products. By presenting PDEs for common E&L substances, the aim is to encourage consistency and harmony in approaches for deriving compound-specific limits.

Genotoxicity assessment of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX).

Hexahydro-1,3,5-trinitro-1,3,5-triazine, a polynitramine compound, commonly known as RDX, has been used as an explosive in military munitions formulations since World War II. There is considerable data available regarding the toxicity and carcinogenicity of RDX. It has been classified as a possible carcinogen (U.S. Environmental Protection Agency, Integrated Risk Information System, 2005, www.epa.gov/IRIS/subst/0313.htm). In order to better understand its gentoxic potential, the authors conducted the in vitro mouse lymphoma forward mutation and the in vivo mouse bone marrow micronucleus assays. Pure RDX (99.99%) at concentrations ranging from 3.93 to 500 microg/ml showed no cytotoxicity and no mutagenicity in forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, with and without metabolic activation. This finding was also confirmed by repeat assays under identical conditions. In addition, RDX did not induce micronuclei in mouse bone marrow cells when tested to the maximum tolerated dose of 250 mg/kg in male mice. These results show that RDX was not mutagenic in these in vitro and in vivo mammalian systems.

Genetic instability favoring transversions associated with ErbB2-induced mammary tumorigenesis.

It has been argued that genetic instability is required to generate the myriad mutations that fuel tumor initiation and progression and, in fact, patients with heritable cancer susceptibility syndromes harbor defects in specific genes that normally maintain DNA integrity. However, the vast majority of human cancers arise sporadically, in the absence of deficiencies in known "mutator" genes. We used a cII-based mutation detection assay to show that the mean frequency of forward mutations in primary mammary adenocarcinomas arising in mouse mammary tumor virus-c-erbB2 transgenic mice harboring multiple copies of the lambda bacteriophage genome was significantly higher than in aged-matched, wild-type mammary tissue. Analysis of the cII mutational spectrum within the mammary tumor genomic DNA demonstrated a >6-fold elevation in transversion mutation frequency, resulting in a highly unusual inversion of the transition/transversion ratio characteristic of normal epithelium; frameshift mutation frequencies were unaltered. Arising oncogenic point mutations within the c-erbB2 transgene of such tumors were predominantly transversions as well. Data from this model system support the notion that elaboration of a mutator phenotype is a consequential event in breast cancer and suggest that a novel DNA replication/repair gene is a relatively early mutational target in c-erbB2-induced mammary tumorigenesis.