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.

26-Week dermal oncogenicity study evaluating pure trans-capsaicin in Tg.AC hemizygous mice (FBV/N).

The objective of this study was to assess the oncogenic potential of trans-capsaicin when administered weekly via topical application to the dorsal skin of Tg.AC mice for 26 weeks. Male and female Tg.AC mice (25 mice/sex/group) received dose formulations containing trans-capsaicin dissolved in diethylene glycol monoethyl ether (DGME). The positive control was tetradecanoylphorbol-13-acetate (TPA) dissolved in DGME. Appropriate controls, including a topical lidocaine local anesthetic pretreatment (4%w/w), were maintained. All groups were dosed once weekly, except for the TPA group, which was dosed twice per week. Analysis of the macroscopic observations after the final sacrifice revealed no noteworthy treatment-related findings, with the exception of dermal masses that were randomly dispersed throughout all treatment groups for both males and females. The frequency of dermal masses in the capsaicin-treated groups (at a dose level of up to 102 mg/kg and an application rate of 25.6 mg/cm2/kg/week) was not elevated in comparison to either concurrent vehicle or untreated controls. In contrast, a notable increase in the frequency of dermal masses was observed in the TPA-treated mice compared to both the concurrent vehicle and untreated controls. Dermal application of capsaicin resulted in no increased incidence of preneoplastic or neoplastic skin lesions. In contrast, over half the male and female mice exposed to TPA had multiple skin papillomas; the majority of the TPA-treated animals either died early or was humanely euthanized due to tumor load. Spontaneously occurring neoplasms were not appreciably increased in capsaicin-treated animals. Capsaicin-related non-neoplastic microscopic findings were seen sporadically in both genders and included acanthosis, hyperkeratosis/parakeratosis (primarily females), epidermal crusts, subepidermal fibrosis, epidermal ulcerations/erosions, and chronic-active inflammation. There was no evidence of a dose response in either the incidence or severity of these findings. The lidocaine- (at a dose level of 162 mg/kg and at an application rate of 40.5 mg/cm2/kg/week) and DGME-treated (at a dose level of 4.0 g/kg and at an application rate of 1 g/cm2/kg/week) control groups also did not display any evidence of increase in dermal masses. Based on these results, trans-capsaicin, lidocaine, and DGME should be considered nononcogenic in the Tg.AC mouse dermal model.

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.