Determining recommended acceptable intake limits for N-nitrosamine impurities in pharmaceuticals: Development and application of the Carcinogenic Potency Categorization Approach (CPCA).

N-Nitrosamine impurities, including nitrosamine drug substance-related impurities (NDSRIs), have challenged pharmaceutical industry and regulators alike and affected the global drug supply over the past 5 years. Nitrosamines are a class of known carcinogens, but NDSRIs have posed additional challenges as many lack empirical data to establish acceptable intake (AI) limits. Read-across analysis from surrogates has been used to identify AI limits in some cases; however, this approach is limited by the availability of robustly-tested surrogates matching the structural features of NDSRIs, which usually contain a diverse array of functional groups. Furthermore, the absence of a surrogate has resulted in conservative AI limits in some cases, posing practical challenges for impurity control. Therefore, a new framework for determining recommended AI limits was urgently needed. Here, the Carcinogenic Potency Categorization Approach (CPCA) and its supporting scientific rationale are presented. The CPCA is a rapidly-applied structure-activity relationship-based method that assigns a nitrosamine to 1 of 5 categories, each with a corresponding AI limit, reflecting predicted carcinogenic potency. The CPCA considers the number and distribution of α-hydrogens at the N-nitroso center and other activating and deactivating structural features of a nitrosamine that affect the α-hydroxylation metabolic activation pathway of carcinogenesis. The CPCA has been adopted internationally by several drug regulatory authorities as a simplified approach and a starting point to determine recommended AI limits for nitrosamines without the need for compound-specific empirical data.

Genotoxicity assessment of eight nitrosamines using 2D and 3D HepaRG cell models.

N-nitrosamine impurities have been increasingly detected in human drugs. This is a safety concern as many nitrosamines are mutagenic in bacteria and carcinogenic in rodent models. Typically, the mutagenic and carcinogenic activity of nitrosamines requires metabolic activation by cytochromes P450 enzymes (CYPs), which in many in vitro models are supplied exogenously using rodent liver homogenates. There are only limited data on the genotoxicity of nitrosamines in human cell systems. In this study, we used metabolically competent human HepaRG cells, whose metabolic capability is comparable to that of primary human hepatocytes, to evaluate the genotoxicity of eight nitrosamines [N-cyclopentyl-4-nitrosopiperazine (CPNP), N-nitrosodibutylamine (NDBA), N-nitrosodiethylamine (NDEA), N-nitrosodimethylamine (NDMA), N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), and N-nitrosomethylphenylamine (NMPA)]. Under the conditions we used to culture HepaRG cells, three-dimensional (3D) spheroids possessed higher levels of CYP activity compared to 2D monolayer cells; thus the genotoxicity of the eight nitrosamines was investigated using 3D HepaRG spheroids in addition to more conventional 2D cultures. Genotoxicity was assessed as DNA damage using the high-throughput CometChip assay and as aneugenicity/clastogenicity in the flow-cytometry-based micronucleus (MN) assay. Following a 24-h treatment, all the nitrosamines induced DNA damage in 3D spheroids, while only three nitrosamines, NDBA, NDEA, and NDMA, produced positive responses in 2D HepaRG cells. In addition, these three nitrosamines also caused significant increases in MN frequency in both 2D and 3D HepaRG models, while NMBA and NMPA were positive only in the 3D HepaRG MN assay. Overall, our results indicate that HepaRG spheroids may provide a sensitive, human-based cell system for evaluating the genotoxicity of nitrosamines.

Revisiting the mutagenicity and genotoxicity of N-nitroso propranolol in bacterial and human in vitro assays.

Propranolol is a widely used ß-blocker that can generate a nitrosated derivative, N-nitroso propranolol (NNP). NNP has been reported to be negative in the bacterial reverse mutation test (the Ames test) but genotoxic in other in vitro assays. In the current study, we systematically examined the in vitro mutagenicity and genotoxicity of NNP using several modifications of the Ames test known to affect the mutagenicity of nitrosamines, as well as a battery of genotoxicity tests using human cells. We found that NNP induced concentration-dependent mutations in the Ames test, both in two tester strains that detect base pair substitutions, TA1535 and TA100, as well as in the TA98 frameshift-detector strain. Although positive results were seen with rat liver S9, the hamster liver S9 fraction was more effective in bio-transforming NNP into a reactive mutagen. NNP also induced micronuclei and gene mutations in human lymphoblastoid TK6 cells in the presence of hamster liver S9. Using a panel of TK6 cell lines that each expresses a different human cytochrome P450 (CYP), CYP2C19 was identified as the most active enzyme in the bioactivation of NNP to a genotoxicant among those tested. NNP also induced concentration-dependent DNA strand breakage in metabolically competent 2-dimensional (2D) and 3D cultures of human HepaRG cells. This study indicates that NNP is genotoxic in a variety of bacterial and mammalian systems. Thus, NNP is a mutagenic and genotoxic nitrosamine and a potential human carcinogen.

Genotoxicity evaluation of nitrosamine impurities using human TK6 cells transduced with cytochrome P450s.

Many nitrosamines are recognized as mutagens and potent rodent carcinogens. Over the past few years, nitrosamine impurities have been detected in various drugs leading to drug recalls. Although nitrosamines are included in a 'cohort of concern' because of their potential human health risks, most of this concern is based on rodent cancer and bacterial mutagenicity data, and there are little data on their genotoxicity in human-based systems. In this study, we employed human lymphoblastoid TK6 cells transduced with human cytochrome P450 (CYP) 2A6 to evaluate the genotoxicity of six nitrosamines that have been identified as impurities in drug products: N-nitrosodiethylamine (NDEA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutanoic acid (NMBA), N-nitrosomethylphenylamine (NMPA), N-nitrosodiisopropylamine (NDIPA), and N-nitrosodibutylamine (NDBA). Using flow cytometry-based assays, we found that 24-h treatment with NDEA, NEIPA, NMBA, and NMPA caused concentration-dependent increases in the phosphorylation of histone H2A.X (γH2A.X) in CYP2A6-expressing TK6 cells. Metabolism of these four nitrosamines by CYP2A6 also caused significant increases in micronucleus frequency as well as G2/M phase cell-cycle arrest. In addition, nuclear P53 activation was found in CYP2A6-expressing TK6 cells exposed to NDEA, NEIPA, and NMPA. Overall, the genotoxic potency of the six nitrosamine impurities in our test system was NMPA > NDEA ≈ NEIPA > NMBA > NDBA ≈ NDIPA. This study provides new information on the genotoxic potential of nitrosamines in human cells, complementing test results generated from traditional assays and partially addressing the issue of the relevance of nitrosamine genotoxicity for humans. The metabolically competent human cell system reported here may be a useful model for risk assessment of nitrosamine impurities found in drugs.

Trends in excipient safety evaluation.

Excipients are used in all drug products and in most food products. New technologies are being tested to increase the amount or rate of absorption of drugs and new and novel excipients may be included among them. New physical approaches such as nanoparticles of drug and excipients or lysosomes may offer better drug delivery especially of hard to absorb or difficult to formulate oral drugs. New excipients may improve or mask the flavor of foods, drugs, and dietary supplements. Recently, impurities in drug products have become subject to greater scrutiny and various international and national guidelines, guidances, and regulations have been proposed and accepted for use; excipient evaluation is included in these efforts. This symposium discussed new developmental concepts, guidelines/guidances and regulations involving impurities in excipients, new drug delivery systems involving excipients, and thoughts for possible improvement to these guidelines to promote faster regulatory acceptance of these substances.

Overview of genotoxic impurities in pharmaceutical development.

This symposium focuses on the management of genotoxic impurities in the synthesis of pharmaceuticals. Recent developments in both Europe and United States require sponsors of new drug applications to develop processes to control the risks of potential genotoxic impurities. Genotoxic impurities represent a special case relative to the International Conference on Harmonisation Q3A/Q3B guidances, because genotoxicity tests used to qualify the drug substance may not be sufficient to demonstrate safety of a potentially genotoxic impurity. The default risk management approach for a genotoxic impurity is the threshold of toxicological concern unless a more specific risk characterization is appropriate. The symposium includes descriptions of industry examples where impurities are introduced and managed in the synthesis of a pharmaceutical. It includes recent regulatory developments such as the "staged threshold of toxicological concern" when administration is of short duration (eg, during clinical trials).