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.

Implementation of the principles of the 3Rs of animal testing at CDER: Past, present and future.

The safety testing of pharmaceutical candidates has traditionally relied on data gathered from studies in animals, and these sources of information remain a vital component of the safety assessment for new drug and biologic products. However, there are clearly ethical implications that attend the use of animals for safety testing, and FDA fully supports the principles of the 3Rs, as it relates to animal usage; these being to replace, reduce and refine. We provide an overview of some of the events and activities (legal and programmatic) that have had, and continue to have, the greatest impact on animal use in pharmaceutical development, and highlight some ongoing efforts to further meet the challenge of achieving our mission as humanely as possible.

An FDA/CDER perspective on nonclinical testing strategies: Classical toxicology approaches and new approach methodologies (NAMs).

Nonclinical testing of human pharmaceuticals is conducted to assess the safety of compounds to be studied in human clinical trials and for marketing of new drugs. Although there is no exact number and type of nonclinical studies required for safety assessments, as there is inherent flexibility for each new compound, the traditional approach is outlined in various FDA and ICH guidance documents and involves a combination of in vitro assays and whole animal testing methods. Recent advances in science have led to the emergence of numerous new approach methodologies (NAMs) for nonclinical testing that are currently being used in various aspects of drug development. Traditional nonclinical testing methods can predict clinical outcomes, although improvements in these methods that can increase predictivity of clinical outcomes are encouraged and needed. This paper discusses FDA/CDER's view on the opportunities and challenges of using NAMs in drug development especially for regulatory purposes, and also includes examples where NAMs are currently being used in nonclinical safety assessments and where they may supplement and/or enhance current testing methods. FDA/CDER also encourages communication with stakeholders regarding NAMs and is committed to exploring the use of NAMs to improve regulatory efficiency and potentially expedite drug development.

A regulatory perspective on issues and approaches in characterizing human metabolites.

This document captures the current thinking within FDA/CDER on the non-clinical safety assessment of human drug metabolites in new drug products. Examples are provided, which define a scientific based approach to the safety evaluation of human metabolites in new drug candidates. A discussion of the need for, and the adequacy of, the assessment of human drug metabolites with specific regard to their potential as mediators of toxicity is presented from a regulatory perspective.