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Genotoxic Impurities - Chemistry, Detection, and Risk Assessment
Genetic Toxicology Association Fall Meeting
Thursday, May 19, 2005
Clayton Hall Conference Center, University of Delaware





Establishing Allowable Concentrations of Genotoxic Impurities – Chemistry, Manufacture and Control Perspective

Dean Ellison (Merck)

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Establishing Allowable Concentrations of Genotoxic Impurities in Drug Substance and Product

Robert Mauthe (Pfizer)

This presentation will outline a procedure for testing, classification, qualification, and toxicological risk assessment of potentially genotoxic impurities in pharmaceutical products as developed by a cross-line task PhRMA force. Referencing accepted principles of cancer risk assessment, this presentation describes a staged Threshold of Toxicological Concern approach for phased implementation of allowable daily intakes of genotoxic impurities. The proposal applies to all clinical routes of administration and to compounds at all stages of clinical development. In addition, certain types of products, such as those for life-threatening indications for which there are no safer alternatives, may require special considerations.

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Approach of One Agro-chemical Company in Addressing the Genotoxic and Toxicological Implications of Process Impurities

Karin Bentley (Dupont)

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I Have an Ames Positive; I Think It's Because of an Impurity. Now What?

Valentine "Skip" O. Wagner III (BioReliance)

The ICH defines an impurity as any component of the new drug substance that is not the chemical entity defined as the new drug substance or an excipient. This can include residual starting materials, intermediates generated during the synthesis, contaminants in the starting materials and degradents of any of the above. An Ames test cannot identify which component is eliciting a positive response without the inclusion of additional controls and reference standards. However, responses that elicit one or more of the following four characteristics may suggest that a response is due to an impurity. (1) Marginal or weak response, generally one that exhibits a 2X to 10X increase; however, no absolute criteria are possible because the response depends upon the potency of the impurity and the quantity present. (2) No structural alert either in the precursor molecules or the resulting molecule. (3) Testing up to 5 mg of test article per plate is also an indication since most pure mutagens are toxic at much lower doses. (4) Lot to lot variation is one of the best indicators of a response that is due to an impurity.

Most companies that investigate such responses test different lots either with just the strain and activation condition of interest or with the full ICH complement of strains. Usually one of the lots that are tested is an ultrapure lot, possibly synthesized by an alternate pathway. The investigative work can be done either GLP or nonGLP.

Two case studies from pharmaceutical companies were presented and each is summarized below. Project 1 exhibited it's original positive responses with TA98 both with and without S9 on a test article that was 95.7% pure (Lot 1). The positive responses were four-fold to five-fold in magnitude and the Sponsor later identified 5 impurities that were thought to be the source of the response. Subsequently, three lots of the test article and a major impurity in Lot 1 were then tested with TA98 both with and without S9. The original lot (1) confirmed positive with four-fold to seven-fold increases. The second lot (2), 99.1% pure, synthesized by a new route, but with impurities 2 and 3 present at comparable concentrations to that found in Lot 1, was negative. A third ultrapure lot (3), >99% pure, was also negative, as was the major impurity in Lot 1. A full GLP study on Lot 2, required by the FDA for each lot being tested in humans, was also negative. Two additional impurities (4 and 5) found in Lot 1 were each negative with TA98 both with and without S9 at concentrations that were up to 10X those in Lot 1. The Sponsor is currently planning additional testing on HPLC fractions of Lot 1. Once the offending fraction is identified, they plan to synthesize the impurity in the fraction and test the impurity alone and spiked into a clean lot (Lot 2).

Project 2, began as a nonGLP screen with TA98 and TA100 both with and without S9 on a lot of test article that was 99.8% pure. The results were negative. About one year later, when conducting the GLP range-finding study on a lot of test article that was 98.4% pure, positive responses (7.1-fold to 19-fold) were observed with TA98 and TA1538 with S9. The results were suspected of being due to an impurity because of the previous negative response, the 7-fold to 19-fold increases and the fact that testing was conducted up to 5 mg per plate. For a three month period, numerous screening Ames tests were conducted with TA98 and TA1538 with S9 until the positive response could be correlated to a peak in the HPLC. The chemist also noted that the positive lots exhibited greater fluorescence. Based on these observations, the impurity was identified and a GLP study was conducted concurrently with the positive reference lot, the impurity alone and a negative lot spike at 20 and 2000 ppm. This design was selected to: establish in-study correlation of the positive lot with a quantified amount of causative agent, establish potency of the contaminant alone and establish lack of interference of test agent on the response of the contaminant. Positive responses were observed with TA98 and TA1538 with S9. The magnitude of the response from the impurity alone correlated to about 20 to 50 ng of impurity present in the original positive lot. The dose response curves from the positive data were super imposable.

In conclusion, the original positive in project 2 was induced by a single positive contaminant at ~4 ppm. While an Ames positive is not a show stopper, it can be timely to resolve. Use of early screening assays can help in later diagnosis and characterization of the test article is critical so that it is known what is being tested.

Acknowledgments were made thanking the two clients for allowing this presentation, the clients' chemists, and the BioReliance participants.

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Characterization of the Role of the Loop Region of DNA Polymerase beta in Polymerization Fidelity

George Lin (Yale University – GTA Student Travel Awardee)

The loop region of DNA polymerase beta is located on the outskirt of the palm domain of the polymerase and it spans fourteen amino acids. It is flexible and amorphous and therefore structural data have not been useful in assigning it a role in polymerization fidelity based on its shape and position. A screen conducted in our laboratory for AZT resistant polymerase beta mutants generated from random mutagenesis identified three AZT resistant mutants in this loop region. Two of these AZT-resistant polymerase mutants were characterized kinetically and shown to be mutator mutants. Mutator mutants are polymerase mutants that are less discriminating than wildtype in terms of selecting the correct nucleotide for polymerization. My goal is to test the hypothesis that the loop region of Pol beta is important for polymerization activity and fidelity and to elucidate its precise role in those processes. A second goal is to determine the nature of the amino acid residues of the loop that are important for activity and fidelity. The approach we have taken is to construct different polymerase beta mutants that contain specific length alterations in the loop region. We have generated several mutant constructs with varying lengths of the loop. The resulting polymerase mutants are then assessed for activity and fidelity using genetic screens. Several of the mutants have been characterized and we have identified two mutator mutants thus far from the group of loop mutants, suggesting that the loop is important for accurate DNA synthesis. Biochemical characterization of the purified variants will assist in the elucidation of the role of the loop domain in accurate DNA synthesis and is currently underway.

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Genetic Toxicology Testing in Assessing Carcinogenic Potential of Impurities in Food Contact Materials

Krista Dobo (Pfizer)

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Starting Materials, Intermediates and Solvents, Oh My!

Bhaskar Gollapudi (Dow)

Starting materials and intermediates used in the synthesis of pharmaceuticals are by necessity reactive in nature and may be present as impurities in active pharmaceutical ingredient (API) used for the conduct of preclinical safety studies as well as human clinical trials. In some cases starting materials and intermediates may be known or suspect mutagens and/or carcinogens. The sensitivity of modern analytical technology allows synthetic impurities to be detected at low ppm levels in API, following development of appropriate analytical methods. Given the linear nature of the dose-response curve associated with genotoxic carcinogens, the ability to demonstrate the presence or absence of impurities at low ppm levels is relevant to human cancer risk assessment. That is, for genotoxic carcinogens the only way to minimize human risk is to minimize exposure. Therefore, it is prudent that during the course of drug development due diligence be applied from two perspectives (1) to understand the potential mutagenic and carcinogenic risk associated with compounds used for API synthesis, (2) to understand the capability of synthetic processes to control compounds identified as genotoxic impurities to low levels in the API. Over the past year, chemical structure-based risk assessments of synthetic routes have been conducted starting with the first lot of API intended for clinical trials. All starting materials and intermediates are classified according to the weight of evidence that a mutagenic and/or carcinogenic risk to humans is associated with the chemical structure. Subsequently, known or suspect carcinogens are evaluated in the context of the synthetic process to evaluate fate in API. This analysis culminates in the prioritization of control and measurement of known or potential impurities in API. An overview of the experience gained in applying this approach will be presented.

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Carcinogenic Impurities in Color Additives

Arthur Lipman (FDA/CFSAN)

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Issues Related to Genotoxic Impurities in Drug Products

Tim McGovern (FDA/CDER)

Genotoxic impurities in drug substances and products can have an impact on pharmaceutical drug development and product approval due to their potential for being carcinogenic to humans. This presentation will provide a regulatory perspective related to genotoxic impurities. In general, a suspected or known genotoxic impurity should either be removed or reduced to a level that conveys no significant increase in cancer risk. The determination of a safe level could be based upon various parameters including, but not necessarily limited to, characterization of the genotoxic profile, structural activity relationships (SARs), risk assessment based on relevant carcinogenicity data, threshold approaches based on appropriate databases, and the proposed use of the drug. An overall safety assessment should consider the relative benefit to be derived from a given drug product versus the risk conveyed by the presence of a genotoxic impurity.

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