Preclinical Development Handbook || In Vitro Mammalian Cell Mutation Assays
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5 IN VITRO MAMMALIAN CELL MUTATION ASSAYS *
C. Anita H. Bigger , 1 Martha M. Moore , 2 and Robert H. Hefl ich 2 1 U.S. Food and Drug Administration, Center for Drug Evaluation and Research, Silver Spring, Maryland 2 U.S. Food and Drug Administration, National Center for Toxicological Research, Jefferson, Arkansas
Preclinical Development Handbook: Toxicology, edited by Shayne Cox GadCopyright 2008 John Wiley & Sons, Inc.
5.1 Introduction 5.2 Regulatory Guidances and Protocol Guidelines for In Vitro Mammalian Cell
Mutation Assays 5.2.1 Regulatory Guidances 5.2.2 Protocol Guidelines
5.3 Endpoints and Unique Characteristics of In Vitro Mammalian Cell Mutation Assays 5.3.1 Differences in the Spectra of Genetic Events Detected by In Vitro Mammalian
Cell Mutation Assays 5.3.2 Differences in the Spectra of Genetic Events Detected by the MLA, the In
Vitro Chromosomal Aberrations Assays, and the Ames Test 5.3.3 Correlation of In Vitro Mammalian Cell Mutation Assays with Rodent
Bioassays 5.4 Criticisms of In Vitro Mammalian Cell Assays
5.4.1 False Positives, High Concentrations, and Cytotoxicity Criteria 5.4.2 Hazard Identifi cation and Risk Assessment
5.5 Protocols for In Vitro Mammalian Cell Mutation Assays 5.5.1 Commonalities and Differences in Protocols for In Vitro Mammalian Cell
Mutation Assays 5.5.2 Detailed Protocols
* The views expressed in this chapter do not necessarily refl ect FDA policy.
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The in vitro mammalian cell mutation assays used for preclinical safety testing detect forward mutations at the following target genes: (1) the thymidine kinase gene ( Tk ), (2) the hypoxanthine guanine phosphoribosyl transferase gene ( Hprt ), and (3) the E. coli xanthine guanine phosphoribosyl transferase gene ( xprt or gpt ), integrated into mammalian cell DNA as a stable bacterial transgene  . All three target genes detect base substitutions, frameshifts, deletions, insertions, and rear-rangements within the locus; however, assays employing the Tk gene and the xprt transgene offer the additional advantage of detecting mutations involving multi-locus deletion. The Tk gene also detects translocation, as well as mutations requiring two alleles, such as mitotic recombination, gene conversion, and chromosome non-disjunction [1 11] .
Commonly used cell lines for in vitro mammalian cell mutation assays are L5178Y mouse lymphoma cells, Chinese hamster ovary (CHO), AS52, and V79 cell lines derived from Chinese hamsters, and TK6 human lymphoblastoid cells  . At this time, by far the most frequently used in vitro cell mutation assay in drug development is the Mouse Lymphoma Assay (MLA), utilizing L5178Y mouse lymphoma cells and the Tk target gene. This is primarily because the MLA, in addition to being capable of detecting the broadest spectrum of types of genetic damage of the in vitro genetic toxicology assays [1 11] , is one of the assays in the standard battery of geno-toxicity tests agreed upon by the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH)  . The ICH Guidance for Industry S2B Genotoxicity: A Standard Battery for Geno-toxicity Testing of Pharmaceuticals (ICH S2B)  also describes conditions under which the Hprt or xprt target genes in any of the cell lines mentioned above might be appropriate for assessment of the genotoxic potential of pharmaceuticals.
This chapter provides the following: (1) discussion of regulatory guidance for the use of in vitro mammalian cell mutation assays, as well as the role of in vitro mam-malian cell mutation assays in the evaluation of pharmaceuticals for genetic toxicity; (2) comments on the validated and internationally accepted protocol guidelines available for these assays; (3) discussion of the advantages and disadvantages of the assays for determining the nature of the genotoxicity elicited by pharmaceuticals; and (4) study designs for the MLA, CHO/ Hprt , and AS52/ xprt assays, including recent changes to the MLA protocol, test criteria, and evaluation of results.
5.2 REGULATORY GUIDANCES AND PROTOCOL GUIDELINES FOR IN VITRO MAMMALIAN CELL MUTATION ASSAYS
5.2.1 Regulatory Guidances
The two primary ICH guidances devoted to genetic toxicology testing of pharma-ceuticals are Guideline for Industry: Specifi c Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (ICH S2A)  and Guidance for Industry S2B Genotox-icity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals (ICH S2B)  .
ICH S2A provides guidance and recommendations on conducting the tests and evaluating the test results. The S2A defi nes the top concentration, cytotoxicity levels,
and a strategy for testing poorly soluble compounds with in vitro mammalian cell mutation assays. ICH S2A also provides general guidance for the evaluation of assay results.
ICH S2B defi nes the standard test battery for pharmaceuticals as: (1) a test for gene mutation in bacteria; (2) an in vitro test for the cytogenetic evaluation of chromosomal damage in mammalian cells or an in vitro MLA measuring Tk gene mutation; and (3) an in vivo test for chromosomal damage using rodent hematopoi-etic cells. Other regulatory agencies also recommend this battery of tests for screen-ing chemicals  . During the early ICH discussions of the standard battery, some parties preferred a four - test battery that included both of the in vitro mammalian assays that are listed in the three - test battery as alternates. The ICH Expert Working Group (EWG), however, reviewed additional studies, some of which were specifi -cally designed to address this issue. The EWG compared results in the two in vitro mammalian cell tests for compounds that were considered genotoxic but gave nega-tive results in the bacterial test. These studies demonstrated a high level of congru-ence between the in vitro mammalian cell assays, leading the EWG to conclude that the MLA and the in vitro mammalian cell chromosomal damage test could be used interchangeably in the standard battery (see ICH S2B III(ii), Notes 3 and 4). Based on this evidence, the EWG compromised with a three - test battery in which either the in vitro mammalian cell cytogenetics assay or the in vitro MLA satisfi ed the second element of the battery.
The ICH S2B also allows modifi cations of the three - test battery (ICH S2B IV) to include in vitro mammalian cell mutation assays other than the MLA. For example, for compounds that are extremely toxic to bacteria or for compounds that interfere with the mammalian cell replication system, the guidance suggests employing two in vitro mammalian cell tests. The two tests should use different cell lines and dif-ferent endpoints, that is, gene mutation and chromosomal damage (ICH S2B IV A). ICH S2B (Note 1) also lists the currently accepted target loci and cell lines for in vitro mammalian cell mutation assays: (1) for the Tk gene, mouse lymphoma L5178Y cells or human lymphoblastoid TK6 cells; (2) for the Hprt gene, CHO cells, V79 cells, or L5178Y cells; and (3) for the xprt gene, AS52 cells.
The ICH S2B defi nes standard procedures for in vitro mammalian cell mutation tests other than the MLA (ICH S2A V) and indicates that a range - fi nding test can substitute for a complete repeat of the assay, as long as certain criteria are met (ICH S2B Note 10). For the MLA, the guidance states that the protocol must include testing with and without exogenous metabolic activation, with appropriate controls, and with treatment for 3 or 4 hours. If the short treatment without metabolic activa-tion is negative, then that arm of the test should be repeated with treatment for 24 hours. An acceptable MLA also includes positive controls that induce small colonies and colony sizing for both positive and negative controls and at least one positive test compound dose, if present.
Both ICH S2A and ICH S2B were fi nalized approximately a decade ago. At the present time, there is discussion of revising these two guidances through the ICH Maintenance Procedure. That process, however, is likely to require years of discus-sion to achieve an agreed upon, fi nalized guidance to replace S2A and S2B. In the interim, possible changes to the guidances are discussed at international genetic toxicology meetings and conclusions are disseminated through publications in the literature. For example, the ICH S2B states that the microwell (or microtiter)
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method for the MLA is preferred over the agar method for use in the standard battery (ICH S2B Note 4). After extensive discussion, an informal agreement was reached between all the parties to the ICH EWG for genetic toxicology that both versions of the assay are acceptable for regulatory purposes. This information was made public through several publications [15, 16] .
Other ICH guidances that govern the regulatory use of in vitro mammalian cell mutation assays are: (1) ICH M3 Guidance: Non - clinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals  ; (2) ICH S1B Guidance: Testing for Carcinogenicity of Pharmaceuticals  ; (3) ICH Q3A Guidance: Impuri-ties in New Drug Substances  ; and (4) ICH Q3B Guidance: Impurities in New Drug Products  .