Genetic Toxicology Testing

A Laboratory Manual

Ray Proudlock

• Publisher: Academic Press
• Year: 2016
• Print ISBN: 9780128007648
• eBook ISBN: 9780128010068
• Topic: Medicine
• Subtopic: Toxicology

Genetic Toxicology Testing: A Laboratory Manual presents a practical guide to genetic toxicology testing of chemicals in a GLP environment. The most commonly used assays are described, from laboratory and test design to results analysis. In a methodical manner, individual test methods are described step-by-step, along with equipment, suggested suppliers, recipes for reagents, and evaluation criteria.An invaluable resource in the lab, this book will help to troubleshoot any assay problems you may encounter to optimise quality and efficiency in your genetic toxicology tests. Genetic Toxicology Testing: A Laboratory Manual is an essential reference for those new to the genetic toxicology laboratory, or anyone involved in setting up their own.- Offers practical and consistent guidance on the most commonly-performed tests and procedures in a genetic toxicology lab- Describes standard genetic toxicology assays, their methodology, reagents, suppliers, and analysis of their results- Includes guidance on general approaches: formulation for in vitro assays, study monitoring, and Good Laboratory Practice (GLP)- Serves as an essential reference for those new to the genetic toxicology laboratory, or anyone involved in setting up their own lab

Chapter 1

A Practical Guide to Genetic Toxicology Testing

Ray Proudlock,    Boone, North Carolina, USA

Abstract

As a result of public concern over chemically induced heritable effects and carcinogenicity, genetic toxicology testing has become a regulatory requirement for all new chemicals and many other materials in virtually every developed country in the world. Regulations exist for testing pharmaceuticals, pharmaceutical degradants/impurities, unique human metabolites of pharmaceuticals, medical devices, agrochemicals, industrial chemicals and intermediates, household chemicals, food additives, unintentional/indirect food additives, and environmental pollutants. Adherence to the methods described and advice given in this book should greatly assist in ensuring the quality, reliability and efficiency of genetic toxicology tests for screening, regulatory submission or a research environment.

Keywords

Chemicals; medical devices; pollutants

Chapter Outline

1.1 Introduction 1

References 3

1.1 Introduction

As a result of public concern over chemically-induced heritable effects and carcinogenicity, genetic toxicology testing has become a regulatory requirement for all new chemicals and many other materials in virtually every developed country in the world. Regulations exist for testing pharmaceuticals, pharmaceutical degradants/impurities, unique human metabolites of pharmaceuticals, medical devices, agrochemicals, industrial chemicals and intermediates, household chemicals, food additives, unintentional/indirect food additives, and environmental media, such as air and water. In practice, this means that genetic toxicity testing is mandatory for virtually any new chemical where significant human exposure is expected.

The purpose of this book is not to describe testing requirements or test strategies for these materials but rather to describe how these genetic toxicology studies can be performed to appropriately high standards on a routine basis in an efficient manner, while producing reliable results. Ideally, study design and performance should be standardized and optimized to minimize risk of failure (and subsequent repeats) while providing results and the draft report in a minimum timeframe. These principles apply equally to formal Good Laboratory Practice (GLP) studies for regulatory submission and to non-GLP tests for screening or investigative purposes. In the latter case, the documentation requirements are generally reduced and there is no QA involvement.

I first started work in the field of genetic toxicology testing in the United Kingdom in 1978, when each industry and country had different testing requirements or expectations. In those days, many of the current test methods were in their infancy and there were no internationally agreed test method guidelines, so we generally followed the recommendations of the innovators of the test methods, particularly as described in a series of volumes edited by Alexander Hollaender and, later, with Frederick de Serres [1] over the period 1971 to 1986 [1]. The first comprehensive handbook of mutagenicity testing procedures [2] covered several of the test systems still in common use today in a single volume. Other practical guides followed, including a second edition of the Kilbey handbook [3] and several compilations coordinated largely through the UK Environmental Mutagenesis Society [4–7], including a consensus on the application of statistical methods for interpretation of results in the major test systems [8].

In the UK in the late 1970s, studies did not follow any standardized protocol or procedure, and records were incomplete, consisting in some cases, of little more than the results and a summary protocol. Staff did not work to any formal instruction or SOPs, and it soon became apparent to me that the quality of much of the work was inadequate. One of my main responsibilities became to improve technical aspects of various study types, primarily the bacterial mutation, in vitro and in vivo cytogenetics, and UDS tests. I took the opportunity to formally document standardized instructions, which allowed entry of specific details and results. GLPs subsequently became a regulatory requirement for all studies and, consequently, these documents became formally incorporated into SOPs. Complete formal instructions and records minimize the chance of errors on a study and facilitate any retrospective investigation of problems or unexpected results.

Experience has repeatedly shown that the effort spent in setting up an assay properly so it produces reliable results will be rewarded many times over when performing routine studies. These characteristics are clearly of paramount importance when test results are often pivotal, either allowing a chemical to proceed to market, or being dropped from further development. My background and technical expertise in a wide range of test types were essential in designing and establishing the Genetic Toxicology Department at Charles River Laboratories (then CTBR) in Montreal in 1999, the largest CRO toxicology facility in the world. Subsequently, I have assisted several test facilities in trouble-shooting or setting up these tests in-house. This experience forms the basis for my recommendation in this and the subsequent chapter titled General Recommendations on Assay Performance.

Although this book is not intended to cover testing strategy, the reader must consider the appropriateness of any test system(s) used when evaluating a test material. It is important to obtain chemical and physical information on the test material, including solubility, volatility, stability, functional groups, and potential metabolites. Much of this information will be available from the chemist responsible for developing the material or, in the case of contract research, will be obtainable from the study monitor. In the case of medical devices and similar “inert” materials, the study director should obtain information on potential leachables and extractables (i.e., amounts and chemical nature). Consideration of these factors and information on related chemicals, will allow the study director to make a preliminary assessment of the relevance of the test and whether there is a need for modifications to the standard test methods, e.g., for volatile materials, sealed systems might be considered for in vitro testing, and the pre-incubation procedure would be preferred over the plate incorporation bacterial mutation test for reasons of sensitivity e.g., [9,10]. For in vivo systems, especially when following up in vitro results, consider the relevance of the endpoint (is it expected to be sensitive to the effect seen in vitro). It particularly important to consider the likely exposure levels (concentration over time) of the target organ to the active chemical and, in the case of genotoxins that require metabolic activation in vitro, any active metabolites. In this case the extrapolated target exposure levels should be compared to the in vitro system where exposure levels can be much higher. Conversely, in vivo systems can be more sensitive to some genotoxins with very low aqueous solubility that require metabolic activation (e.g., some polyaromatic hydrocarbons). Other examples where in vivo systems are more sensitive or can give more relevant results have been discussed by the IWGT (InternationalWorkshop on Genotoxicity Testing) [11].

References

1. Hollaender A., de Serres F.J., editors. Chemical mutagens: principles and methods for their detection, vols. 1–10. New York, NY: Plenum Press; 1971–86.

2. Kilbey BJ, Legator M, Nichols W, Ramel C, eds. Handbook of mutagenicity test procedures. Amsterdam: Elsevier; 1977.

3. Kilbey BJ, Legator M, Nichols W, Ramel C, eds. Handbook of mutagenicity test procedures. 2nd edition Amsterdam: Elsevier; 1984.

4. Venitt S, Parry JM, eds. Mutagenicity testing: a practical approach. Washington, DC: IRL Press; 1984.

5. Kirkland DJ, ed. Basic mutagenicity tests: UKEMS Recommended Procedures. Cambridge: Cambridge University Press; 1990.

6. Kirkland DJ, Fox M, eds. Supplementary mutagenicity tests: UKEMS recommended procedures. Cambridge: Cambridge University Press; 1993.

7. Parry JM, Parry EM, eds. Genetic toxicology: principles and methods. New York: Humana Press (Springer); 2012.

8. Kirkland DJ, ed. Statistical evaluation of mutagenicity test data. Cambridge: Cambridge University Press; 1990.

9. Araki A, Kamigaito N, Sasaki T, Matsushima T. Mutagenicity of carbon tetrachloride and chloroform in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, using a gas exposure method. Environ Mol Mutagen. 2004;43(2):128–133.

10. O’Donovan MR, Mee CD. Formaldehyde is a bacterial mutagen in a range of Salmonella and Escherichia indicator strains. Mutagenesis. 1993;8(6):577–581.

11. Tweats DJ, Blakey D, Heflich RH, et al. IWGT Working Group Report of the IWGT working group on strategy/interpretation for regulatory in vivo tests II Identification of in vivo-only positive compounds in the bone marrow micronucleus test. Mutat Res. 2007;627(1):92–105.

Chapter 2

General Recommendations

Ray Proudlock, Boone, North Carolina, USA

Abstract

This chapter outlines the recommended approach to establishing a new assay within a laboratory and ensuring quality and efficiency while subsequently minimizing timelines. The advice applies equally to all genotoxicity test systems, whether they are investigative, prescreening, GLP, in vitro, or in vivo. Documentation, internal validation, and auditing of routine tests and facilities are also addressed. Suggestions given in the chapter are based primarily on my own experience and view of GLP, which will not coincide with those of your own test facility and QA unit. It is particularly important when establishing new procedures and associated documentation to meet directly with your QA unit and get their formal buy-in; this will save a lot of effort and annoyance later. Although method set-up and validation steps are virtually identical to those of non-GLP and GLP procedures, there is no requirement to involve the QA unit if the test system will never be used for regulatory submission.

Keywords

Laboratory; plate; protocol; procedures

Chapter Outline

2.1 Establishing a New Assay 6

2.1.1 Method Set-Up 6

2.1.2 Method Validation 8

2.2 Spreadsheets and Manipulation of Results 10

2.3 Laboratory Historical Control Databases 11

2.3.1 Vehicle/Negative Control Database 11

2.3.2 Positive Control Database 13

2.3.3 Spreadsheet Calculations 13

2.4 Use of Computer Systems 13

2.5 Study Design 15

2.6 Evaluation Criteria 17

2.6.1 Valid Assay 17

2.6.2 Criteria for Interpretation of Results 18

2.6.3 Statistical Analysis 19

2.7 Organization of SOPs 20

2.8 Planning a Study 22

2.9 Preparing a Protocol Complying with GLP...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• List of Contributors
• Foreword
• Preface
• Chapter 1. A Practical Guide to Genetic Toxicology Testing
• Chapter 2. General Recommendations
• Chapter 3. Formulation of Test Articles
• Chapter 4. The Bacterial Reverse Mutation Test
• Chapter 5. The Mouse Lymphoma TK Assay
• Chapter 6. The In Vitro Micronucleus Assay
• Chapter 7. The In Vitro Chromosome Aberration Test
• Chapter 8. The In Vivo Rodent Micronucleus Assay
• Chapter 9. The Rodent Bone Marrow Chromosomal Aberration Test
• Chapter 10. The In Vivo Comet Assay Test
• Chapter 11. The Pig-a Endogenous Gene Mutation Assay
• Index