Mutagenesis, Vol. 17, No. 6, 451-455,
November 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
The role of the UKEMS in the development of testing guidelines
Covance Laboratories Ltd, Otley Road, Harrogate HG3 1PY, UK
 |
Abstract |
|---|
 Top Abstract IntroductionFirst trial; first guidelinesImpact of these first...Statistics recommendationsRevisions to Parts I...The impact on international...International Workshops on...The future?SummaryReferences |
|---|
Twenty years ago UKEMS established a sub-committee to determine the minimal professional criteria that should be achieved to comply with mutagenicity testing requirements in the UK. Recommendations on the conduct of basic and supplementary tests were published in 1983 and 1984, respectively. Despite their local distribution, these recommendations had an impact around the world. Further guidelines for statistical evaluation of mutagenicity test data and revisions to the first two volumes followed. By the early 1990s the mood was for international harmonization rather than national or regional isolation. The processes by which UKEMS had achieved its testing recommendations in the 1980s and early 1990s were successfully employed in the International Workshops for Genotoxicity Testing, of which three have now been held, and made a significant impact on OECD guidelines and ICH guidance. Summary outcomes from the latest meeting (2002 Plymouth Workshop) are given.
|
Introduction |
|---|
|
TopAbstractIntroductionFirst trial; first guidelinesImpact of these first...Statistics recommendationsRevisions to Parts I...The impact on international...International Workshops on...The future?SummaryReferences |
|---|
The UK was not the first region to establish an Environmental Mutagen Society, the USA was first. Nor is the UKEMS the largest society. In its early days, therefore, the Society probably did not realise what an impact it would have on the global stage and in the advancement of the science.
|
First trial; first guidelines |
|---|
|
TopAbstractIntroductionFirst trial; first guidelinesImpact of these first...Statistics recommendationsRevisions to Parts I...The impact on international...International Workshops on...The future?SummaryReferences |
|---|
The ability of mutagenicity tests to predict carcinogenicity has been of interest since the famous paper of Ames et al. (1973)
that declared that ‘Carcinogens are mutagens...’. However, as data from the MRC/ICI/NIEHS International Collaborative Study (sometimes known as the ‘42-compound trial’) were being decoded (later published by de Serres and Ashby, 1981), it was becoming clear that no single mutagenicity test would detect all classes and examples of chemical carcinogens. Some UKEMS members were concerned that a more quantitative approach to genotoxicity testing may be useful and thus the first UKEMS collaborative trial was initiated. The selected chemicals for test were benzylchloride (BC), 4-chloromethylbiphenyl (4CMB) and 4-hydroxymethylbiphenyl (4HMB).
Participating laboratories were encouraged to use whatever test system they liked, to accomplish whatever testing they had the time and resources to complete, but there was no review or harmonization of protocols. Thus the participating laboratories followed whatever was their favourite protocol. In the late 1970s and early 1980s there were very few published guidelines on how to conduct tests. OECD guidelines did not appear for the first time until 1983. The Committee on Mutagenicity of the UK Department of Health was preparing guidelines (DHSS, 1981) on testing at the time this UKEMS trial was being conducted. There was little knowledge of the content until after the trial was complete, but in any case the DHSS guidelines were concerned with strategy (which tests to conduct) and not method.
When the trial was over and the data decoded (Parry, 1982) it became clear that many different methods had been used by different laboratories for the same test system (for the Ames test, for example, different combinations of strains, different concentrations of S9, different top doses and dose intervals, etc.). Some methods produced positive results with BC and 4CMB whereas others gave negative results. This was particularly noticeable in the mammalian cell tests for chromosomal aberrations and mutations. Yet all of these methods would have fitted the strategy published in the 1981 DHSS guidelines. It seemed clear that some guidance on methods was needed.
The question arose, therefore, as to whether the scientific knowledge existing within UKEMS could be used to provide more detailed methodological guidance to laboratory staff conducting experiments at the bench. Could the Society provide some kind of ‘handbook’ on the critical aspects of method that would be likely to affect the overall outcome of a study and make recommendations on best approaches to follow?
As a result of these questions, UKEMS established a sub-committee in March 1982 to ‘determine minimal professional criteria that should be achieved to comply with mutagenicity testing requirements in the United Kingdom’. The sub-committee was chaired by Brian Dean and consisted of a steering group which was representative of the main sections of the Society (academia, government and industry) and a series of working groups, each consisting of a leader and between two and five members.
The terms of reference of the sub-committee were:
- to define the minimal criteria, i.e. the minimum basic experimental design required to perform current test procedures to professionally acceptable standards (with due regard to the good laboratory practice guidelines) of relevant authorities;
- to define criteria necessary to constitute a positive result in each procedure;
- to define the criteria and extent of testing required to identify a material as negative in each procedure;
- to describe modifications to standard test procedures that may be required to meet specific circumstances or to answer specific problems;
- to prepare proposals for regular updating of the test systems and protocols as envisaged by the DHSS guidelines and those of other bodies with regard to accepted technical advances supported by up to date and appropriate references;
- to prepare recommendations for a framework of testing procedures subsequent to the initial battery, i.e. which supplementary tests should be carried out and in what circumstances.
The first UKEMS guideline report (Part I) was published in 1983 (UKEMS, 1983). It addressed the most commonly used tests at that time, namely:
- bacterial reverse mutation tests;
- chromosomal aberration tests in cells in vitro;
- gene mutation tests in cultured mammalian cells;
- in vivo cytogenetics assays;
- mutation tests in Drosophila;
- dominant lethal tests.
This publication was quickly followed by Part II (UKEMS, 1984), which offered guidance on some of the tests considered non-core (or supplementary) at the time, namely:
- assays for DNA repair in bacteria;
- genotoxicity studies using yeast cultures;
- unscheduled DNA synthesis;
- sister chromatid exchange in cultured cells;
- in vitro cell transformation assays;
- mammalian germ cell cytogenetics.
This volume also contained recommendations for:
- bacterial assays for mutagens in food;
- mutagens in urine, faeces and body fluids;
- bacterial mutation assays with nitrosation products.
The wide range of tests in Parts I and II reflects the climate of the early 1980s. As we shall see later, this list would be pruned down somewhat into a much smaller range of useful tests by the early 1990s.
|
Impact of these first UKEMS reports |
|---|
|
TopAbstractIntroductionFirst trial; first guidelinesImpact of these first...Statistics recommendationsRevisions to Parts I...The impact on international...International Workshops on...The future?SummaryReferences |
|---|
It was quite surprising to those of us involved in the production of these recommendations what an impact they had within and outside the UK. Most of the feedback is personal and anecdotal, but practitioners at the bench found the books extremely helpful; they were so heavily used that the softback format was not durable enough and hardly any copies of Part I (UKEMS, 1983
) are left. UKEMS recommendations in Parts I and II also had a significant impact on the development of guidelines in Canada (HPB, 1992), where they were widely referenced. |
Statistics recommendations |
|---|
|
TopAbstractIntroductionFirst trial; first guidelinesImpact of these first...Statistics recommendationsRevisions to Parts I...The impact on international...International Workshops on...The future?SummaryReferences |
|---|
Throughout the development of Parts I and II, recommendations for data analysis were frequently truncated and, rather like the early OECD guidelines (OECD, 1983
), indicated that ‘appropriate statistical methods should be used’, without describing what they were. UKEMS decided it was time to make some detailed recommendations on the role of statistics in experimental design and data analysis, but this turned out to be a much more difficult project than producing the first two books. The chairmanship of the Guidelines Sub-committee had passed to David Kirkland, but the approaches of the former chairman continued to be followed. The one new development was a decision to publish the reports through a recognized international publisher, in order to achieve wider international exposure than could be achieved locally through the UKEMS office. Thus terms were agreed with Cambridge University Press for this and subsequent guideline publications.
As before, specialist workgroups of genetic toxicologists and statisticians from academia, government and industry were assembled to address each of the main test systems, namely:
- analysis of data from microbial colony assays;
- mammalian cell gene mutation assays based on colony formation;
- statistical evaluation of bacterial/mammalian fluctuation tests;
- analysis of data from in vitro cytogenetic assays;
- statistical methods for sister chromatid exchange experiments;
- statistical analysis of in vivo cytogenetic assays;
- statistical methods for the dominant lethal assay;
- statistical methods for the design and analysis of mutation experiments with the fruit fly Drosophila melanogaster.
Whereas the timetable for guideline reports Parts I and II had been short and strictly adhered to (to reflect the state of the science at a point in time), the development of statistical recommendations took considerably longer (1985–1988). It had become necessary to allow a period of familiarization during which the genetic toxicologists and statisticians in the various groups learned more of each other’s disciplines and languages, such that communication could be effective.
The statistics recommendations were eventually published in 1989 (UKEMS, 1989) and received mixed acceptance. In the USA, where empirical judgement of positive and negative calls was the tradition, these recommendations were largely overlooked. In Japan, however, they were received with considerable enthusiasm. Although these recommendations did not achieve the acclaim of Parts I and II, they are referenced in the OECD Guidelines revisions (OECD, 1997) and a number of (now) widely used data analysis software packages were developed on the basis of the UKEMS recommendations.
|
Revisions to Parts I and II |
|---|
|
TopAbstractIntroductionFirst trial; first guidelinesImpact of these first...Statistics recommendationsRevisions to Parts I...The impact on international...International Workshops on...The future?SummaryReferences |
|---|
An objective of the UKEMS Guidelines Sub-committee established in 1982 was that the reports should reflect the current state of knowledge of the field of mutagenesis. It was therefore envisaged that the reports would need to be revised, and the UKEMS recommended they be reviewed every 5 years. By 1988 the field had moved on, guidelines for mutagenicity testing had been published by several other organizations and countries and the UK Department of Health was revising its 1981 guidelines; the new DOH guidelines were published in 1989 (DOH, 1989
). It was therefore time for the original UKEMS recommendations to be revised.
Between 1983 and 1988 some tests that had been used quite widely fell into disfavour. Thus, when revisions to the basic test battery were made (UKEMS, 1990), the number of ‘core’ tests had reduced from six to four, namely:
- bacterial mutation assays;
- metaphase chromosome aberration assays in vitro;
- gene mutation assays in cultured mammalian cells;
- in vivo cytogenetics assays.
Tests for bacterial DNA repair, SCE, cell transformation and in Drosophila were not even considered as ‘supplementary’ tests by the time revisions to Part II were underway in the early 1990s. The dominant lethal test moved into the ‘supplementary tests’ volume and, along with procedures for yeast tests, unscheduled DNA synthesis (UDS), DNA binding and germ cell cytogenetics, new recommendations were published in 1993 (UKEMS, 1993).
|
The impact on international harmonization |
|---|
|
TopAbstractIntroductionFirst trial; first guidelinesImpact of these first...Statistics recommendationsRevisions to Parts I...The impact on international...International Workshops on...The future?SummaryReferences |
|---|
In the early 1990s, the differences between recommendations for testing in different guidelines were recognized. For example, Japanese guidelines included Escherichia coli in the bacterial test whereas in the USA only Salmonella strains were used. For in vitro chromosomal aberration tests, recommendations in the UK and Japan were for two different sampling times (which, in fact, were different!), whereas in the USA there was generally only one. Instead of persisting with different national and regional approaches it was agreed that there was a need to try to reach international consensus, if at all possible. The process by which this would be achieved was based on the working group methods that had been so successful for UKEMS.
|
International Workshops on Genotoxicity Testing (IWGT) |
|---|
|
TopAbstractIntroductionFirst trial; first guidelinesImpact of these first...Statistics recommendationsRevisions to Parts I...The impact on international...International Workshops on...The future?SummaryReferences |
|---|
The first IWGT was held in Melbourne, Australia in 1993 as a satellite to the 6th International Conference on Environmental Mutagens. Workgroups were established to resolve differences in approaches to the following commonly used tests, for which guidelines already existed:
- bacterial mutation assays;
- mammalian cell gene mutation assays;
- in vitro tests for chromosomal aberrations;
- UDS tests in vitro and in vivo;
- rodent erythrocyte micronucleus assay;
- in vivo mammalian bone marrow chromosomal aberration test;
- mammalian germ cell tests.
Experts in these ‘core’ tests from academia, government and industry debated the science behind the tests and used data to drive consensus recommendations that all could accept. The reports were published the following year (Galloway, 1994). These consensus statements had a significant impact on the OECD guidelines, which were being revised at the time (OECD, 1997).
Since the success of the Melbourne Workshop, two further workshops have been held. In 1999, workgroups assembled in Washington, DC (as a satellite to the annual EMS meeting) to try to provide consensus recommendations on a number of up and coming tests for which few, if any, guidelines existed. These tests were:
- in vitro micronucleus assay;
- photochemical genotoxicity;
- single cell gel/Comet assay in vitro and in vivo;
- DNA adduct determination;
- transgenic mutation assays.
In addition, due to new developments, the following assays/aspects were revisited:
- mouse lymphoma tk mutation assay;
- cytotoxicity and chromosome aberrations in vitro;
- in vivo rodent erythrocyte micronucleus assay (repeated treatments, integration with toxicity tests and automated scoring).
A statistics group was also included and assisted all of the working groups. These reports were published the following year (Kirkland, 2000).
The latest workshop was held as a prelude to the 25th Anniversary Meeting of UKEMS, in Plymouth, 2002. Some of the assays discussed in Washington had not reached full consensus and were revisited, namely:
- in vitro micronucleus assay;
- transgenic mutation assays;
- mouse lymphoma tk mutation assay.
However, two new topics were introduced:
- genetic and molecular analysis of tumour and non-tumour tissue in p53-deficient and Hras2 transgenic tumour models;
- classification of genotoxins and a strategy for risk assessment.
Many important consensus decisions were made, and can be summarised as follows.
In vitro micronucleus assay
It was agreed that cell proliferation should be demonstrated in both control and treated cells. Cytochalasin B can be used with cell lines (optional), but must be used for human lymphocytes and to measure non-disjunction. In cell lines without cytochalasin B, demonstrating a greater number of cells at harvest than at the start of treatment will verify proliferation.
For a valid study, at least three dose levels should be used, at intervals of no more than ÷10. The highest concentration should produce at least 60% toxicity; this is needed to detect aneugens.
Detailed treatment and sampling protocols have been recommended both for cell lines (with and without cytochalasin B) and for human lymphocytes. It is also recommended that 1000 target cells per culture are scored for micronuclei, with parallel scoring of 1000 mononucleated cells in those cultures with cytochalasin B, where the target cells will be 1000 binucleates.
Transgenic mutation assays
The recommendations for treatment and sampling times that were based on theory rather than data in Washington were reviewed. From a database of 143 compounds it was clear that treatment for 28 days with sampling 3 days later will detect all mutagens. Unless existing information suggests otherwise, at least one rapidly proliferating and one slowly proliferating tissue should be sampled.
Sequencing of mutants is not necessary for a clearly negative or clearly positive result. However, sequencing data are useful to identify and correct for ‘jackpot’ mutations and to investigate high variability in controls. It may also be useful to resolve equivocal results.
Mouse lymphoma assay
This group has been working tirelessly since the Washington workshop and had achieved a number of consensus agreements prior to the latest Plymouth workshop, e.g. confirming the usefulness of the 24 h treatment protocol. Data from 550 experiments on 170 chemicals tested in 10 different laboratories have been reviewed. This has allowed consensus agreement that the standard measure for toxicity should be relative total growth (RTG). These data also allowed the group to recommend acceptability criteria (ranges for spontaneous mutant frequency, cloning efficiency and suspension growth) for both the agar and microwell methods.
Recommendations for positive controls were more difficult to agree. A wide variety of positive control chemicals have been used at many different doses. The working group proposes to select (what is hoped will be) universally acceptable positive controls and to define what some of their properties should be.
For statistical evaluation, 398 data sets and 29 different statistical methods were reviewed. No single statistical method can be recommended to define mouse lymphoma results as positive or negative, although a variety of methods are acceptable for analysis of dose–response relationships. The group proposes to try to define a certain value above which an induced mutant frequency would need to rise before triggering statistical methods and on the basis of both biological and statistical approaches a decision on the result of the study would be made.
Genetic and molecular analysis of tumour and non-tumour tissue in p53-deficient and Hras2 transgenic tumour models
The working group reviewed the experimental data from the p53 and Hras2 tumour models on about 100 chemicals so far tested and also reviewed the data with the Tg.AC model for reference. They were able to make a number of recommendations related to characterization of the strains, such as genotype, integration site and copy number (for Hras2) and genotype, expression and allelic markers (for p53).
For analysis of tumours, codons 12, 13 and 61 are the ‘hot-spots’ for Hras2, and quantitative PCR should be carried out for gene expression. In the p53 model, in the absence of loss of heterozygosity, mutation analysis is needed.
Classification of genotoxins and a strategy for risk assessment
Earlier IWGT workshops focused on recommendations for the best ways to conduct tests. This new workgroup would aim to suggest ways in which the tests can be used strategically, and it was anticipated this would be a long process. Prior to the meeting it was envisaged that a classification scheme for hazard would be a good place to start, as this should lead to definition of the minimum tests needed to achieve a particular classification. This should then lead to a strategic approach to testing which could be applied similarly to industrial chemicals, pharmaceuticals, agrochemicals, food additives, etc. However, it soon became apparent that there was no consensus for a classification scheme. There was serious concern that classification would lead to ‘labels’ that could have serious commercial consequences.
The working group therefore decided to focus on the development of a universally acceptable, step-wise process for genotoxicity hazard evaluation and risk assessment. In particular, there is a need to encompass the different approaches to hazard evaluation and to remove the inconsistencies in regulatory interpretation.
The group agreed a mission statement and some consensus statements on hazard evaluation, mainly concerning an elementary data set on which hazard evaluation could be based. The group defined a number of future activities and will be meeting regularly to review progress.
Many UKEMS members have participated in all three workshops. The international workshop activity has now been incorporated into a Foundation of the International Association of Environmental Mutagen Societies and more workshops will be held. Although recommendations for methods will need to be revisited from time to time, the trend may well be towards strategies for testing and the usefulness of combinations of tests.
|
The future? |
|---|
|
TopAbstractIntroductionFirst trial; first guidelinesImpact of these first...Statistics recommendationsRevisions to Parts I...The impact on international...International Workshops on...The future?SummaryReferences |
|---|
The future of guideline development is probably going to be international rather than national or regional. This trend began in the 1990s with the International Conferences on Harmonization in the pharmaceutical industry (Müller et al., 1999
) and the International Workshops on Genotoxicity Testing. Whilst I believe individual organizations and countries will continue to discuss and propose approaches to genotoxicity testing (for example the latest UK Committee on Mutagenicity guidelines; Parry, 2000) and such guidelines will stimulate important debate, isolation will not be sustainable. The contact between scientists of different nations, the abundance of international scientific meetings and the ease of international travel mean that multinational and multidisciplinary discussions are inevitable. Thus, whilst the UKEMS was pioneering in establishing its Guidelines Sub-committee back in 1982, I do not believe such an exercise would be successful in the climate of today. The processes of the UKEMS guidelines activities have been carried forward into the international arena and UK genetic toxicologists are involved in these international activities. I hope that the science of genetic toxicology will remain sufficiently strong in the UK to guarantee their continued involvement. |
Summary |
|---|
|
TopAbstractIntroductionFirst trial; first guidelinesImpact of these first...Statistics recommendationsRevisions to Parts I...The impact on international...International Workshops on...The future?SummaryReferences |
|---|
As the UKEMS celebrates 25 years, the Guidelines Sub-committee celebrates 20 years since its foundation. Its first reports had a significant impact on ‘best practices’ and ensured that UK scientists would be invited to a seat at the table of harmonization. The processes by which UKEMS working groups reached their recommendations has been successfully carried forward into the international arena in the shape of the IWGT workshops. The Society should be justly proud of these achievements.
|
References |
|---|
|
TopAbstractIntroductionFirst trial; first guidelinesImpact of these first...Statistics recommendationsRevisions to Parts I...The impact on international...International Workshops on...The future?SummaryReferences |
|---|
-
Ames,B.N., Durston,W.E., Yamasaki,E. and Lee,F.D. (1973) Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl Acad. Sci. USA, 70, 2281–2285.
De Serres,F.J. and Ashby,J. (1981) Short-Term Tests for Carcinogens: Results of the International Collaborative Study. Elsevier/North Holland, Amsterdam, The Netherlands.
DHSS (1981) Guidelines for the Testing of Chemicals for Mutagenicity, Report on Health and Social Subjects no. 24. HM Stationery Office, London, UK.
DOH (1989) Guidelines for the Testing of Chemicals for Mutagenicity, Report on Health and Social Subjects no. 35. HM Stationery Office, London, UK.
Galloway,S.M. (ed.) (1994) Report of the International Workshop on Standardisation of Genotoxicity Test Procedures. Mutat. Res., 312, 195–322.
HPB (1992) Conduct and Reporting of Mutagenicity Tests. Assays Recommended by the Health Protection Branch, Final Draft. Department of National Health and Welfare, Ottawa, Canada.
Kirkland,D.J. (ed.) (2000) The International Workshop on Genotoxicity Test Procedures. Washington D.C., March 25–26, 1999. Environ. Mol. Mutagen., 35, 159–263.
Müller,L., Kikuchi,Y., Probst,G., Schechtman,L., Shimada,H., Sofuni,T. and Tweats,D. (1999) ICH-harmonised guidances on genotoxicity testing of pharmaceuticals: evolution, reasoning and impact. Mutat. Res., 436, 195–225.[Web of Science][Medline]
OECD (1983) OECD Guideline for Testing of Chemicals. Genetic Toxicology, nos 471–474. Organisation for Economic Cooperation and Development, Paris, France.
OECD (1997) OECD Guideline for Testing of Chemicals. Genetic Toxicology, nos 471–476, 483, and 486. Organisation for Economic Cooperation and Development, Paris, France.
Parry,J.M. (ed.) (1982) The UKEMS Genotoxicity Trial 1981. Mutat. Res., 100, 1–433.
Parry,J.M. (ed.) (2002) Guidance on a Strategy for Testing of Chemicals for Mutagenicity. UK Department of Health Committee on Mutagenicity, London, UK.
UKEMS (1983) UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part I. Basic Test Battery, Dean,B.J. (ed.). United Kingdom Environmental Mutagen Society, Swansea, UK.
UKEMS (1984) UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Part II. Supplementary Tests, Dean,B.J. (ed.). United Kingdom Environmental Mutagen Society, Swansea, UK.
UKEMS (1989) UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Part III. Statistical Evaluation of Mutagenicity Test Data, Kirkland,D.J. (ed.). Cambridge University Press, Cambridge, UK.
UKEMS (1990) Basic Mutagenicity Tests: UKEMS Recommended Procedures, Kirkland,D.J. (ed.). Cambridge University Press, Cambridge, UK.
UKEMS (1993) Supplementary Mutagenicity Tests: UKEMS Recommended Procedures. Kirkland,D.J. and Fox,M. (eds). Cambridge University Press, Cambridge, UK.
Received on June 10, 2002; revised on July 12, 2002; accepted on July 12, 2002.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||