SUMMARY REPORT ON THE
EVALUATION OF SHORT-TERM TESTS FOR CARCINOGENS
(COLLABORATIVE STUDY ON IN VITRO TESTS)
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INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
ENVIRONMENTAL HEALTH CRITERIA 47
SUMMARY REPORT ON THE EVALUATION OF SHORT-TERM TESTS FOR CARCINOGENS (COLLABORATIVE STUDY ON IN VITRO TESTS)
This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organisation, or the World Health Organization.
Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization World Health Orgnization
Geneva, 1985
The International Programme on Chemical Safety (IPCS) is a joint venture of the United Nations Environment Programme, the International Labour Organisation, and the World Health
Organization. The main objective of the IPCS is to carry out and disseminate evaluations of the effects of chemicals on human health and the quality of the environment. Supporting activities include the development of epidemiological, experimental laboratory, and risk-assessment methods that could produce internationally
comparable results, and the development of manpower in the field of toxicology. Other activities carried out by the IPCS include the development of know-how for coping with chemical accidents,
coordination of laboratory testing and epidemiological studies, and promotion of research on the mechanisms of the biological action of chemicals.
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CONTENTS
SYNOPSIS - THE INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY (IPCS):
COLLABORATIVE STUDY ON THE ASSESSMENT AND VALIDATION OF SHORT-TERM TESTS FOR CARCINOGENS
1. SUMMARY REPORT ON IN VITRO TESTS 1.1. Introduction
2. THE COLLABORATIVE STUDY ON SHORT-TERM TESTS (CSSTT) 1981-83 3. CRITERIA FOR SELECTION OF THE TEST CHEMICALS
4. PURITY OF THE TEST CHEMICALS
5. CRITERIA FOR THE DEFINITION OF COMPLEMENTARY IN VITRO ASSAYS FOR THE DETECTION OF POTENTIAL CARCINOGENS
6. ASSAYS AND END-POINTS 6.1. Bacteria
6.2. Fungi
6.3. Drosophila
6.4. Cultured mammalian cells 7. RESULTS
8. CONFIRMATION OF THE NON-MUTAGENICITY OF THE TEST CHEMICALS FOR SALMONELLA
9. ASSESSMENT OF THE PERFORMANCE OF ASSAYS ON THE REDUCED LIST 9.1. Gene mutation in yeast
9.2. Drosophila somatic cell mutation assays
9.3. Assays for DNA damage SSB (single-strand breaks) and UDS (detected via autoradiography or scintillation counting)
9.4. Assays for the induction of aneuploidy 9.5. Mammalian cell gene-mutation assays 9.6. Chromosomal-aberration assays
9.7. Assays for polyploidy induction
9.8. Sister chromatid exchange (SCE) assays 9.9. Transformation assays
10. SELECTION OF A PREFERRED COMPLEMENTARY ASSAY 11. CONCLUSIONS
REFERENCES
LIST OF PARTICIPANTS
Dr D. Amacher, Pfizer Central Research, Groton, Connecticut Dr P. Arni, Ciba-Geigy, Basle, Switzerland
Dr J. Ashby, Central Toxicology Laboratory, Imperial Chemical Industries, Ltd, Macclesfield, Cheshire, United Kingdom Dr R. Baker, School of Public Health and Tropical Medicine, University of Sydney, Sydney NSW, Australia
Dr J.C. Barrett, Laboratory of Pulmonary Function and Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA
Dr R.H. Barrett, The Boots Company Industrial Division, Nottingham, United Kingdom
Dr M.O. Bradley, Merck, Sharp & Dohme, West Point, Pennsylvania, USA
Dr T. Brooks, Shell Research, Ltd, Tunstall Laboratory, Kent, United Kingdom
Dr A. Carere, Higher Institute of Health, Rome, Italy
Dr W. Caspary, National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA
Dr D.V. Chitavichus, Institute of Medical Genetics, Moscow, USSR Dr C.L. Crespi, Gentest Corporation, Woburn, Massachusetts
Dr N. Danford, Department of Genetics, University College of Swansea, Swansea, United Kingdom
Dr B.J. Dean, Shell Research, Ltd, Tunstall Laboratory, Kent, United Kingdom
Dr G. Delow, Paterson Laboratories, Christie Hospital & Holt Radium Institute, Manchester, United Kingdom
Dr F.J. de Serres, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA Dr G.R. Douglas, Environmental Health Centre, Department of
National Health and Welfare, Tunney's Pasture, Ottawa, Ontario, Canada
Dr M. Draper, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland
LIST OF PARTICIPANTS (contd.)
Dr E. Elmore, Northrop Services, Inc., Research Triangle Park, North Carolina, USA
Dr L.R. Ferguson, Cancer Research Laboratory, Pathology Department, The Medical School, Auckland, New Zealand Dr K. Fujikawa, Drug Safety Evaluation Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., Osaka, Japan
Dr R.C. Garner, Cancer Research Unit, University of York, York, United Kingdom
Dr H. Glauert, Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin, USA
Dr D.K. Gulati, EHRT, Inc., Lexington, Kentucky, USA
Dr G. Hatch, Northrop Services, Inc., Research Triangle Park, North Carolina, USA
Dr J. Heinisch, Institute for Microbiology, Darmstadt, Federal Republic of Germany
Dr C. Howard, Central Toxicology Laboratory, Imperial Chemical Industries, Ltd, Macclesfield, Cheshire, United Kingdom Dr S. Inge-Vechtemov, Department of Genetics and Breeding, Leningrad State University, Leningrad, USSR
Dr M. Ishidate, Division of Mutagenesis, National Institute of Hygienic Sciences, Tokyo, Japan
Dr A. Knaap, Laboratory of Carcinogenesis and Mutagenesis, National Institute of Public Health, Bilthoven, The Netherlands
Dr T. Lakhanisky, Institut d'Hygiene et d'Epidemiologie, Brussels, Belgium
Mr C.G. Lee, Chemical Defence Establishment, Porton Down, Wiltshire, United Kingdom
Prof N. Loprieno, Institute of Biochemistry, Biophysics, and Genetics, University of Pisa, Pisa, Italy
Dr D. McGregor, Development Toxicology, Inveresk Research Int., Ltd, Musselborough, Scotland
Dr B. Margolin, Biometry and Risk Assessment Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina
LIST OF PARTICIPANTS (contd.)
Dr C. Martin, Cancer Research Unit, University of York, York, United Kingdom
Dr M. Mercier, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland
Dr B.C. Myhr, Department of Genetics and Cell Biology, Litton
Bionetics, Inc., Kensington, Maryland
Dr A.J. Nelmes, Gallaher Limited, London, United Kingdom
Dr S. Nesnow, Carcinogenesis and Metabolism Branch, Health Effects Research Laboratory, US Environmental Protection Agency,
Research Triangle Park, North Carolina
Dr E.R. Nestmann, Environmental Health Centre, Department of
National Health and Welfare, Tunney's Pasture, Ottawa, Ontario, Canada
Dr G. Obe, Institute of General Genetics of the Free University of Berlin, Berlin (West)
Dr T.J. Oberly, Lilly Research Laboratories, Greenfield Laboratory, Greenfield, Indiana
Dr F. Palitti, Evolutionary Genetics Centre, Institute of Genetics, Citta Universitaria, Rome, Italy
Dr S. Parodi, Scientific Institute for Tumours, University of Genoa, Genoa, Italy
Dr J. Parry, Department of Genetics, University College of Swansea, Singleton Park, Swansea, Wales, United Kingdom
Dr B.J. Phillips, British Industrial Biological Research Association, Carshalton, Surrey, United Kingdom
Dr G. Probst, Lilly Research Laboratories, Greenfield Laboratory, Greenfield, Indiana, USA
Mr C.R. Richardson, Central Toxicology Laboratory, Imperial
Chemical Industries, Ltd, Macclesfield, Cheshire, United Kingdom Dr E. Matthews, Department of Molecular Biology, Litton Bionetics, Inc., Kensington, Maryland
Dr T. Sanner, Laboratory for Environmental and Occupational Cancer, Norsk Hydro's Institute for Cancer Research, Oslo, Norway
Dr M.D. Shelby, National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina
LIST OF PARTICIPANTS (contd.)
Dr J.W.I.M. Simons, State University of Leiden, Leiden, The Netherlands
Dr J. Styles, Central Toxicology Laboratory, Imperial Chemical Industries, Ltd, Macclesfield, Cheshire, United Kingdom Dr W.A. Suk, Northrop Services, Inc., Research Triangle Park, North Carolina
Dr G.F. van Went, Division of Toxicology and Chemical Analysis of Foodstuffs, National Institute of Public Health, Bilthoven, The Netherlands
Dr S. Venitt, Chemical Carcinogenesis Division, Pollards Wood Research Station, Bucks, United Kingdom
Dr E. Vogel, Department of Radiation Genetics and Chemical
Mutagenesis, State University of Leiden, Leiden, The Netherlands Dr R.C. von Borstel, Department of Genetics, The University of Alberta, Alberta, Canada
Dr M.D. Waters, Genetic Toxicology Division, Health Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, North Carolina, USA
Dr G. Williams, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, New York, USA
Dr F. Würgler, Institute of Toxicology, University of Zurich, Schwerzenbach, Switzerland
Dr M.Z. Zdzienicka, Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Leiden, The Netherlands Dr E. Zeiger, Toxicology Research and Testing Program, National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA
NOTE TO READERS OF THE CRITERIA DOCUMENTS
While every effort has been made to present information in the criteria documents as accurately as possible without unduly
delaying their publication, mistakes might have occurred and are likely to occur in the future. In the interest of all users of the environmental health criteria documents, readers are kindly
requested to communicate any errors found to the Manager of the International Programme on Chemical Safety, World Health
Organization, Geneva, Switzerland, in order that they may be included in corrigenda, which will appear in subsequent volumes.
In addition, experts in any particular field dealt with in the criteria documents are kindly requested to make available to the WHO Secretariat any important published information that may have inadvertently been omitted so that it may be considered in the event of updating of the criteria document.
* * *
Partial financial support for the publication of this criteria document was kindly provided by the United States Department of Health and Human Services, through a contract from the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA - a WHO Collaborating Centre for Environmental Health Effects.
SYNOPSIS - THE INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY (IPCS):
COLLABORATIVE STUDY ON THE ASSESSMENT AND VALIDATION OF SHORT-TERM TESTS FOR CARCINOGENS
The first part of this project, dealing with in vitro studies, has already been published by Elsevier, Amsterdam. The second part, concerning in vivo studies, is expected to be completed and evaluated by early 1985, with publication about one year later.
The rationale for the collaborative study was derived, to a
large extent, from the major findings of the "International Program for the Evaluation of Short-Term Tests for Carcinogens" (IPESTTC) (de Serres & Ashby, 1981). This study, in turn, arose from the necessity to evaluate the efficacy of different short-term assays proposed for supplementing the traditional long-term assay in the rodent. The results of the IPESTTC clearly confirmed the value of Salmonella reversion assays as suitable primary tests for potential carcinogens and mutagens. However, it was also confirmed that some known rodent carcinogens were either not detected, or only detected with considerable difficulty, by such assays. The IPESTTC study did not succeed in defining any complementary eukaryotic assay that could be used to detect carcinogens, found to be negative in the standard Salmonella reversion assay. Several assays showed promise, but none could be recommended, because it was considered that the supporting data base was too small.
It was against this background that the Collaborative Study on the Assessment and Validation of Short-Term Tests for Genotoxicity and Carcinogenicity (CSSTT) was proposed by the International Programme on Chemical Safety (IPCS) and the National Institute of Environmental Health Sciences (NIEHS) of the USA, as a
Participating Institution in the IPCS. The objective of the study was to generate a wide range of test results, using a small group of carefully-selected chemicals, which would contribute to an empirical basis for the selection of one or more in vitro short- term tests to complement the widely-used Salmonella test, developed by Professor Bruce Ames.
Some 60 investigators presented nearly 90 individual sets of assay results to the collaboration study, generating in all some 2500 dose-response relationships. Most of the currently available in vitro eukaryotic assay systems were represented. The following 8 organic carcinogens known to be either inactive or difficult to detect in the Salmonella assay were chosen: o-toluidine,
hexamethylphosphoramide (HMPA), safrole, acrylonitrile, benzene, diethylhexylphthalate, phenobarbital, and diethylstilboestrol, together with 2 chemicals, caprolactam and benzoin, for which there was no evidence of carcinogenicity in 2-year, 2-species rodent bioassays.
The results of the study were evaluated at a meeting of the investigators, held at St. Simon's Island, Georgia, USA, on 22 - 28 October, 1983. Each group of assays was chaired by a coordinator, and all the original data were discussed, evaluated, and agreed. At these assay group meetings, important protocol deficiencies were identified and these findings constitute one of the most significant developments arising from the collaborative study.
The findings indicate that carcinogens that are inactive or difficult to detect in the Salmonella assay fall into 2 distinct groups. The first group includes genotoxins that are probably non- mutagenic to Salmonella because of deficiencies in the available metabolic capacity of the assay system. Thus, HMPA, o-toluidine, safrole, and acrylonitrile were detected by most of the eukaryotic assays studied, indicating that there is a range of assays that can complement the Salmonella mutation assay to a limited extent.
The other group of carcinogens, benzene, DEHP, DES, and phenobarbital, displayed a more selective range of genotoxic activities, and none of the assays was selectively sensitive to them.
Of the assays studied, only the induction of chromosomal aberrations, cell transformation, and gene mutation in mammalian cells, and aneuploidy in yeast gave encouraging overall
performances for the 8 carcinogens, and, with the exception of the first, formidable protocol deficiencies have to be remedied.
The 3 carcinogens, DES, phenobarbital, and DEHP, chosen to represent the class of chemicals believed to induce tumours in rodents without first modifying the integrity of the nuclear DNA, each displayed a range of genotoxic activity. Thus, the term "non- genotoxic" should be used only when a sufficiently large
genotoxicity data base has been established.
The collaborative study has provided considerable evidence to support the view that in vitro assays should be classified as confirmatory, complementary, and supplementary. This understanding of the potential of an assay should provide a basis for the
elimination of redundancies in proposed combinations of test procedures.
The major conclusion of the study was that the use of
chromosomal aberration assays, preferably in an agreed cell type, in conjunction with an adequate assessment of the mutagenicity of a chemical for Salmonella, might provide an efficient primary screen for possible new carcinogens. A first priority should be the
application of resources to establish a generally acceptable and applicable protocol for the conduct of this type of assay. The adoption of a chromosomal-aberration assay as a common
complementary test has additional advantages in that it would allow easy comparison of data, ready extension to supplementary
cytogenetic assays, and the provision of data derived from an independent endpoint from the gene mutations of the Salmonella assay. Finally, looking back at the developments in this field of toxicology, it seems clear that to establish an assay at the level of international acceptance requires about a decade of meticulous scientific endeavour and international collaboration.
1. SUMMARY REPORT ON IN VITRO TESTS 1.1. Introduction
It was discovered in the late 1960s and early 1970s that many chemicals underwent metabolic changes before they were capable of inducing the processes leading to cancer. This led to the
development of in vitro techniques for bringing about such
metabolic transformations and, consequently, enormous progress was made in the fields of mutagenesis and carcinogenesis. It soon emerged that there was a strong correlation between the
carcinogenic activity of a chemical, particularly in rodents, and its mutagenic properties, as demonstrated in a wide variety of in vitro and in vivo experimental systems involving bacteria, yeasts, insects, rodents, and mammalian cells in tissue culture. At the same time, there was a growing awareness that some chemicals, for example, vinyl chloride monomer, posed hitherto unsuspected health dangers. The fact that increasing numbers of chemicals were being shown to have toxic properties in the evolving in vitro
genotoxicity test systems, together with a series of disasters associated with chemicals that resulted in considerable mortality and morbidity, brought about a realization that appropriate
legislative control of chemicals was needed to ensure adequate protection of human health. Thus, in the latter part of the 1970s, there was an unprecedented amount of activity, both nationally and internationally, in the field of chemical safety.
Public perception about the inadvertent exposure of human beings to chemical carcinogens was greatly heightened by the development of one particular assay system for the detection of mutagens. This was the Salmonella typhimurium reversion test incorporating a metabolic activation system, pioneered by Professor Bruce Ames and his colleagues, and now known universally as the Salmonella assay, or the Ames test. This assay system, which is based on fundamental genetic and molecular biological principles, produced test results within a week, and it was soon adopted by scientists throughout the world, but more particularly in North America and Europe. As a result, many hundreds of chemicals were tested and pronounced on, as is now known, without full
appreciation of the technical difficulties of the test and the biological significance of the results. The fact that many
chemicals in common use, ranging from food additives and cosmetics to household products, were claimed to have mutagenic properties and, hence, by implication could be carcinogens, received a considerable amount of uncritical attention in the general scientific and lay press.
It is generally accepted, and was so for the purposes of this study, that the strongest evidence that a chemical is a carcinogen is derived from either chemical or epidemiological findings in human beings showing an unequivocal relationship between exposure and the induction of malignant disease, supported by appropriate animal studies; or, in the absence of adequate human exposure data, the experimental induction in several rodent species of malignant tumours following carefully-controlled systematic exposures to the chemical for most of their life span. These rodent bioassays require about 3 years of experimental effort to produce a conclusion and are extremely costly. Thus, the prospect of
obtaining apparently equivalent information in a far shorter time and at a fraction of the cost was immensely appealing. These hopes were reinforced by the claims of confirmatory evidence provided by an increasing number of different assay systems. It was these developments that gave rise to the term "short-term tests" to refer to assay systems that were believed to indicate carcinogenic
properties. It was even hoped that, in time, these assays would replace, at least in part, the rodent bioassay. Although,
understandably, this idea received much uncritical support, many scientists were sceptical about some of the claims made for these assay systems, pointing out that there were many discrepancies in the findings, so-called false positives and false negatives, and there was much debate concerning the true predictive nature of assay systems, singly and in combination, for rodent carcinogens and the relevance of the findings for human disease.
By the early 1980s, many nations had adopted legislation to control toxic chemicals, incorporating various test requirements for acute and chronic effects and other preventive measures, such as adequate labelling. However, the issue of a legal requirement for specific short-term tests for mutagenic and carcinogenic properties was usually avoided. In some cases, the problem was recognized by the formulation of a discretionary set of
recommendations involving data from a battery or tier of assays in which the Salmonella assay was a basic requirement. The enormous international trade in chemicals and the realization that, for most of the 50 000 or so chemicals in common use, little systematic toxicological data existed, brought about an early appreciation of the need to use scarce toxicological resources with maximum
efficiency and under international agreement. Because of the profound implications for international trade in chemicals, the Organization of Economic Cooperation and Development (OECD)
addressed the problem of ensuring that toxicological data developed in one member state could be accepted in all member states. To this end, an extensive series of guidelines for toxicity and other testing was developed by international experts. Further, and of great importance, a set of procedures was formulated to ensure good laboratory practice and quality assurance. This major
international collaboration identified and codified the tests that were believed to be necessary to provide sufficient data to ensure safety in the use of a chemical. Obligatory measures and
procedures were set out to ensure that the tests were carried out according to the highest standards, as laid down by international agreement. The implementation of these guidelines and procedures has proved to be a difficult and onerous task, even for well-
established toxicological laboratories. The attempt to set some of the newly-developed short-term tests into a similar legislative framework revealed many uncertainties, which it now appears can only be resolved by international collaborative efforts on a scale that has few precedents.
In the fields of genetics and molecular biology, from which the science of mutagenesis has evolved, and which now have assumed great importance for the understanding of carcinogenesis,
scientists have shown great interest and ingenuity in adapting the particular biological systems they use for their research studies to assay systems of possible general use. Unfortunately, what can be a powerful and flexible tool in the hands of an experienced research worker cannot easily be transformed into the somewhat inflexible procedure that is required for a test system for routine use throughout the world. Studies to evaluate the efficiency of different short-term tests, which were started in a number of countries more or less in parallel with the development of the systems, were mostly concerned with the validity of the test procedure. By the time legislative measures were being consolidated in many countries, notably the Toxic Substances Control Act, 1976, in the USA, legislators were faced with a
plethora of some 40 short-term tests, all claiming some promise for revealing mutagenic or carcinogenic potential. Of considerable concern to industry, at this time, was the desire of legislators to have clear-cut criteria for yes/no decisions, even in a rapidly- evolving subject such as carcinogenesis. If the Delaney clause (United States Public Law, 1958), which states, in part, "that no additive shall be deemed to be safe if it is found to induce cancer when ingested by man or animal, or if it is found after tests which are appropriate for evaluation of safety of food additives to
induce cancer in man or animals", were strictly applied to the wider field of chemicals, the consequences for a society so dependent on chemicals could be most serious.
It is understandable that, against this background, there was considerable incentive and support for cooperative efforts to resolve these important issues. The project, which is the subject of this report, follows on from an earlier international study that was based on the realization that, as a first step, the
effectiveness of any short-term test in discriminating between carcinogens and noncarcinogens had to be established using, as reference, chemicals for which extensive rodent bioassay results were available.
The original project, called the International Programme for the Evaluation of Short-Term Tests for Carcinogenicity (IPESTTC) was carried out between 1977 and 1979 and 42 coded chemicals were tested by over 60 scientists using some 30 assay systems. The project arose as a result of initiatives by the Health and Safety Executive and the Medical Research Council of the United Kingdom
and the National Institute of Environmental Health Sciences of the USA. At the time of the planning of the IPESTTC, there was general agreement concerning the value of the Ames test, which by this time had a generally-accepted protocol. However, it had also become apparent that certain carcinogens or classes of carcinogens failed to mutate Salmonella; hence, there was a need to identify other test systems that could complement the Ames assay. Thus, there were 3 basic objectives in the programme. The first was to obtain more systematic knowledge concerning the carcinogens that the Ames test failed to detect. The second was to examine the ability of selected test systems to discriminate between carcinogens and noncarcinogens, and the third was to define assays that
complemented the bacterial mutation assays. To this end, 14
carcinogen/noncarcinogen pairs, together with 11 other carcinogens and 3 other chemically-unrelated noncarcinogens, were specially prepared with defined purity and distributed "blind" to the investigators.
The principal results of the IPSSTTC, which have been published in full (de Serres & Ashby, 1981), clearly confirmed the value of the Salmonella assay as a suitable primary test for the detection of potential mutagens and carcinogens. However, it was also
confirmed that some known rodent carcinogens were either not detected, or only detected with difficulty, by this assay. The study did not succeed in arriving at clear-cut conclusions
concerning a single complementary eukaryotic assay that was capable of giving a positive response for the carcinogens found negative in the standard Salmonella assay. Several assays that might serve in this capacity were identified, but none was recommended for general adoption, because it was considered that the supporting data base was too small. An important practical aspect of the IPESTTC came about through the meetings of investigators, where each assay group discussed their results with immediate access to the raw data.
These discussions resolved discrepancies in the findings and produced not only consensus views on the findings, but also
extremely valuable indications of protocol deficiencies, even for the well-established Salmonella assay.
2. THE COLLABORATIVE STUDY ON SHORT-TERM TESTS (CSSTT) 1981-83 The IPESTTC was remarkably successful in attracting the
voluntary participation of a large number of scientists, together with additional support from scientific institutions. The
conclusions from this study indicated clearly that priority should be given to the identification of assay systems to complement the Ames test. By 1981, the validity and usefulness of the Ames test had been well substantiated, but it had also been established that a number of important carcinogens were not detected, or detected only with difficulty using this assay. It was, thus, generally accepted that no single assay system could be relied on to detect all carcinogens. This led to the proposal of the adoption of testing schemes involving multiple in vitro assays in various configurations such as batteries, tiers, or combinations of the two. The basis for the selection and deployment of these multiple tests was, and remains, theoretical prudence rather than empirical evidence. That is, a variety of genetic end-points and organisms representing different phylogenetic levels were selected with the intent of not missing end-points or phylum-specific chemical activity. Furthermore, in general, reliance was placed on the deployment of genotoxicity assays, even though, by this time, other factors were assuming importance in the biological etiology of natural and chemically-induced cancer. Thus, the molecular targets for investigation were no longer dominated by observations of
readily discernible changes in the sequence or integrity of nuclear DNA, but involved consideration of subtle changes in chromosome, gene, or oncogene function or expression (Klein, 1981; Reddy et al., 1982; Tabin et al., 1982; Weiss, 1982). This implies that some of the genetic end-points monitored in assay systems may ultimately be shown not to be directly related to the critical events in the etiology of some chemically-induced cancers (Cairns, 1981). Amidst these scientific controversies about the reliability and biological significance of many short-term tests, registration and health authorities were endeavouring to assess the genotoxic data from the same short-term tests without adequate scientific guidance for the interpretation of the all too frequently
discordant data.
It was against this background that the Collaborative Study on Short-Term Tests for Genotoxicity and Carcinogenicity (CSSTT) was proposed by the International Programme on Chemical Safety (IPCS) and the National Institute of Environmental Health Sciences of the USA, as a Participating Institution in the programme. The general goals and designs of the study were outlined by an ad hoc Working Groupa, which met at the invitation of the IPCS in Geneva, on 30 April - 1 May 1981. The plans were consolidated by an IPCS Working Groupb, which met in Geneva, on 13 - 14 November 1981. The ---
a Participants: Dr J. Ashby, Professor N.P. Bochkov, Dr B.E. Matter, Professor T Matsushima, Dr F.J. de Serres, Dr M. Shelby, and Professor F.H. Sobels.
b Participants: Dr J. Ashby, Dr G.R. Douglas, Dr M. Ishidate, Dr A. Leonard, Dr Loprieno, Dr B.E. Matter, Professor T. Matsushima, Dr R. Montesano, Dr F.J. de Dr M. Shelby, Professor F.H. Sobels, Dr M. Stoltz and Dr M. Waters.
subsequent coordination of the collaborative study was the
responsibility of a Steering Committee derived primarily from the Working Group (J. Ashby; F. de Serres, Chairman; M. Ishidate Jr;
B. Margolin; B. Matter; M. Shelby; and M.H. Draper, IPCS).
The financial burden of the organization of this study was met largely by the IPCS, together with some of its Participating
Institutions, particularly the National Institute of Environmental Health Sciences in the USA. As in the IPESTTC project (de Serres &
Ashby, 1981), the funding of the assay work was provided, in the majority of cases, by the individual investigators managing to incorporate the work into their research programmes. This could only occur with the goodwill and belief in the project of the senior managements of the approximately 50 involved laboratories from universities, research institutes, and industrial research facilities, throughout the world. In addition, a number of
governments that support the IPCS provided financial assistance for this study. These include the governments of Belgium, Italy, the Netherlands, and, in particular, the United Kingdom.
The experience gained from the conduct of the IPESTTC and the goodwill of the participants in that study were extensively drawn on in the planning and organization of the CSSTT. The major objective was defined as the generation of a wide range of test results for a small group of carefully-selected rodent carcinogens that would contribute to an empirical basis for selecting one or more in vitro short-term tests as complementary to the Ames test.
The number of chemicals was kept to a minimum, because of the experience of handling the 42 chemicals in the IPESSTC study. It was argued that it was better to aim for an extensive data base for a few chemicals than a reduced and patchy data base for a large number. The chemicals were selected with much care, and those chosen were all known to be particularly difficult to detect in assay systems and, thus, would be expected to expose weaknesses and
inconsistencies in both the assay system and the protocols. This indeed proved to be the case.
Some 60 investigators participating in the project carried out nearly 90 individual sets of assays, generating, in all, some 2500 dose-response relationships. Most of the in vitro eukaryotic tests, currently available, were represented. The 8 organic carcinogens chosen as either inactive, or difficult to detect as positive in the Salmonella assay, were: o-toluidine,
hexamethylphosphoramide, safrole, acrylonitrile, benzene, diethylhexylphthalate, phenobarbital, and diethylstilboestrol, together with 2 chemicals, caprolactam (Huff, 1982) and benzoin (NTP, 1980), which had not shown any evidence of carcinogenicity in 2-year rodent bioassays. The criteria for the selection of these chemicals is an important matter and the reasons for each inclusion are given in the following section.
3. CRITERIA FOR THE SELECTION OF THE TEST CHEMICALS
Eleven carcinogens were defined as either difficult or
impossible to detect as bacterial mutagens in the IPESTTC study, and 4 of these were selected for the CSSTT. These were
hexamethylphosphoramide, safrole, diethylstilboestrol, and
o-toluidine. The carcinogenicity of these agents for rodents is well-established but only a small proportion of the Salmonella assays conducted in the IPESTTC study detected them as mutagenic (1/15, 4/17, 1/17, and 3/16, respectively). However, several of these responses were not reproducible, and each was weak. This led to the compounds being regarded as essentially non-mutagenic for Salmonella in this study.
Hexamethylphosphoramide (HMPA)
Hexamethylphosphoramide (HMPA) is among the most potent of animal carcinogens producing metastasizing nasal tumours in rats exposed by inhalation.
There is a wealth of data indicating it to be non-mutagenic for Salmonella, yet the results of the IPESTTC study suggested that it was a general genotoxin in eukaryotic assays. One possible mode of action for this agent is via the enzyme-mediated formation of
formaldehyde (Ashby & Lefevre, 1983). This is also a rat nasal carcinogen that is difficult to detect as mutagenic in the Salmonella assay but is a gene mutagen in human cells (Ashby &
Lefevre, 1983; Goldmacher & Thilly, 1983).
o-Toluidine
o-Toluidine is a relatively weak rodent hepatocarcinogen. Its activity in this respect is interesting because it weakens the earlier assumption that single ring aromatic amines, as opposed to multiple ring arylamines such as 2-naphthylamine and 4-
aminobiphenyl, are non-carcinogenic. o-Toluidine was established as difficult or impossible to detect in the Salmonella assay in the IPESTTC study, which also showed that it was mutagenic to these bacteria, if evaluated in the presence of norharman. These
collected findings suggested o-toluidine to be a general
genotoxin, which requires specific metabolic activation, rather than an agent showing specificity of genetic action.
Safrole
Safrole is a weak rodent liver carcinogen and has been studied extensively in the Salmonella assay. Although certain
investigators have reported it to be mutagenic, it is generally found inactive in this assay. Both the alpha-acetoxy and the sidechain epoxide derivatives are mutagenic, and these have been suggested as the metabolites responsible for the carcinogenic
action observed. Safrole may, therefore, be a further example of a general genotoxin that requires specific metabolic activation. Set against this is the fact that it appears devoid of genetic activity in vivo; thus, it gave a negative response in both the mouse bone- marrow micronucleus assay and the in vivo rat liver unscheduled DNA synthesis (UDS) assay. Consequently, the possibility cannot be excluded that the tumours produced by this agent may be mediated via some disturbance of normal homeostasis in the test animals (i.e., by a non-genotoxic mechanism), despite its ability to induce genetic changes in some in vitro test systems.
Diethylstilboestrol (DES)
Diethylstilboestrol (DES) is carcinogenic in both human beings and experimental animals. It could have been selected for this study simply on the basis of a recent paper that showed it to be capable of transforming cells and inducing chromosomal damage in the apparent absence of gene mutations (Barrett et al., 1981).
This finding was supported by the fact that, in the IPESTTC study, DES was regarded by the investigators as a clastogen that was non- mutagenic for Salmonella. Therefore, DES, together with benzene (see below), were included in the study as agents that could possibly demonstrate the reality of the genetic specificity of action of some chemical carcinogens.
Benzene
Benzene is a unique carcinogen. Its possible leukaemogenic activity in man has been discussed for many years, yet this effect has been difficult to reproduce in animals. The compound is
nonetheless generally regarded as carcinogenic and extensive data exist on its clastogenicity, particularly when evaluated in vivo.
Dean (1978) reviewed the literature on the genotoxicity of this agent in short-term tests, and this, together with subsequent studies, clearly defined it as non-mutagenic for bacteria. The possibility of its complete inability to induce gene mutations in vitro is implied in some papers, but its gene mutagenicity in vivo has not yet been assessed.
Acrylonitrile
Similarities in structure between acrylonitrile and the
carcinogen vinyl chloride led Venitt to evaluate it for bacterial mutagenicity. The debate that ensued in Mutation Research (Milvy &
Wolff, 1977; Venitt et al., 1977) regarding the mutagenic activity of this agent in Salmonella and Escherichia coli can be summarized by describing acrylonitrile as a chemical that could easily be
found non-mutagenic in a routine screening programme that employed only bacteria as marker cells. The carcinogenicity of this agent has been subsequently defined and reviewed. The question of whether acrylonitrile interacts directly with DNA via a Michael reaction, or via the intermediate metabolic formation of an epoxide derivative, heightens interest in this agent.
Diethylhexylphthalate (DEHP)
Diethylhexylphthalate (DEHP) has been shown to produce hepatomas in the rodent liver, yet the majority of experimental data indicate it to be non-mutagenic for bacteria. It has been
proposed that the carcinogenicity of this agent is associated with its ability to proliferate peroxisome microbodies in the rodent liver (Moody & Reddy, 1978). This explanation would not require DEHP itself to interact with nuclear DNA. The carcinogenicity of DEHP has, therefore, been considered as possibly "epigenetic" in origin, which increases the need to determine accurately its
genotoxic status in vitro. The extent to which DEHP is hydrolysed to the corresponding mono-acid derivative (MEHP) could influence the outcome of certain assays as the latter chemical, unlike the former, is reported to be a clastogen and SCE-inducing agent in vitro (Phillips et al., 1982; Tomita et al., 1982; Ashby, 1983).
Phenobarbital
Phenobarbital, although active as a rodent liver carcinogen, also has significant tumour-promoting properties in the rodent liver. In fact, the issue of whether phenobarbital is a pure promoting agent devoid of cancer-initiating activity is of great current interest. In contrast to DES, the rodent carcinogenicity of phenobarbital appears not to be reflected in man, despite the extensive and controlled exposure of epileptic patients (Clemmesen
& Hjalgrim-Jensen, 1977). Although this chemical is generally regarded as non-genotoxic, limited evidence exists for its ability to induce SCEs in vitro (Athanasiou & Kryrtopoulos, 1982; Ashby, 1983). This property may be related to its ionic composition (cf.
sodium saccharin, MEHP above, lacchaic acid, sodium benzoate, etc., for similar activity profiles) (Ashby, 1983). An additional point of interest in this chemical is that Williams has presented data to support the claim that phenobarbital is an example of an epigenetic carcinogen (Williams, 1981).
The non-carcinogens caprolactam and benzoin
The selection of non-carcinogens suitable for use in the evaluation of short-term tests has presented a stumbling block in all validation exercises. In the early validation studies, non- carcinogens were simply selected from compounds commonly regarded as being non-carcinogenic. In some cases, no data existed
regarding their carcinogenicity, and this was taken as indicative of inactivity. In the IPESTTC study, the non-carcinogens selected were graded according to the extent and quality of the negative data, and although this was an advance, the interpretation of unexpected positive assay responses was difficult. This issue is particularly important in relation to the widespread reference to false-positive responses occurring in short-term tests; the
credibility that can be accorded to a false-positive response is directly proportional to the certainty associated with the
compound's classification as a non-carcinogen. The fact that some assumed non-carcinogens may eventually be classified as either weak or organ-, strain-, sex-, or species-specific carcinogens might lead to the re-evaluation of many previous examples of false- positive assay responses.
In order to circumvent this problem in the current
collaborative study, particular attention was paid to the selection of the 2 chemicals required to act as negative controls. The
agents selected were benzoin and caprolactam. The major criterion for their selection was inactivity in recent cancer bioassays conducted as part of the US National Toxicology Program. In the reports of these studies (US NTP, 1980, 1982), it was concluded that neither compound was carcinogenic in male or female Fischer 344 rats or B6C3Fl mice dosed at levels up to the maximum tolerated dose over their lifetimes. These 2 studies were taken as
definitive as they represented the most detailed cancer bioassay protocols currently in use. In addition, these agents were devoid of overtly DNA-reactive substituents and were known to be non- mutagenic for bacteria.
The 10 chemicals selected covered a wide range of structural types and could, therefore, be considered representative of agents encountered in the environment and chemical industries. In
addition, several of the carcinogens selected had been associated, by other investigators, with possible mechanisms of cancer
induction other than the DNA-reaction/somatic mutation theory.
Finally, the 2 non-carcinogens were sufficiently well supported by negative carcinogenicity data to ensure that clear decisions could be made, regarding the significance of their genotoxic activity, in vitro.
4. PURITY OF THE TEST CHEMICALS
As analytical techniques improve, it is possible to find trace impurities in materials, formerly considered pure. Set against this is the practical need to obtain large supplies of pure chemicals for a study, such as the present collaborative study, without inordinate costs and delays. This dilemma is heightened by the history of the conduct of cancer bioassays where the test
chemical was often, if not usually, assumed to be pure in the absence of appropriate analytical data. Many chemicals bioassayed for carcinogenicity have been of technical quality and, therefore, probably not more than 95% pure. Normally, this would not matter, but when the cancer bioassay data are to be the ultimate reference point, as in the present study, then the relative purity of the in vitro test chemical becomes of importance. A 5% impurity, at high doses, could be of biological significance. At one extreme, it can be argued that material of similar purity (or impurity) to that employed in the cancer bioassay should be assayed, but this may lead to a further confounding of the total data base. At the other extreme, it can be suggested that only absolutely pure materials should be employed in vitro, whatever the cost and inconvenience incurred in their preparation. This approach carries the penalty that the carcinogenic response observed in mammals may have been produced by impurities, in which case, activities observed in vitro may not be correlated with carcinogenic activity (or inactivity).
This consideration is particularly relevant for benzene. The most convincing carcinogenicity data for benzene were derived from human beings exposed to it together with other chemicals, the number and type of which varied from situation to situation. The fact that the carcinogenicity of this chemical is difficult to define in rodents has led to the suspicion that it may not be benzene, but the chemicals used in association with it, that are carcinogenic. Pure benzene was used in this study; a risk was taken by doing so.
The purity criteria adopted for the present study entailed the following assays of chemical purity:
a) One batch of each chemical of the highest grade commercial samples available, usually 99% or more pure, was obtained.
b) The proton nuclear magnetic resonance spectrum, mass
spectrum, and infrared spectrum were determined and checked for consistency with the proposed structure and for the possible presence of impurities.
c) The elemental analysis (C, H, and N) was determined for
both liquids and solids; each was within 0.4% of the theoretical value.
d) The melting point was determined and compared with previously reported values for all solids. Because of differences in thermometer calibrations, variations of less than 4 °C were hard to interpret.
e) In 2 cases (safrole and o-toluidine), high-pressure liquid chromatography (HPLC) was employed to evaluate trace
impurities seen by earlier assay methods.
f) Thin-layer chromatography (TLC) was undertaken on each material, as appropriate. A variety of eluants and detection systems was employed.
On the basis of the above determinations, the present chemicals were deemed to be pure to a level of 99%. These techniques cannot eliminate the chance that some activities observed for some of the agents (both carcinogens and non-carcinogens) were due to
impurities. This admission is necessary, but is not exceptional, given the paucity of analytical data usual in such studies,
including the reference cancer bioassays. Nonetheless, trace impurities may have contributed to some activities, the weak gene mutagenicity of phenobarbital in Salmonella being an example of where further purification and reassaying in vitro might yield useful additional data. Genotoxic impurities should not, however, be too easily invoked to explain unexpected genotoxic responses.
First, similar concerns should apply to positive responses observed in vitro for mammalian carcinogens, and second, such uncertainties reflect equally on previous studies, the findings of which
constitute most of the established data base of this science. The chemicals, made up in 5-g lots, were labelled and distributed to the investigators in specially-sealed double containers. As these chemicals were carcinogens and for various other reasons, they were not distributed "blind".
5. CRITERIA FOR THE DEFINITION OF COMPLEMENTARY IN VITRO ASSAYS FOR THE DETECTION OF POTENTIAL CARCINOGENS
In order to qualify as a complementary assay for routine use in conjunction with the Salmonella plate-incorporation assay, a test must have fulfilled the following requirements (Ashby et al., 1983):
(a) It should have been successfully employed as a short-term test in a number of laboratories, and should be
substantially represented in the literature.
(b) It should have performed well in the detection of the present 8 carcinogens, while concomitantly finding both of the non-carcinogens negative.
(c) Positive responses obtained with the 8 carcinogens tested should have been unambiguous, dose-related, and
reproducible.
(d) Similar qualitative responses should have been observed by the majority of the laboratories using the same assay.
(e) It should be appropriate for routine screening purposes, i.e., not unduly demanding as far as resources and
technical facilities are concerned.
Four categories of assay may thus be defined:
(1) Assays suitable for general use in conjunction with the Salmonella assay;
(2) Promising assays, i.e., assays that may be capable of fulfilling criteria (a)-(e), but for which data are not available for all of the test chemicals, or where repeat studies are not available;
(3) Relatively new assays that, while not meeting criterion (a), have performed well in the collaborative study, and for which the present 10 chemicals form the greater part of the available data base; these cases would best be handled by the rapid and coordinated acquisition of further information.
(4) Assays that are clearly inappropriate for routine use in testing for potential carcinogens, i.e., that do not meet criteria (b)-(e).
6. ASSAYS AND END-POINTS
As discussed above, the design of the present collaborative study reflected the primary purpose of attempting to identify in vitro eukaryotic assays, which are capable of detecting chemical carcinogens, not readily detectable using bacterial assays. At the organism level, 4 categories of assays were employed: bacteria, yeast, fruit flies, and cultured mammalian cells. Within each of these groups of organisms, a variety of test end-points were used.
Organisms and end-points will be described briefly and are
presented in Table 1. Full details are available in the published assay working group reports and the reports of individual
investigators (Ashby et al., 1985).
Table 1. IPCS CSSTT test systems
--- I. Bacteria
Salmonella typhimurium
TA97, TA98, TA100, TA102, HIS- HIS+ TA1535, TA1537, TA1538
TM677 AZAS AZAR II. Fungi
Mutation:
Saccharomyces cerevisiae
XV185-14C ARG- ARG+; TRP- TRP+ HIS- HIS+; HOM- HOM+ RM52 HIS- HIS+
D7 ILV- ILV+
D6 and D61-M ADE- ADE+ ILV- ILV+
D5 small colonies due to mitochondrial mutations
Schizosaccharomyces pombe
P1 red white colonies (ADE) Aspergillus nidulans
35 methionine metabolism mutants
--- Table 1. (contd.)
--- II. Fungi (contd.)
Recombination:
Saccaromyces cerevisiae
JD1 gene conversion, tryptophan or histidine prototrophy
D7 and D7-144 crossing-over, red and pink colonies (ADE) gene conversion, tryptophan prototrophy
PV-2 and PV-3 crossing-over, canavanine
resistance gene conversion, lysine protrophy
D6 and D61-M crossing-over, cycloheximide resistance
Aspergillus nidulans
P1 crossing over, green yellow colonies
Aneuploidy:
Saccharomyces cerevisiae
D6 and D61-M red, cycloheximide sensitive white cycloheximide resist
Aspergillus nidulans
P1 yellow sectors in green colonies Illegitimate mating:
Saccharomyces cerevisiae
PV-4a and PV-4b mating type a III. Drosophila
Somatic cell mutations
wing-mosaicism wing spots from mutations, deletions, chromosome breakage, mitotic recombination or aneuploidy white-zeste eye eye spots from mutations or
mosaicism deletions
---
Table 1. (contd.)
--- III. Drosophila (contd.)
white/white coral eye spots (same events as wing eye mosaicism spots above)
IV. Cultured mammalian cells Metabolic cooperation
V79 survival of HGPRT- cells Transformation
SHE colony assay C3H10T1/2 focus assay BALB/c 3T3 focus assay
SHE/SA7 viral enhancement of chemical transformation-focus assay RLV/FRE enhanced survival of Rauscher leukaemia
virus-infected rat embryo cells CHO invasive growth in agar
DNA damage
single-strand breaks
CHO alkaline sucrose sedimentation rat hepatocytes alkaline elution
Unscheduled DNA synthesis
HeLa S3 scintillation counting-extraced DNA
rat hepatocytes scintillation counting -DNA extracted from isolated nuclei rat hepatocytes autoradiography
Cytogenetic damage
chromosomal aberrations
CHO structural aberrations; micronuclei Chinese hamster lung, structural aberrations; polyploidy CHL
Chinese hamster, structural aberrations; polyploidy liver,CHl-L aneuploidy
rat liver, RL4 structural aberrations; polyploidy human lymphocytes structural aberrations
--- Table 1. (contd.)
--- Sister chromatid exchange
CHO V79
rat liver, RL4 Gene mutations
L5178Y TK+/- TK-/- OUAS OUAR V79 HGPRT+ HGPRT- V79 OUAS OUAR CHO HGPRT+ HGPRT- Human lymphoblasts TK6 TK+/- TK-/- AHH HGPRT+ HGPRT-
--- 6.1. Bacteria
The carcinogens included in the collaborative study were selected on the basis of previously-published results indicating their lack of activity in routinely-conducted Salmonella
mutagenicity tests. Five sets of Salmonella data were obtained in this study to confirm the previous results and to provide bacterial mutagenicity data on the batches of chemicals used in the current study. Test data are reported for Salmonella typhimurium strains TA97, TA98, TA100, TA102, and TA1535 in both pre-incubation and plate incorporation protocols and TA1537 and TA1538 in the plate assay only. S. typhimurium strain TM677 was used to detect azaguanine resistant forward mutants, employing a treat and plate method.
6.2. Fungi
Fungal systems, which offer the advantages of being both microbial and eukaryotic, were used to evaluate a wide range of genetic end-points. Test results from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Aspergillus nidulans are reported in relation to the following genetic end-points: nuclear gene
mutation (both forward and reverse), mitochondrial mutation, gene conversion, mitotic crossing over, and aneuploidy.
6.3. Drosophila
Three separate laboratories reported test results from 3 newly- developed assays for detecting genetic damage induced in somatic cells of Drosophila. The white-zeste eye mosaicism test detects eye spots resulting from mutations or deletions, while the wing mosaicism and white/white coral eye mosaicism tests detect wing or eye spots resulting from mutations, deletions, chromosome breakage, mitotic recombination, or aneuploidy.
6.4. Cultured Mammalian Cells
Five major categories of chemically-induced effects were reported for cultured mammalian cells: inhibition of metabolic cooperation, transformation, DNA damage, cytogenetic effects, and gene mutations.
Test data on the inhibition of metabolic cooperation, an assay intended to detect promoting agents, as evidenced by increased survival of HGPRT- V79 cells in the presence of an excess of HGPRT+ cells and 8-azaguanine or 6-thioguanine, were reported by 3
laboratories.
Six distinct transformation assays were reported including those from 2 laboratories using Syrian hamster embryo (SHE) cells, 2 using C3H10T1/2 mouse cells, and single laboratories using the Syrian hamster embryo/Simian adenovirus-7 and Rauscher leukaemia virus-infected rat embryo cell assays. In addition, data for 5 compounds derived from a new assay in which the end-point was invasive growth of CHO cells in soft agar were considered. Two investigators, who had offered to generate data using the BHK21 transformation assay, withdrew from the study because of lack of adequate time. This was disappointing as they had presented the prospect of a link with the IPESTTC study in which the BHK21 assay represented the sole transformation end-point.
The chemical induction of DNA single-strand breaks was
determined by assessing single-strand breaks using alkaline elution or alkaline sucrose sedimentation. Tests for unscheduled DNA
synthesis were reported using protocols involving both scintillation counting and autoradiography.
A large body of test data was reported for the 2 most commonly used cytogenetic end-points, structural aberrations, and sister chromatid exchanges. In addition, limited results were reported for the induction of micronuclei, aneuploidy, and polyploidy.
Gene-mutation induction data were reported for 3 loci:
thymidine kinase (TK), hypoxanthine guanine phosphoribosyl transferase (HGPRT), and NA+, K+ ATPase (Ouabain resistance) in mouse, Chinese hamster, or human cells. These studies included 7 sets of test results from the L5178Y TK+/- system.
7. RESULTS
The investigators met at St. Simon's Island, Georgia, USA from 23 - 28 October 1983. During this meeting, each group of assay participants, with the assay coordinator as chairman, discussed the results with the raw data in front of them and individual results were agreed. The group then formulated a consensus report on the response of each chemical in the assay. These decisions were incorporated into the coordinators' report, prepared during group discussions on the overall performance of the assay and any defects discovered. The coordinator's reports were presented and discussed at plenary sessions, during which the conclusions and
recommendations of the study were developed. The coordinators subsequently finalized their reports after further consultations with the members of the group. The reports have been incorporated into the text of the publication, which includes all the individual reports of results as well as an editorial overview of the study and a number of technical appendices (Ashby et al., 1985).
Table 2 includes, in summary form, all the agreed results for each chemical in each test system in the study, as established in the assay group discussions at St. Simon's Island. In order to make some attempt at an overall assessment of assay performances, the editorial group proceeded along the following lines. First, the qualitative responses displayed in Table 2 were assumed to be correct. Some of these results were unconfirmed and may, therefore, represent false-positive or false-negative observations. Second, it was decided that an assay should be capable of detecting at least 2 of the selected carcinogenic test agents as positive, before it could be assessed for possible use as a complementary test. The extent to which inadequacies of individual test
protocols, as opposed to the insensitivity of the particular assay
or its genetic end-point, were responsible for negative responses could only be discussed in cases where the same assay had been conducted in 2 or more laboratories. Third, statistical
comparisons of the overall performance of assays were not
undertaken because, with the present rather unusual set of test chemicals, this could yield meaningless if not misleading
conclusions, unless undertaken in depth. The entire data base was entered into a computer file at NIEHS and detailed statistical analyses may be undertaken, as appropriate.
In developing the discussion, it was further accepted that in vitro assays are, by their constitution, only appropriate for the identification of potential carcinogens; that is, in vitro tests can be used to predict possible carcinogens, but not to define
them; at present, this can only be attempted by in vivo techniques.
It is, therefore, to be expected that certain agents will show activity in vitro, but will be unable to express this potential in vivo, because of their non-absorption, rapid excretion,
preferential detoxification, inappropriate partitioning, etc., in mammals. In vitro assays cannot, and should not, be expected to reveal these possibilities, which are, by definition, unique to living animals. Activity seen in vitro for the present 2 non- carcinogens was not used when assessing the overall performance of the assays in question (Table 3), but rather, was used to emphasize the true role and generic predictive weaknesses of in vitro assays.
On occasions, the assimilation of the large data base was made easier by considering 4 carcinogens selected for the study as a group (HMPA, o-toluidine, safrole, and acrylonitrile). This was because it was known, at the outset, that they were more likely to be detected by most assays, as each had already been established as being genotoxic, though they were usually inactive in the
Salmonella mutation assay. The remaining carcinogens, with the possible exception of benzene, were loosely regarded as non-
genotoxic, prior to this study. The collaborative study data base generally supported the segregation of these 2 groups of
carcinogens and, thus, enabled a selective assessment of each assay to be made. Some assays performed well with the first 4
carcinogens but poorly with the others, and some were insensitive to this division and performed either generally well or poorly. A possible further subdivision of the second 4 carcinogens became evident as the review progressed and this will be discussed in the section dealing in more detail with the assays.
Table 2. IPCS CSSTT in vitro study: summary of qualitative resultsa,b
--- ASSAY ACN TOL HMPA SAF DES BEN PB DEHP ZOIN CAP --- 1 BACTERIA
1.1.1 Salmonella ? N N N N N P N N N 1.1.2 Salmonella P N N N N N N N N N 1.1.3 Salmonella N N N N N N N N N N 1.1.4 Salmonella P P N N N N P N P N 1.1.5 Salmonella N N N N N N P N N N 2 FUNGI
2.1 Mutation
2.1.1 D7 N N P N N N N N N N 2.1.2 Asper 35 N N N N
2.1.3 D7 N N N N N P N N N N 2.1.4 XV185 P P P P P P P P P 2.1.5 XV185 N P P P P 2.1.6 P1 N N N P N N N N N N 2.1.7 D6 P N P N N N N N N N 2.1.8 D61-M P P P P N N N N N N
2.1.9 Mito. D5 P P N P N P N N N N 2.2 Gene conversion
2.2.1 D7 P N P N N N N P N N 2.2.2 D7 P N N P N N N N N N 2.2.3 D7-144 P P P P P P P P P 2.2.4 PV-3 N N N N N N N N N N 2.2.5 PV-2 N N N N N N N N N N 2.2.6 JD-1 P N N N N N N N N N 2.2.7 D7 P N P N N N N P N N 2.3 Crossing-over
2.3.1 D7 N N N N N N N N N N 2.3.2 Asper. 35 N N N N N N N N N N 2.3.3 D6 P N N N N P N N N --- Table 2 (contd.)
--- ASSAY ACN TOL HMPA SAF DES BEN PB DEHP ZOIN CAP --- 2.3.4 D61-M P N N N N N N N N N 2.3.5 D61-M P N N N N N N N N N 2.3.6 D7 N N N P N N N P P N 2.4 Aneuploidy
2.4.1 D6 P P P P P P P P N N 2.4.2 D61-M N N N N P P N N N ? 2.4.3 D61-M P P P P P P P P N N 2.4.4 Asper. 35 P N N P N N N N N N 3. DROSOPHILA SOMATIC CELLS
3.1.1 Wing spots P P P P N P N N N P 3.1.2 Eye spots P N P N N N N ? N P 3.1.3 Eye spots P P P P ? ? N ? N P 4. CULTURED MAMMALIAN CELLS (endpoints other than gene mutation)
4.1 Metabolic cooperation
4.1.1 V79 P P N ? N N ? P ? N 4.1.2 V79 P N N N N 4.1.3 V79 P N N ? N N N N N N 4.2 Transformation
4.2.1 BALB/C N N N N N N N N N N 4.2.2 C3H P P P P P ? ? P P P 4.2.3 C3H P ? p ? N 4.2.4 SHE P P P ? N P P P N ? 4.2.5 SHE P P P P P P N P N P 4.2.6 SHE/SA7 P ? N ? N ? N 4.2.7 Rl-FRE ? P N P P N N 4.2.8 CHO N ? N N N 4.3 Single-strand breaks
4.3.1 Rat Hepat. P P N P P N N N P N 4.3.2 CHO N P N P P P N N N 4.3.3 CHO P P N N P N N N N N 4.4 Unscheduled DNA synthesis (UDS)
4.4.1 Rat Hepat. (autorad) N N P N N N N N N N 4.4.2 Rat Hepat. (autorad) N N N N N N N N N N 4.4.3 Rat Hepat. (scint.) P P P P N P N P P N 4.4.4 HeLa (scint.) N P P P N N N N N N 4.4.5 HeLa (scint.) P ? P N N 4.5 Chromosomal aberrations
4.5.1 CHO P P N N P N P N N N 4.5.2 CHO N N P P N 4.5.3 CHO P N N P N 4.5.4 LYM P P P N P 4.5.5 CH1-L P P P N P N P N N N 4.5.6 CHL P P P P N P ? N P P
