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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY


    ENVIRONMENTAL HEALTH CRITERIA 109






    SUMMARY REPORT ON THE EVALUATION OF
    SHORT-TERM TESTS FOR CARCINOGENS 
    (COLLABORATIVE STUDY ON  IN VIVO 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, 1990


         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.

    WHO Library Cataloguing in Publication Data

    Summary report on the evaluation of short-term tests for
    carcinogens : (collaborative study on  in vivo  tests).

        (Environmental health criteria ; 109)

        1.Carcinogens - analysis 2.Mutagens - analysis 
        3.Mutagenicity tests 4. Evaluation studies I.Series

        ISBN 92 4 157109 8        (NLM Classification: QZ 202)
        ISSN 0250-863X

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CONTENTS

    SYNOPSIS        

1. INTRODUCTION 

2. THE COLLABORATIVE STUDY ON SHORT-TERM  IN VIVO  TESTS FOR MUTAGENS AND 
    CARCINOGENS (CSSTT/2) 1983-85  

3. OVERALL AIMS OF THE STUDY AND CRITERIA FOR THE SELECTION OF AN 
    APPROPRIATE SHORT-TERM  IN VIVO TEST   

    3.1. The use of short-term tests for the primary identification of 
         genotoxic chemicals  
    3.2. The use of short term  in vivo assays for assessing the hazard
         associated with exposure to  in vitro genotoxins    
    3.3. The role of short-term  in vitro tests in research into the
         mechanisms of cancer
    3.4. Assays for the detection of germ cell mutagens 

4. CRITERIA FOR THE SELECTION OF THE FOUR TEST CHEMICALS   

    4.1. Activity of the four test chemicals in short-term  in vitro tests
    4.2. Summary of carcinogenicity data on the test chemicals  

5. SOURCE AND PURITY OF THE TEST CHEMICALS 

6. SHORT-TERM  IN VIVO ASSAYS   

    6.1. Cytogenetic assays 
    6.2. Assays in rodent liver cells   
    6.3. Miscellaneous assays   
    6.4. The mouse spot test    
    6.5. Mammalian germ cell studies    
    6.6. Drosophila assays  

7. RESULTS

    7.1. Benzo [a] pyrene and pyrene  
         7.1.1. Cytogenetic studies    
         7.1.2. Liver-specific assays  
         7.1.3. Miscellaneous assays   
         7.1.4. Mouse spot tests   
         7.1.5. Mammalian germ cell assays 
         7.1.6. Drosophila assays  
    7.2. 2-Acetylaminofluorene and 4-acetylaminofluorene    
         7.2.1. Cytogenetic studies    
         7.2.2. Liver-specific assays  
         7.2.3. Miscellaneous assays   
         7.2.4. Mouse spot tests   
         7.2.5. Mammalian germ cell assays 
         7.2.6. Drosophila assays
    7.3. Summary of the  in vivo genotoxicity of the four chemicals

8. ASSESSMENT OF THE PERFORMANCE OF THE ASSAYS 

    8.1. Cytogenetic assays 
         8.1.1. Chromosome aberrations 
         8.1.2. Micronuclei    
         8.1.3. Sister chromatid exchange  
    8.2. Liver assays   
         8.2.1. Initiation and promotion   
         8.2.2. Unscheduled DNA synthesis and S-phase analysis 
         8.2.3. DNA strand breaks  
         8.2.4. Cytogenetics   
    8.3. Miscellaneous assays   
         8.3.1. Specific carcinogenicity assays    
         8.3.2. Supplementary assays   
         8.3.3. Immunotoxicity assays  
         8.3.4. Host-mediated assays and urine mutagenicity tests  
    8.4. Mouse spot tests   
    8.5. Assays in mammalian germ cells 
         8.5.1. Dominant lethal and unscheduled DNA synthesis assay    
         8.5.2. Sperm abnormality tests    

    8.6. Drosophila assays

9. SELECTION OF THE MOST EFFECTIVE  IN VIVO ASSAYS IN RELATION TO 
    THEIR PERFORMANCE    

    9.1. Assays that are not considered appropriate for routine  in vivo 
         testing of chemicals for genotoxic activity    
    9.2. Assays that satisfy some or all of the criteria for an acceptable 
          in vivo short-term test     
         9.2.1. Assays currently in general use    
         9.2.2. Assays that show promise forfuture development 
    9.3. The detection of germ cell mutagens    
    9.4. Influence of route of administration of the test chemicals 

10. CONCLUSIONS  

REFERENCES          

ETUDE COLLECTIVE POUR L'EVALUATION ET LA VALIDATION DES EPREUVES DE COURTE 
DUREE RELATIVES AUX CANCEROGENES      

ESTUDIO EN COLABORACION SOBRE EVALUACION Y COMPROBACION DE PRUEBAS A CORTO 
PLAZO PARA SUSTANCIAS CARCINOGENAS 

PARTICIPANTS IN THE COLLABORATIVE STUDY

Dr I. Adler, Mammalian Genetics Institute, Association for
   Radiation and Environmental Research, Neuherberg, Federal
   Republic of Germany
Dr R. Albanese, Pharmaceuticals Division, Imperial Chemical
   Industries PLC, Macclesfield, Cheshire, England
Dr J.W. Allen, Genetic Toxicology Division, US Environmental
   Protection Agency, Research Triangle Park, North
   Carolina, USA
Dr J.A. Allen, Department of Mutagenesis and Cell Biology,
   Huntingdon Research Centre Ltd., Huntingdon, Cambridge-
   shire, England
Dr O. Andersen, Odense University, Institute of Community
   Health, Department of Environmental Medicine, Odense,
   Denmark
Dr D. Anderson, British Industrial Biological Research
   Association, Carshalton, Surrey, United Kingdom
Dr J. Arany, Institut d'Hygiène et d'Epidémiologie,
   Brussels, Belgium
Dr J. Ashby, Central Toxicology Laboratory, Imperial Chemi-
   cal Industries PLC, Macclesfield, Cheshire, United
   Kingdom
Dr R.A. Baan, Medical Biological Laboratory TNO, Rijswijk,
   Netherlands
Dr P. Bannasch, Cytopathology Department, Institute of
   Experimental Pathology, German Cancer Research Centre,
   Heidelberg, Federal Republic of Germany
Dr G.C. Becking, International Programme on Chemical Safety,
   World Health Organization, Research Triangle Park, North
   Carolina, USA
Dr B. Beije, Department of Genetic and Cellular Toxicology,
   Wallenberg Laboratory, Stockholm University, Stockholm,
   Sweden
Dr J. Benes, Institute of Nuclear Biology and Radiochemis-
   try, Prague, Czechoslovakia
Dr E. Bermudez, Department of Genetic Toxicology, Chemical
   Industry Institute of Toxicology, Research Triangle Park,
   North Carolina, USA
Dr H.C. Birnboim, Department of Experimental Oncology,
   Ottawa Regional Cancer Centre, Ottawa, Ontario, Canada
Dr J.B. Bishop, Cellular and Genetic Toxicology Branch,
   Toxicology Research and Testing Program, National Insti-
   tute of Environmental Health Sciences, Research Triangle
   Park, North Carolina, USA
Dr D.H. Blakey, Mutagenesis Section, Environmental Health
   Centre, Department of National Health and Welfare,
   Tunney's Pasture, Ottawa, Ontario, Canada
Dr R. Braum, Central Institute for Genetics and for Research
   on Cultivated Plants, Academy of Science of the German
   Democratic Republic, Gatersleben, German Democratic
   Republic
Dr G. Bronzetti, National Research Council, Institute of
   Mutagenesis and Differentiation, Pisa, Italy
Dr B.E. Butterworth, Chemical Industry Institute of Toxi-
   cology, Research Triangle Park, North Carolina, USA

Dr P.S. Chauhan, Bio-Medical Group, Bhabha Atomic Research
   Centre, Bombay, India
Dr I. Chouroulinkov, Unité de Cancérogénèse Expérimentale et
   de Toxicologie Génétique (E.R. 304) I.R.S.C.-C.N.R.S.,
   Villejuif, France
Dr M.G. Clare, Shell Research Ltd, Sittingbourne, Kent,
   United Kingdom
Dr R.D. Combes, School of Biological Sciences, Portsmouth
   Polytechnic, Portsmouth, United Kingdom
Dr C. Coton, Mammalian Genetics Laboratory, Department of
   Biology, European Nuclear Centre, Mol, Belgium
Dr R. Crebelli, Higher Institute of Health, Viale Regina
   Elena, Rome, Italy
Dr B.J. Dean, Upchurch, Sittingbourne, Kent, United Kingdom
Dr G.M. Decad, Department of Materials Toxicology, IBM
   Corporation, San Jose, California, USA
Dr F.J. de Serres, National Institute of Environmental
   Health Sciences, Research Triangle Park, North Carolina,
   USA
Dr D.J. Doolittle, Toxicology Research, Bowman Gray
   Technical Center, R.J. Reynolds Co., Winston-Salem, North
   Carolina, USA
Dr U.H. Ehling, Mammalian Genetics Institute, Association
   for Radiation and Environmental Research, Neuherberg,
   Federal Republic of Germany
Dr B.M. Elliott, Genetic Toxicology Section, Imperial Chemi-
   cal Industries PLC, Macclesfield, Cheshire, United
   Kingdom
Dr R. Fahrig, Fraunhofer Institute for Research on Toxi-
   cology and Aerosols, Hannover, Federal Republic of
   Germany
Dr R. Forster, Life Science Research, Rome Toxicology
   Centre, Pomezia, Rome, Italy
Dr K. Fujikawa, Drug Safety Evaluation Laboratories, Central
   Research Division, Takeda Chemical Industries Ltd, Osaka,
   Japan
Dr C. Furihata, Department of Molecular Oncology, Institute
   of Medical Science, University of Tokyo, Tokyo, Japan
Dr S.M. Galloway, Merck Sharp & Dohme Research Labora-
   tories, West Point, Pennsylvania, USA
Dr W.M. Generoso, Biology Division, Oak Ridge National
   Laboratory, Oak Ridge, Tennessee, USA
Dr H.P. Glauert, McArdle Laboratory for Cancer Research,
   University of Wisconsin, Madison, Wisconsin, USA
Dr U. Graf, Toxicology Institute, Zurich Federal Polytechnic
   and University, Zurich, Switzerland
Dr B.L. Harper, Division of Environmental Toxicology, Uni-
   versity of Texas Medical Branch, Galveston, Texas, USA
Dr G.G. Hatch, Toxicology Division, Northrop Services Inc.,
   Environmental Sciences, Research Triangle Park, North
   Carolina, USA
Dr M. Hayashi, Biological Safety Research Centre, National
   Institute of Hygienic Sciences, Tokyo 158, Japan
Dr R.M. Hicks, School of Pathology, Middlesex Hospital
   Medical School, London, United Kingdom

Dr J.M. Hunt, Department of Pathology and Laboratory Medi-
   cine, University of Texas Medical School, Houston, Texas,
   USA
Dr N. Inui, Biological Research Centre, Japan Tobacco Inc.,
   Kanagawa, Japan
Dr M. Ishidate, Jr., Biological Safety Research Centre,
   National Institute of Hygienic Sciences, Tokyo, Japan
Dr V.I. Ivanov, Institute of Medical Genetics, Academy of
   Medical Sciences, Moscow, USSR
Dr J.C. Jensen, National Food Institute, Institute of Toxi-
   cology, Copenhagen, Denmark
Dr D. Jenssen, Department of Genetic Toxicology, Wallenberg
   Laboratory, University of Stockholm, Stockholm, Sweden
Dr B.J. Kilbey, Institute of Animal Genetics, University of
   Edinburgh, Edinburgh, United Kingdom
Dr I. Kimber, Central Toxicology Laboratory, Imperial Chemi-
   cal Industries PLC, Macclesfield, Cheshire, United King-
   dom
Dr U. Kliesch, Mammalian Genetics Institute, Association for
   Radiation and Environmental Research, Neuherberg, Federal
   Republic of Germany
Dr A.D. Kligerman, Environmental Health Research and
   Testing Inc., Research Triangle Park, North Carolina,
   USA
Dr D. Kornbrust, Merck Sharp & Dohme Research Laboratories,
   Department of Safety Assessment, West Point,
   Pennsylvania, USA
Dr C. Lasne, Unité de Cancérogénèse Expérimentale et de
   Toxicologie Génétique (ER-304) I.R.S.C.-C.N.R.S.,
   Villejuif, France
Dr A. Léonard, Mammalian Genetics Laboratory, Department
   of Biology, European Nuclear Centre, Mol, Belgium
Dr C.A. Luke, Medical Department, Brookhaven National Lab-
   oratory, Upton, New York, USA
Dr J.T. MacGregor, US Department of Agriculture, Western
   Regional Research Center, Berkeley, California, USA
Dr A.M. Malashenko, Scientific Research Laboratory of Exper-
   imental Biological Models of the Academy of Medical
   Sciences of the USSR, Moscow Region, USSR
Dr C. Malaveille, International Agency for Research on
   Cancer, Lyon, France
Dr B.H. Margolin, Biometry and Risk Assessment Program,
   National Institute of Environmental Health Sciences,
   Research Triangle Park, North Carolina, USA
Dr D. McGregor, Developmental Toxicology, Inveresk Research
   International Ltd, Musselburgh, United Kingdom
Dr A.L. Meyer, Shell Research Ltd, Sittingbourne, Kent,
   United Kingdom
Dr J.C. Mirsalis, Cellular and Genetic Toxicology Depart-
   ment, SRI International, Menlo Park, California, USA
Dr N. Nashed, Johann Wolfgang Goethe-Universität, Frankfurt
   am Main, Federal Republic of Germany
Dr S.B. Neal, Toxicology Division, Lilly Research Labora-
   tory, Greenfield, Indiana, USA

Dr A. Neuhäuser-Klaus, Mammalian Genetics Institute,
   Association for Radiation and Environmental Research,
   Neuherberg, Federal Republic of Germany
Dr D.A. Pagano, Cellular and Genetic Toxicology Branch,
   Toxicology Research and Testing Program, National Insti-
   tute of Environmental Health Sciences, Research Triangle
   Park, North Carolina, USA
Dr F. Palitti, Evolutionary Genetics Centre of the National
   Research Council, Genetics and Molecular Biology Depart-
   ment, University City, Rome, Italy
Dr S. Parodi, Chemical Carcinogenesis Laboratory, National
   Cancer Research Institute, Genoa, Italy
Dr M. Pereira, Health Effects Research Laboratory, US
   Environmental Protection Agency, Cincinnati, Ohio, USA
Dr J. Pot-Deprun, Unité de Cancérogénèse Expérimentale et
   Toxicologie Génétique (ER-304) I.R.S.C.- C.N.R.S.
   Laboratoires de Recherche Appliquée sur le Cancer,
   Villejuif, France
Dr G.S. Probst, Toxicology Division, Lilly Research Labora-
   tories, Greenfield, Indiana, USA
Dr C. Ramel, Wallenberg Laboratory, University of Stockholm,
   Stockholm, Sweden
Dr K. Randerath, Baylor College of Medicine, Department of
   Pharmacology, Houston, Texas, USA
Dr H.S. Rosenkranz, Department of Environmental Health
   Sciences, Case Western Reserve University, Cleveland,
   Ohio, USA
Dr P. Russo, National Cancer Research Institute, Genoa,
   Italy
Dr M.F. Salamone, Moe-Biohazard Laboratory, Rexdale,
   Ontario, Canada
Dr C.B. Salocks, Department of Materials Toxicology, IBM
   Corporation, San Jose, California, USA
Dr J. Schöneich, Central Institute for Genetics and for
   Research on Cultivated Plants, Academy of Science of the
   German Democratic Republic, Gatersleben, German
   Democratic Republic
Dr A.G. Searle, Medical Research Council Radiobiology Unit,
   Harwell, Didcot, United Kingdom
Dr G.A Sega, Biology Division, Oak Ridge National Labora-
   tory, Oak Ridge, Tennessee, USA
Dr M.D. Shelby, Cellular and Genetic Toxicology Branch,
   Toxicology Research and Testing Program, National Insti-
   tute of Environmental Health Sciences, Research Triangle
   Park, North Carolina, USA
Dr H. Shibuya, Laboratory of Genetic Toxicology, Hatano
   Research Institute, Food and Drug Safety Center,
   Kanagawa, Japan
Dr T. Shibuya, Laboratory of Genetics, Hatano Research
   Institute, Food and Drug Safety Center, Kanagawa, Japan
Dr R.H. Stevens, Radiation Research Laboratory, Department
   of Radiation, University of Iowa, Iowa, USA
Dr G.D. Stoner, Department of Pathology, Medical College of
   Ohio, Toledo, Ohio, USA

Dr J.A. Styles, Central Toxicology Laboratory, Imperial
   Chemical Industries PLC, Macclesfield, Cheshire, United
   Kingdom
Dr K.E. Suter, Preclinical Research, Toxicology Department,
   Sandoz Limited, Basel, Switzerland
Dr A. D. Tates, Department of Radiation Genetics and
   Chemical Mutagenesis, State University of Leiden, Leiden,
   Netherlands
Dr R.R. Tice, Medical Department, Brookhaven National
   Laboratory, Upton, New York, USA
Dr H. Tsuda, First Department of Pathology, Nagoya City,
   University Medical School, Nagoya, Japan
Dr R. Valencia, Department of Zoology, University of
   Wisconsin, Zoology Research Building, Madison, Wisconsin,
   USA
Dr A. Vlachos, Haskell Laboratory for Toxicology and Indus-
   trial Medicine, E.I. Du Pont de Nemours & Co. Inc.,
   Newark, Delaware, USA
Dr E.W. Vogel, Department of Radiation Genetics and Chemical
   Mutagenesis, State University of Leiden, Leiden, Nether-
   lands
Dr P.A. Watkins, Pharmaceuticals Division, Imperial Chemical
   Industries PLC, Macclesfield, Cheshire, United Kingdom
Dr G.A. Wickramaratne, Central Toxicology Laboratory,
   Imperial Chemical Industries PLC, Macclesfield, Cheshire,
   United Kingdom
Dr D. Wild, Pharmacology and Toxicology Institute, Würzburg
   University, Würzburg, Federal Republic of Germany
Dr P.K. Working, Department of Genetic Toxicology, Chemical
   Industry Institute of Toxicology, Research Triangle Park,
   North Carolina, USA
Dr V.S. Zhurkov, A. N. Sysin Institute of General and
   Environmental Hygiene, Moscow, USSR

NOTE TO READERS OF THE CRITERIA DOCUMENTS


    Every effort has been made to present information in
the criteria documents as accurately as possible without
unduly delaying their publication.  In the interest of all
users of the environmental health criteria documents,
readers are kindly requested to communicate any errors
that may have occurred 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.

ABBREVIATIONS


AAF       Acetylaminofluorene

BP        Benzo [a] pyrene

CSSTT/1   Collaborative study on short-term tests for
          genotoxicity and carcinogenicity

CSSTT/2   Collaborative study on short-term  in vivo
          tests for mutagens and carcinogens

GGT       Gamma-glutamyltranspeptidase

IPESTTC   International Collaborative Programme for the
          Evaluation of Short-Term Tests for Carcinogens

NK        Natural killer

PYR       Pyrene

SCE       Sister chromatid exchange

SSB       Single strand breaks

UDS       Unscheduled DNA synthesis

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,   was published in 1985 (Ashby et al., 1985)  and
was  summarized in Environmental Health  Criteria 47 (WHO,
1985).  The second  part, which  is the  subject  of  this
report, was published in 1988 (Ashby et al., 1988a).

      The need for inter-laboratory  collaborative studies
on  an  international scale  arose  from the  necessity to
investigate  the value of  short-term tests for  detecting
mutagenic  and carcinogenic chemicals.   Short-term assays
were  proposed as alternatives or supplementary procedures
to  traditional long-term rodent bioassays.  Concern about
the choice of short-term tests and their  reliability  and
sensitivity  led  to the  instigation  of the  first major
international  collaborative  exercise, the  International
Collaborative  Programme for the Evaluation  of Short-Term
Tests for Carcinogens (IPESTTC) (de Serres & Ashby, 1981).
The  results of  this study  confirmed the  value  of  the
salmonella  mutation  test  as a  reliable and practicable
assay  for the primary  identification of carcinogens  and
mutagens.  It was also  observed that, in  the  salmonella
test,  some  known  rodent  carcinogens  were  either  not
detected  or  only detected  with considerable difficulty.
Several other assays represented in the IPESTTC study were
able  to detect some of  the rodent carcinogens that  were
negative  in  the  salmonella assay.   The supporting data
base  was too small, however, to permit the recommendation
of  an assay that would complement the salmonella mutation
test.

    It  was  apparent, from  the  results of  the  IPESTTC
study,  that  a  further collaborative  exercise  would be
required to establish a) the most effective combination of
 in  vitro  assays for primary  screening of chemicals  for
genotoxic  activity and b)  the most useful  short-term  in
 vivo tests  for confirming mammalian genotoxicity and car-
cinogenic   potential.  The  Collaborative  Study  on  the
Assessment  and Validation of  Short-Term Tests for  Geno-
toxicity  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  (a Participating Institution  of  the
IPCS).   Because of the complexity of the organization and
the  magnitude of  the project,  it was  divided into  two
discrete  studies:  the Collaborative  Study on Short-Term
Tests  for Genotoxicity and Carcinogenicity  (CSSTT/1) and
the  Collaborative  Study on  Short-Term  In Vivo Tests for
Mutagens and Carcinogens (CSSTT/2).

    In  CSSTT/1, a comprehensive  data base was  assembled
from  a wide range  of  in vitro assays conducted  with ten
carefully selected organic chemicals. These included eight
established  rodent carcinogens that were  either negative
or  difficult to detect  in the salmonella  assay and  two
chemicals  that  were regarded  as non-carcinogenic.  Data
were  evaluated from almost 90 sets of assays conducted by
some  60 participating scientists.   Four types of  assays
performed well enough to be considered as  possible  comp-
lementary  tests to the salmonella  assay.  These included
tests for chromosomal aberrations, gene mutations and neo-
plastic transformation in cultured mammalian cells, and an
assay  for aneuploidy in yeast.  With the exception of the
chromosomal  aberration assay, it was apparent that proto-
cols  in  general use  for  these assays  required further
evaluation  before they could be  considered fully accept-
able.

    The  major conclusion of the CSSTT/1 study on  in vitro
assays   was that the use of chromosomal aberration assays
in  conjunction with the salmonella mutation test may pro-
vide an efficient primary screen for possible new carcino-
gens.

    In the IPESTTC study, seven of the  fourteen  presumed
non-carcinogens  gave positive results  in many of  the  in
 vitro assays. The limited  in vivo data available from that
study  suggested that these seven  chemicals were inactive
in  in vivo short-term tests. The non-carcinogens in two of
the  carcinogen/non-carcinogen  pairs  in  IPESTTC,   i.e.
benzo [a] pyrene/pyrene (BP/PYR) and 2-acetylamino-fluorene/
4-acetylaminofluorene  (2AAF/4AAF), provided good examples
of these different responses, and these pairs of chemicals
were  selected for the   in vivo part of the  collaborative
study  (CSSTT/2).  There  was, however,  a  question  mark
against  the presumed non-carcinogenicity  of 4AAF, and  a
vital  part of the study  was the initiation of  long-term
cancer bioassays of 2AAF and 4AAF in rats.

    The objective of CSSTT/2, therefore, was to generate a
comprehensive  data profile from  a broad range  of short-
term  in vivo tests as a means of understanding how various
genetic  endpoints in key target tissues respond to chemi-
cals defined as genotoxic  in vitro.  The ultimate goal was
to identify which  in vivo assays could be used  to  deter-
mine the  in vivo activity of established genotoxins.

    Ninety-seven investigators from sixteen countries par-
ticipated  in the  in vivo project and  data were presented
from  some fifty separate  in vivo techniques.  The results
were evaluated at a meeting of investigators held  at  Cap
d'Agde,  France, in May  1985.  A series  of reports  were
prepared comprising an assessment of each group of assays,
summary  reports on the  germ cell assays  and the  liver-
specific tests, and summary reports on the total data base

on each pair of chemicals.  Subsequently, an  overview  of
the  whole   in vivo study  was prepared  in  readiness for
final publication.

    The criteria defining an acceptable short-term  in vivo
test   were satisfied by  only a small  proportion of  the
assays   represented  in  CSSTT/2.   However,  the  assays
included  in the  study were  not limited  to  those  most
likely  to meet the  criteria.  Thus, although  data  were
submitted  from assays not designed  primarily to identify
 in   vivo   genotoxicity,  they provided  information on a
broad   spectrum  of  biological   effects  of  the   four
chemicals.  Most of the   in vivo somatic cell  assays  for
genotoxicity discriminated between the two carcinogen/non-
carcinogen  pairs although, in  some cases, weak  activity
was   detected   in   tests  with   the   non-carcinogens,
particularly  with 4AAF.  The insensitivity of some assays
to  one or other of the two pairs of chemicals support the
concept that negative  in vivo data should be obtained from
at least two assays in different tissues before a chemical
can be accepted as non-genotoxic  in vivo. 

    The  mouse bone marrow micronucleus test was confirmed
as  a robust, sensitive,  and reproducible assay  and  was
recommended for primary  in vivo testing of  in vitro  geno-
toxins. The overall performance of the rat liver assay for
unscheduled DNA synthesis suggested that it could be comp-
lementary  to  the  micronucleus  test,  although  certain
aspects of the sensitivity and selectivity of  this  assay
require  additional investigation.  Some  widely advocated
assays  including the host-mediated and urine mutagenicity
assays  and tests using  drosophila were concluded  to  be
inappropriate for hazard-assessment purposes.

    The results of the CSSTT/2 study confirmed that short-
term   in vivo tests have a  vital role to  play in  hazard
assessment  and that this role is to identify those chemi-
cals,  shown to be genotoxic  in vitro,  that are active  in
 vivo and, thus, are most likely to present a carcinogenic/
mutagenic hazard to mammals, including humans.

1.  INTRODUCTION

    For  many years it has been known that  some  environ-
mental  chemicals are associated  with an increased  inci-
dence of cancer in humans. The danger posed by exposure to
established human carcinogens such as  ß-naphthylamine and
vinyl  chloride  was  originally recognized  from epidemi-
ological  evidence.   The  main  purpose  of investigating
chemical  carcinogenesis, however, is to  prevent environ-
mentally  induced  cancer  by identifying  such  chemicals
before they are released into the environment.   Over  the
past  two  or  three decades,  carcinogenic  activity  has
usually  been determined by the  ability of a chemical  to
produce  tumours  in  laboratory animals  during  lifetime
exposure  to  the  chemical.  Long-term  animal studies of
this  kind  may last  for two or  three years and  utilize
scarce  resources  and  expertise.  In  consequence, it is
only feasible to test a very small proportion of  the  new
chemicals produced each year in animal bioassays.

    Many  attempts were made in  the late 1960s and  early
1970s  to  detect  potentially carcinogenic  chemicals  in
tests  using bacteria or cultured mammalian cells.  It was
suspected that many cancers resulted from changes  to  the
informational  macromolecules  of  cells, i.e.  deoxyribo-
nucleic  acid (DNA), and, in general, the tests were based
on the induction of genetic changes to the test cells such
as  gene  mutations  and chromosomal  aberrations.  Little
progress  was made until it was realized that the majority
of  carcinogenic  chemicals required  biotransformation by
mammalian  enzymes before they  were in a  molecular  form
capable  of interaction with DNA.  This observation led to
the  development,  by Professor  Bruce  Ames and  his col-
leagues  at the University of  California, USA, of a  bac-
terial  test  for  mutagens  that  incorporated  essential
aspects  of mammalian metabolism in the form of an enzyme-
rich fraction of mammalian liver.  In this  system,  known
as the "salmonella assay" or the "Ames test",  it  was
shown that a number of chemicals, known to be carcinogenic
in  laboratory  animal  studies, were  metabolized  by the
incorporated  mammalian enzymes to reactive molecules that
induced  mutations  in the  Salmonella  typhimurium tester
strains.   In  a series  of  validation studies  with  the
salmonella   assay  conducted  during  the  mid-1970s  and
totalling  about  500  chemicals,  a  high  percentage  of
carcinogenic  chemicals induced mutations in  the bacteria
and a high proportion of non-carcinogens were negative.

    Considering  that  the salmonella  assay could produce
results  within a  week (compared  with the  two or  three
years for a rodent bioassay), it is not surprising that it
was soon being used extensively throughout the world. Many
hundreds  of  chemicals  of diverse  structure were tested
and,  in  many cases,  interpreted  in human  health terms
without  full  comprehension of  the biological principles

involved  in  extrapolating  data from  a simple bacterial
assay  to a complex organism like man.  It became apparent
at  this time that a number of established animal carcino-
gens  consistently gave negative results in the salmonella
assay  and,  similarly,  some chemicals,  considered to be
non-carcinogenic,  were shown to be mutagenic in bacterial
tests.   This observation confirmed that  no single short-
term  assay could be relied  on to detect all  carcinogens
and  also confirmed  the value  of the  practice of  using
short-term   in vitro and  in vivo tests in batteries  or in
tier  systems.  The variety of such packages proliferated,
leading  to a great deal of confusion and conflict regard-
ing  the most appropriate  tests to investigate  chemicals
for possible carcinogenic activity.

      Concern about the  reliability, sensitivity and  the
choice  of short-term tests resulted in the instigation of
the  International Collaborative Programme for  the Evalu-
ation of Short-Term Tests for Carcinogens (IPESTTC).  This
project,  completed  in 1981,  involved investigators from
more than 50 laboratories, and some 30  in vitro and  in vivo
assays   were evaluated for their  ability to discriminate
between  carcinogenic  and non-carcinogenic  chemicals (de
Serres and Ashby, 1981). Twenty-five known carcinogens and
17  chemicals considered to be non-carcinogenic, including
14  pairs  of  carcinogen/non-carcinogen  analogues,  were
tested in most of the assays.  An evaluation of  the  data
indicated that the salmonella mutation assay gave the best
overall performance, producing reliable results in a large
number  of laboratories.  Other assays  that also appeared
to  discriminate  between carcinogens  and non-carcinogens
included   in vitro tests  for chromosomal  aberrations and
unscheduled  DNA  synthesis and,  although fewer chemicals
were tested, results from drosophila tests, mammalian cell
gene  mutation assays, and rodent  bone marrow cytogenetic
studies  suggested that they, too,  were useful components
of  a testing battery.  Following a critical evaluation of
the  data from the IPESTTC project, it was concluded that,
although  the value of the salmonella assay was confirmed,
there  were still some rodent carcinogens that were either
reproducibly  negative in this assay or detected only with
difficulty.  A proportion of these chemicals were detected
in  some  of  the  other  assays  but  the data  base  was
insufficient to identify which  in vitro or  in vivo test(s)
were  the  most  effective complementary  assay(s)  to the
salmonella test.

    It  was apparent from the IPESTTC study that a further
collaborative  exercise would be necessary to establish a)
the most effective combination of  in vitro assays for pri-
mary screening of chemicals for carcinogenic potential and
b)  the most useful short-term  in vivo procedures for con-
firming mammalian genotoxicity and carcinogenic potential.
A  collaborative  programme designed  to investigate these
two  questions was proposed by the International Programme
on  Chemical Safety (IPCS)  and the National  Institute of

Environmental Health Sciences (NIEHS) of the USA.  Because
of  the  logistical  problems involved  in  organizing and
managing  international collaborative projects,  this pro-
gramme  was divided into two discrete studies.  The first,
referred to as the Collaborative Study on Short-Term Tests
for  Genotoxicity  and Carcinogenicity  (CSSTT/1) was pub-
lished  in 1985  (Ashby et  al., 1985)  and summarized  in
Environmental  Health Criteria 47 (WHO, 1985).  The second
part  of  the programme  was  the Collaborative  Study  on
Short-Term   In Vivo Tests  for  Mutagens  and  Carcinogens
(CSSTT/2) and is the subject of this report.

    It  was decided very early in the planning stage that,
whereas  42  chemicals  were investigated  in  the IPESTTC
project, the CSSTT/1 study would concentrate on generating
a  more comprehensive  data base  on a  smaller number  of
chemicals  rather than an  incomplete set of  data from  a
large  number.  Thus, ten organic  chemicals were selected
comprising  eight established carcinogens that were either
negative  or difficult to  detect in the  salmonella assay
and  two chemicals that had not shown any evidence of car-
cinogenicity  in rodent cancer bioassays.   The results of
the  study were evaluated at a meeting of investigators in
1983  at which  data from  nearly 90  individual  sets  of
assays  conducted by some 60 participating scientists were
scrutinised. Almost all the  in vitro assays in use at that
time were represented and four assays, in particular, per-
formed well enough to be considered as possible complemen-
tary  tests to the salmonella assay.  These were tests for
chromosomal  aberrations,   gene mutations  and neoplastic
transformation  in cultured mammalian cells,  and an assay
for aneuploidy induction in yeast.  An important component
of  the project was  to identify protocol  variations that
might explain differences in results between investigators
using,  ostensibly, the same assay.  This assessment indi-
cated  that the protocols in general use for gene mutation
and   transformation   assays   in  mammalian   cells  and
aneuploidy in yeast require further evaluation before they
can be considered fully reproducible between laboratories.
The  data provided by the  investigators using chromosomal
aberration  assays allowed the  working group to  identify
certain critical factors in the protocols that appeared to
influence  sensitivity  and   selectivity in  response  to
carcinogens. The  in vitro test for chromosomal aberrations
was,  therefore,  considered  to be  the  most appropriate
complementary assay to the salmonella test and it was con-
cluded  that a combination of these two assays may provide
an  efficient primary screen for  possible new carcinogens
and mammalian mutagens.

2.  THE COLLABORATIVE STUDY ON SHORT-TERM  IN VIVO TESTS
FOR MUTAGENS AND CARCINOGENS (CSSTT/2) 1983-1985

    In   general terms, the  in vitro part of the collabor-
ative study (CSSTT/1) achieved its targets by indicating a
small group of assays that show promise  as  complementary
tests to the salmonella mutation assay.  Detailed scrutiny
of the data also identified critical aspects of the proto-
cols of these assays that required modification  in  order
to  reach the levels of  reproducibility, sensitivity, and
selectivity  already  achieved  by the  salmonella  assay.
Even so, the IPESTTC study had shown that 7 of 14 presumed
non-carcinogens elicited positive responses in many of the
 in   vitro assays. In the same study, these seven non-car-
cinogens were predominantly inactive in a series of short-
term    in vivo  tests.  The  in vivo test data presented in
the  IPESTTC study were  limited, i.e. only  five investi-
gators  provided results from mammalian   in vivo tests and
only  a proportion of  the chemicals were  tested by  each
investigator.   The results suggested that,  although some
non-carcinogens  were positive  in vitro,  they  were inac-
tive  in   in vivo short-term tests. The non-carcinogens in
two  of  the carcinogen/non-carcinogen  pairs, i.e. benzo-
 [a] pyrene/pyrene   (BP/PYR)  and 2-acetylaminofluorene/4-
acetylaminofluorene (2AAF/4AAF), provided good examples of
these different responses.

FIGURE 1

    These  observations  were  the origin  of  the present
study, the object of which was to generate a comprehensive
data  profile  from a  broad  range of  short-term  in vivo
assays   as a means  of understanding how  various genetic
end-points  in  key  target tissues  respond  to chemicals
defined as genotoxic  in vitro.   The objectives and design
of  the  collaborative  study on  short-term  in vivo tests

(CSSTT/2) were outlined by an  ad hoc Working Groupa   at a
meeting  organized by the IPCS in Geneva on 30 April 1981,
and  the  plans  were  consolidated  by  an  IPCS  Working
Groupb    in  Geneva, on  13-14  November 1981.   The sub-
sequent  coordination of the  collaborative study was  the
responsibility  of a Steering Committeec   derived primar-
ily from the Working Group.

    The four test chemicals selected for the  in vivo  pro-
ject  were  the two  carcinogen/non-carcinogen pairs, i.e.
BP/PYR and 2AAF/4AAF, that provided the initial impetus to
the  study,  and the  first  samples were  distributed  to
investigators  in March 1983. During 1983, additional par-
ticipants  joined the study to provide data on new  in vivo
assays   and nine coordinatorsd were  appointed to oversee
the  work  being  conducted  by  investigators  performing
identical or similar assays. In all, 97 investigators from
16   countries  participated  in  CSSTT/2.   Progress  was
reviewed  at a  meeting of  the Steering  Group  with  the
coordinators held in Brussels on 12-13 January  1984.   At
this meeting a plan was developed to enter the  data  from
the study into a computer at NIEHS.  It was envisaged that
such   a  plan  would allow  a  comprehensive  statistical
analysis using common statistical techniques for each kind
of   assay  and  provide  a  comparison  of  results  from
different   laboratories   performing   the  same   assay.
Initially, it was expected that all the studies  would  be
completed  by October 1984, but  by September 1984 it  was
evident that additional time would be required for many of
the investigators to complete their evaluation of the four
chemicals.   At a second meeting of the Steering Group and
coordinators,  held on 14-17 November 1984, status reports
prepared by the coordinators were reviewed in  detail  and
deadlines were set for the provision of final reports from
each  investigator.  Two additional  coordinators were ap-
pointed  at this meeting  to oversee the  rodent  dominant
lethal  assay (Dr W. Generoso)  and the mouse coat  colour
spot  test (Dr R. Fahrig). Following this progress review,
a  meet-ing of investigators was planned for May 1985, for
the presentation, collation, and assessment of the results
and the preparation of final reports on the study.

--------------------------------------------------------------------------------
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, Jr., Dr A.
   Leonard, Dr N. Loprieno, Dr B.E. Matter, Professor T. Matsushima, Dr R.
   Montesano, Dr F.J. de Serres, Dr M. Shelby, Professor F.H. Sobels, Dr M.
   Stoltz, and Dr M. Waters.
c  Steering Committee: Dr F.J. de Serres (Chairman), Dr J. Ashby, Dr. M.
   Ishidate, Jr., Dr B. Margolin, Dr M. Shelby, Dr M. Draper, and Dr G.C.
   Becking.
d  Coordinators: Dr W. Vogel, Dr W.M. Generoso, Dr I.-D. Adler, Dr D. Wild,
   Dr. T. Tice, Dr B.E. Butterworth, Dr D. McGregor, Dr M. Salamone, and 
   Dr R. Fahrig.

    The meeting of investigators was held during 15-21 May
l985 at Cap d'Agde, France. Representatives from each par-
ticipating laboratory met to prepare a series  of  reports
comprising  a) an assessment of  each group of assays,  b)
summary  reports on the  germ cell assays  and the  liver-
specific  assays, and c) summary reports on the total data
base  on each  pair of  chemicals, i.e.  BP/PYR and  2AAF/
4AAF.  During the meeting, draft reports were prepared and
were  to be finalized during the following two months.  At
a meeting of the Editorsa held  at NIEHS from 24 July to 6
August  1985,  the  work group  and investigators' reports
were reviewed, and a time-table was developed for the com-
pletion  of all  reports and  for the  preparation of  the
Introduction  and Overview of  the study in  readiness for
final publication of the  in vivo study.

    A  feature of both the  CSSTT studies and the  earlier
IPESTTC project was the voluntary participation of a large
number  of  scientists  together with  support  from their
parent   institutions.   The  organization of  CSSTT/1 and
CSSTT/2  was financed largely by IPCS and some of its par-
ticipating  institutions.  In most  cases, funding of  the
experimental work was by individual investigators, many of
whom  incorporated  the  studies into  their research pro-
grammes.  This,  of  course,  required  the  goodwill  and
support  of senior management of the participating labora-
tories  from universities, research institutions,  and in-
dustrial  research  facilities throughout  the world.  Ad-
ditional  financial assistance was provided by a number of
governments  that support IPCS, including  Belgium, Italy,
Japan, The Netherlands, the United Kingdom, and  the  USA.
The  United Kingdom Department of Health and Social Secur-
ity funded the rat carcinogenicity studies with  2AAF  and
4AAF.   The Belgian government and  its National Institute
of  Hygiene  and  Epidemiology financed  meetings  of  the
Steering  Committee  and  coordinators in  Brussels.   The
meeting  of investigators held in Cap d'Agde was organized
by  the French government  and cosponsored by  the  French
Ministry   of  Health  and  the   Commission  of  European
Communities.

--------------------------------------------------------------------------------
a  Editors:  Dr J. Ashby, Dr F.J. de Serres, Dr M.D. Shelby, Dr B.H. 
   Margolin, Dr M. Ishidate, Jr., and Dr G.C. Becking.

3.  OVERALL AIMS OF THE STUDY AND CRITERIA FOR THE SELECTION OF
APPROPRIATE SHORT-TERM  IN VIVO TESTS

    Because of their relative simplicity, reproducibility,
and reliability, short-term  in vitro tests are the methods
of choice for the initial testing of chemicals  for  geno-
toxic  activity.  The role and  usefulness of genotoxicity
assays   using  whole  animals,  i.e.  short-term   in vivo
tests,   are less clearly  defined despite the  fact  that
they  are  widely used  and are an  integral part of  most
legislative  guidelines  for  the conduct  of mutagenicity
tests.   In  general,   in vivo tests  are  more  resource-
consuming  than their  in vitro counterparts and the use of
animals for experiments for which there is  an  acceptable
 in   vitro  alternative  is  to  be discouraged.  As these
observations suggest,  in vivo assays should be designed to
answer  questions  that cannot  be investigated adequately
with  in vitro tests.

3.1  The use of short-term tests for the primary identification
of genotoxic chemicals

    Certain  short-term  in vivo assays, such as the rodent
bone  marrow chromosome assay, the  rodent dominant lethal
test,  and the host-mediated assay, were used in the early
1970s in primary screens for the identification  of  muta-
genic  chemicals.  With the  introduction of reliable  and
valid  short-term  in vitro tests for mutagens  in the mid-
1970s,  the use of  in vivo procedures  for initial screen-
ing  of chemicals declined considerably. Certain legislat-
ive  authorities still recommend a combination of  in vitro
tests   reserved for confirmatory or  supplementary use or
for providing data for hazard assessment.  These different
roles  for whole animal procedures have led to the accept-
ance  of test protocols  of varying complexity,  i.e. less
rigorous protocols for screening modes and more comprehen-
sive  protocols when the test is required to assess hazard
potential.

    Implicit in the concept of the two IPCS studies is the
assumption  that chemicals shown  to be genotoxic   in vivo
also   exhibit genotoxic activity  in a properly  designed
and  conducted  in vitro primary screen.  This principle is
well-established in the scientific literature and was con-
firmed in the IPESTTC and CSSTT/1 studies.  Thus,  it  was
an integral principle in the study design that the primary
screening  of chemicals could  be adequately served  by  in
 vitro tests  alone and that short-term  in vivo assays have
no  role to play when screening for genotoxic activity.  In
 vivo procedures  will,  therefore,  be reserved  for  more
specific  applications such as investigating  the activity
of  in vitro genotoxins in the whole animal and  to  assist
in the assessment of the mutagenic and carcinogenic poten-
tial  associated with exposure of humans to  in vitro geno-
toxins.

3.2  The use of short-term  in vivo assays for assessing the 
hazard associated with exposure to  in vitro genotoxins

    It  is apparent from the last paragraph that the major
objective  of the study was to investigate the activity in
the  whole mammal of chemicals  identified as genotoxic  in
 vitro and  to establish which   in vivo tests are the  most
useful for this purpose.  The major criterion for  an  ac-
ceptable  short-term   in vivo test, therefore,  rests with
its ability to differentiate between carcinogenic and non-
carcinogenic  chemicals, particularly those that have been
identified as genotoxic in an  in vitro primary screen.  To
extend  this criterion further, an acceptable assay should
be  capable  of separating  carcinogen/non-carcinogen ana-
logues,  e.g., 2AAF/4AAF, in  which, in some  cases, small
differences  in  chemical  structure are  responsible  for
dramatic  differences  in  carcinogenic potential.   Other
important  criteria,  some  of  which  were  more  clearly
characterized  during the course of the study, include the
following:

*   data  should be reproducible between different labora-
    tories,

*   the  assays should not  require too high  a degree  of
    technical  and scientific expertise to be conducted on
    a routine, every-day basis,

*   the  genotoxic  changes  or end-points  of  the assays
    should be clear and unambiguous,

*   there  should be agreed, valid  statistical techniques
    to differentiate between positive and negative data.

    It  is relevant to the performance of  in vivo tests to
consider  the  metabolic  fate of  carcinogenic chemicals.
As a generalization and depending on the route of exposure
of  the animal to  the chemicals, reactive  metabolites of
many  carcinogens are generated mainly  in the liver as  a
by-product  of a predominantly detoxifying process.  These
metabolites may be capable of interaction with the genetic
material, DNA, in the liver cells, they may be transported
in an active form to other tissues, or they may be further
modified in those tissues to forms able to  interact  with
DNA. Thus, a study of the genotoxic activity of a chemical
in the whole animal should be able to detect the genotoxic
effects  of chemicals or their metabolites in tissues out-
side  the liver and  of those whose  main genotoxic  reac-
tivity  may be confined to the liver itself.  For example,
assays based on genetic alterations to cells in  the  bone
marrow,  which is readily accessible to chemicals or their
metabolites  circulating in the  blood, respond to  a wide
range  of  chemical carcinogens.   However, where reactive
metabolites  are not readily  transported from the  liver,

bone marrow-type tests would be of little value and assays
capable  of  detecting  the genotoxicity  in  liver  cells
should be available.

3.3  The role of short-term  in vivo tests in research
into the mechanisms of cancer

    Chemical  carcinogenesis is generally recognized  as a
multistage  process  that begins  with initiation, usually
considered  to involve changes in DNA structure leading to
mutations, followed by promotion of the lesion to  a  pre-
malignant state and progression to overt cancer. Thus, the
majority  of current short-term tests,  designed to detect
the  consequences of DNA interaction, will only respond to
chemicals that may induce tumours by a predominantly geno-
toxic  mechanism or induce the  initial phase of the  car-
cinogenic process.

    A  feature of the  literature on short-term  tests for
chemical carcinogens is the occasional reference to chemi-
cals, shown to be associated with the induction of tumours
in  laboratory animals, that  consistently fail to  be de-
tected  in short-term  in vitro or  in vivo assays for geno-
toxicity. Such negative observations with established car-
cinogens have been explained by a lack of  sensitivity  of
the  particular assays.  If these chemicals (diethylhexyl-
phthalate  is an example) are truly non-DNA-reactive, how-
ever,  then negative data in assays for genotoxicity would
be the expected result.  The acceptance of  the  existence
of  so-called "non-genotoxic carcinogens" is of critical
importance  to the future  of short-term testing  and  one
consideration  during the assessment of  the CSSTT/2 study
was  the identification of  in vivo procedures  that may be
useful  for investigating such chemicals with the eventual
objective of developing specific short-term tests for non-
DNA-reactive carcinogens.

3.4  Assays for the detection of germ cell mutagens

    As  a general principle of genetic toxicology, genetic
damage  induced by chemicals  in somatic cells  results in
hazard  only  to  the affected  individual,  while genetic
effects in male or female germ cells may  cause  heritable
disease  or malformation in  the immediate progeny  or  in
future  descendants of the  affected individual.  The  ma-
jority of the assays represented in the CSSTT/2 study were
conducted  in cells from  somatic tissues and  are, there-
fore,  only  directly  interpretable in  terms  of somatic
mutation  and carcinogenic potential.  Although there have
been  attempts  to  extrapolate data  derived from somatic
cell assays to assessment of the probability of a chemical
inducing  germ  cell  mutations,  there  are  a  number of
assays, some of which were represented in this study, that
measure  genetic damage directly  in the germ  cells.  The

objectives behind the inclusion of germ cell assays in the
present study were to determine:

*   which  kind of germ cell  procedure effectively ident-
    ifies germ cell mutagens;

*   what  is  the  relationship between  the  induction of
    changes  in sperm morphology and  unscheduled DNA syn-
    thesis in germ cells and the formation of  true  heri-
    table mutations;

*   which  of the four  in vitro genotoxins  induce genetic
    damage in mammalian germ cells.

4.  CRITERIA FOR THE SELECTION OF THE FOUR TEST CHEMICALS

    Selection of the two pairs of chemicals, benzo [a] pyrene/
pyrene  (BP/PYR)  and 2-acetylaminofluorene/4-acetylamino-
fluorene  (2AAF/4AAF), was based, primarily, on their per-
formance  in the IPESTTC study.   In that study, seven  of
the  fourteen  chemicals  believed to  be  non-carcinogens
exhibited  a  high  frequency of  positive responses in  in
 vitro tests  while being predominantly or totally negative
in  a series of short-term  in vivo tests.  Although the  in
 vivo data  base  was  limited, the  non-carcinogens in the
BP/PYR  and 2AAF/4AAF pairs provided good examples of such
differences  in  response  and these  four  chemicals were
selected  as useful representative candidates for investi-
gating  the  in vivo behaviour of chemicals  known to be  in
 vitro genotoxins.

4.1  Activity of the four test chemicals in short-term  in vitro tests

    The majority of chemical carcinogens, including BP and
2AAF,  require some form of  enzyme-mediated biotransform-
ation  for  the  formation of  reactive metabolites.  Most
target  cells,  whether  bacteria, yeast  or cultured mam-
malian  cells, used in  in vitro tests lack the appropriate
enzyme activity and this is usually provided in  the  form
of an enzyme-rich fraction derived from homogenates of rat
liver,  referred  to as  the  S9 fraction.   Liver  enzyme
activity  is  usually  stimulated by  pre-treatment of the
animals with an enzyme-inducer such as Aroclor 1254.

    Both  the carcinogens, BP  and 2AAF, are  consistently
positive in bacterial assays and other  in vitro short-term
tests  and, with the  exception of certain  mammalian cell
assays,  demonstration of genotoxic activity  requires the
incorporation  of a rodent liver  enzyme system.  Positive
results have also be reported for the two non-carcinogens,
PYR and 4AAF, in a number of  in vitro systems but  not  on
the  scale nor  usually with  the same  potency  as  their
carcinogenic  analogues.  Thus, both  PYR and 4AAF  induce
mutations  in  bacteria,  gene conversion  in  yeast,  and
unscheduled  DNA  synthesis  (UDS) and  gene  mutation  in
cultured mammalian cells.  They do not, apparently, induce
mutations  in yeast or chromosome  aberrations in cultured
mammalian  cells.  4AAF, but  not PYR, can  induce UDS  in
cultured primary hepatocytes.

    In  the salmonella mutation  assay, separation of  the
carcinogen/non-carcinogen  pairs  is  influenced  signifi-
cantly by the nature of the rodent liver activation system
employed in the test.  When the liver enzyme suspension is
derived  from uninduced rodents, BP and 2AAF give positive
results  and the two  non-carcinogens, PYR and  4AAF,  are
generally  negative.  If Aroclor-induced animals  are used
as a source of the activation system, however, the ability
of the assay to discriminate between the  carcinogens  and
non-carcinogens is lost.

    A  comprehensive tabulation of  the activities of  the
four chemicals in short-term  in vitro tests is provided by
McGregor (1988b).

4.2  Summary of carcinogenicity data on the test chemicals

    The  rodent  carcinogenicity  data for  the  four test
chemicals is reviewed in Hicks et al. (1988).

    Both  BP  and 2AAF  are  well established  and  potent
carcinogens in rodents.  Fewer studies have been conducted
with the non-carcinogens, PYR and 4AAF, and their presumed
non-carcinogenicity,  although derived from limited rodent
bioassays,  must remain tentative pending  more comprehen-
sive testing.

    BP  is a locally-acting  carcinogen in rats  and mice,
producing   skin  tumours  after  dermal  application  and
tumours of the stomach when administered orally.  In mouse
skin,  it  has  been shown  to  have  both initiating  and
promoting  activity.  BP is also a lung carcinogen in mice
and  induces mammary gland tumours  in rats.  There is  no
evidence  that BP is hepatocarcinogenic in rats and only a
single,  unconfirmed report of the  induction of hepatomas
in mice.  On balance, the available evidence suggests that
BP does not induce liver tumours in rodents.

    PYR has not been tested for carcinogenic  activity  in
any  species other than the  mouse and even then,  only by
dermal  application.  These studies have shown fairly con-
clusively  that  PYR is  not  carcinogenic by  this route.
There  is some evidence from  promotion/initiation studies
on mouse skin that it may have weak  initiating  activity.
Because data on systemic carcinogenesis in the  mouse  and
information  from other species are  lacking, the carcino-
genicity  of PYR is somewhat equivocal.  However, the fact
that its analogue, BP, is such a potent  skin  carcinogen,
whereas the data obtained with PYR in fairly comprehensive
mouse  skin  studies  are  negative,  suggests  that   the
presumed non-carcinogenic status of PYR is justified.

    2AAF is a potent carcinogen in both rats and mice.  It
produces  tumours in the liver and bladder in both species
after  oral administration and in  the rat it is  carcino-
genic  in  Zymbal's  glands and  mammary  glands.   Little
information  is available about its activity when adminis-
tered by other routes.

    4AAF  has only been tested in rats by incorporation in
the diet, and its activity in mice has not  been  investi-
gated.  In rat feeding experiments, 4AAF has been consist-
ently  non-carcinogenic though there  are limited data  to
suggest  that  its  metabolite,  N -OH-4AAF,  may  have some
weak  carcinogenic  activity.  If  4AAF  does prove  to be
carcinogenic, its activity is clearly far less potent than
that  of 2AAF and  further long-term studies  are required
before  4AAF can be unequivocally regarded as non-carcino-
genic.  The question may be resolved when the  results  of
the  rat carcinogenicity study,  initiated as part  of the
CSSTT/2 project, are made available.

5.  SOURCE AND PURITY OF THE TEST CHEMICALS

    Samples  of the four test chemicals were obtained from
commercial  sources: BP, 2AAF,  and 4AAF were  supplied by
Lancaster  Synthesis Ltd., Eastgate, Whiteland, Morecambe,
Lancashire,  United  Kingdom,  and PYR  was  obtained from
Aldrich  Chemicals,  Gillingham,  Dorset, United  Kingdom.
4AAF was synthesized by a route that avoided contamination
with  2AAF.  The purity of the chemicals was determined by
the   supplying   laboratories  before   dispatch  to  the
participants.   All the chemicals were  greater than 99.5%
pure and full analytical details are provided by Paton and
Ashby (1988).

6.  SHORT-TERM  IN VIVO ASSAYS

    For  convenience  the  assays are divided into six groups:

*   cytogenetic (chromosome) assays conducted on cells from
    rodent bone marrow or other non-hepatic tissues;

*   assays investigating a variety of effects in cells from
    rodent liver;

*   miscellaneous assays that utilize other tissues or body
    fluids;

*   mouse coat colour spot tests;

*   assays in mammalian germ cells;

*   tests utilizing the fruit fly,  Drosophila melanogaster.

6.1  Cytogenetic assays

    Assays  for  the induction  of chromosome aberrations,
i.e.,  microscopically  visible alterations  in chromosome
structure,  were conducted in bone marrow cells from rats,
mice,  and  Chinese hamsters  and  also in  mouse  ascites
tumour cells.

    Micronucleus  tests were conducted  on cells from  rat
and mouse bone marrow and on erythrocytes  in  circulating
blood  from mice.  Micronuclei are chromosome fragments or
intact  chromosomes excluded from the  cell nucleus during
mitosis.   They are considered  to be evidence  of induced
chromosome  breakage  or  chromosome loss  and are usually
analysed in developing or mature erythrocytes.

    Sister  chromatid exchanges (SCE) can  be demonstrated
in  metaphase chromosomes by a differential staining tech-
nique and occur as a consequence of the exchange of repli-
cating  DNA  between  chromatids at  apparently homologous
loci.  Although considered to result from DNA breakage and
reunion,  the mechanism  of the  formation of  SCE is  not
fully  understood.  Assays for  the induction of  SCE were
conducted  in  bone  marrow  cells  from  rats,  mice, and
Chinese  hamsters,  in  circulating blood  leucocytes from
mice, and in Chinese hamster intestinal epithelium.

Table 1.  Assays employed in the CSSTT/2 study
-------------------------------------------------------------------------
1.    Cytogenetic studies

            Chromosome aberrations
                  Mouse bone marrow
                  Mouse ascites tumour cells
                  Rat bone marrow
                  Chinese hamster bone marrow
            Micronucleus tests
                  Mouse bone marrow
                  Mouse circulation blood cells
                  Rat bone marrow
            Sister chromatid exchanges
                  Mouse bone marrow
                  Mouse circulating blood lymphocytes
                  Rat bone marrow
                  Chinese hamster bone marrow
                  Chinese hamster intestinal epithelial cells

2.    Liver-specific assays

            Tests for initiation/promotion: altered enzyme foci
            Unscheduled DNA synthesis (UDS)
                  UDS in mouse liver
                  UDS in neonatal, weanling and adult rat liver
            Frequency of S-phase hepatocytes
                  S-phase in mouse liver
                  S-phase in weanling and adult rat liver
            Cytogenetic tests
                  Aberrations in rat liver epithelial-like cells
                  SCE in rat liver epithelial-like cells
                  Micronuclei in hepatocytes
                  Diploid/tetraploid ratio in rat and mouse hepatocytes
            Primary changes in DNA
                  Alkaline elution assay for DNA strand damage
                  DNA/protein cross-links
                  DNA unwinding assay for strand damage

3.    Miscellaneous assays

            Specific carcinogenicity assays
                  Two-year oral dosing study in rats
                  Mouse lung adenoma assay
                  Quail egg tumour-induction
            UDS in rat fore-stomach
            Sebaceous gland suppression assay
            Observation of dermal epithelial hyperplasia
            Transformation of rat peritoneal macrophages
            6-Thioguanine-resistant mutations in Syrian hamster lung cells
            Measurement of DNA adducts
                  32P-post-labelling in rat and mouse tissues
                  Immunochemical detection of DNA adducts
                  Radiolabelled test chemicals - rat liver
-----------------------------------------------------------------------------

Table 1. (contd.)
-----------------------------------------------------------------------------
3.    Miscellaneous assays (contd.)

            Immunotoxicity assays
                  Natural Killer (NK) cell and T-cell cytotoxicity in rats
            Host-mediated assays
                  Mutation of salmonella in mouse tissues
                  Genetic changes in yeast cells in mouse tissues
            Urine mutagenicity tests in rats, mice and guinea pigs

4.    Mouse spot tests

            Mouse coat colour spot test
            Mouse melanocyte assay

5.    Mammalian germ cell studies

            Dominant lethal assays with male and female mice and male rats
            Morphological abnormalities in mouse and rat spermatozoa
            Unscheduled DNA synthesis in rat and mouse male germ cells

6.    Drosophila assays

            Sex-linked recessive lethal mutations in germ cells
            Chromosome loss in germ cells
            Somatic mutation and recombination: mosaic spots in eyes 
              and wings
-----------------------------------------------------------------------------

6.2  Assays in rodent liver cells

    A variety of liver-specific assays were represented in
the  study, including the demonstration of enzyme changes,
a  number  of  different  tests  on  DNA,  and cytogenetic
assays.

    The  rat liver assay for altered enzyme foci employs a
histochemical technique that identifies groups of cells or
foci that have elevated levels of the marker enzyme  gamma-
glutamyltranspeptidase   (GGT).   The observation  of GGT-
positive  foci often precedes the appearance of liver car-
cinoma and the test is used to attempt to  identify  early
stages of carcinogenesis in the liver. Protocols have been
devised  to  investigate  both  initiation  and  promotion
stages of carcinogenesis.

    A  number  of  investigators used  assays that measure
directly  or indirectly the  response of DNA  to  chemical
damage.  These included tests for breaks in single strands
of  DNA using the alkaline elution technique, an assay for
the induction of crosslinks between DNA and  protein  mol-
ecules, and a measure of single strand breaks based on the
degree  of unwinding of DNA molecules.  One consequence of

DNA  breakage  is  the initiation  of  the enzyme-mediated
repair  process which involves the synthesis of new, rela-
tively short strands of DNA to repair the break. This type
of repair is referred to as unscheduled DNA  synthesis  or
UDS  (to differentiate from the normal or S-phase DNA syn-
thesis that occurs during cell replication). DNA synthesis
can  be measured by observing the uptake of tritiated thy-
midine  by the newly synthesized  strands using autoradio-
graphical techniques.  Assays for the induction of UDS and
to  measure changes  in the  numbers of  S-phase cells  in
rodent liver were included in the study.

    The cytogenetic methods included the analysis of meta-
phase  chromosome aberrations, micronuclei and  SCE in rat
liver cells, and the determination of the ratio of diploid
to tetraploid cells in rat and mouse liver.

6.3  Miscellaneous assays

    The  first group of assays to be considered under this
heading  are those that are directly related to the induc-
tion of tumours in rodents.  The most important  of  these
is  a 2-year oral dosing study in rats to compare the car-
cinogenicity of 2AAF and 4AAF.  Although the study was not
completed at the time of this report,  preliminary  infor-
mation from a small group of rats  examined after 26 weeks
of dosing is available.  Also included in this  group  was
a) the mouse lung tumour assay that determined the effects
of  each of the  four test chemicals  on the incidence  of
lung  adenoma in mice and  b) a test in  Japanese quail in
which  the test chemicals  were introduced into  the  yolk
sacs  of quail  eggs.  The  birds were  then examined  for
evidence of tumours at various intervals after hatching.

    The sebaceous gland suppression test is based  on  the
observation  of  morphological  changes to  the  sebaceous
glands  in histological preparations  of mouse skin  after
dermal  exposure to the  test chemicals.  The  presence of
epidermal  hyperplasia can also be detected histologically
and both methods have been shown to respond to  skin  car-
cinogens, particularly polycyclic hydrocarbons.

    Genotoxic  carcinogens can bind covalently to biologi-
cal  macromolecules  such as  DNA  either before  or after
metabolic  biotransformation.  The products of DNA-binding
are  referred to as DNA  adducts and a number  of investi-
gators provided data on the detection and  measurement  of
DNA adducts with the test chemicals in tissues  from  rats
and mice. Three different methods were represented:

*   the enzyme-linked immunoabsorbent assay (ELISA);

*   the 32P-post-labelling assay;

*   an assay for radiolabelled chemicals bound to DNA.

    Two similar assays for the investigation of the immune
response of animals to exposure to carcinogens  were  rep-
resented.   Natural Killer (NK) cells  are leucocytes with
the  ability to lyse a  variety of `foreign' cells,  e.g.,
those with a malignant phenotype.  The NK cell  assay  was
used to investigate the capacity of NK cells, derived from
the  splenic mononuclear leucocytes  of rats treated  with
2AAF  or  4AAF,  to lyse  human  erythroleukaemic cells  in
 vitro.  The basis of the T-cell assay is  that  immunocom-
petent T-cells are able to react to tissue changes induced
by  carcinogenic chemicals in rats.  The changes in T-cell
activity  can be determined by  their cytotoxicity towards
target cells derived from rat intestinal adenocarcinoma.

    The  rat peritoneal cell transformation  test is based
on  the  observation  that  mitogen-stimulated  peritoneal
cells, harvested from rats dosed with carcinogens, undergo
a  form of transformation that enables them to proliferate
and produce colonies in soft agar culture.

    Host-mediated  assays were used  fairly widely in  the
early  l970s  as  primary screening  tests  for  mutagenic
chemicals. In its original form, a suspension of microbial
target  cells, i.e. yeast or bacteria, was introduced into
the  peritoneal cavities of  mice.  The mice  were treated
with the suspect chemical and, after an appropriate inter-
val,  the target cells were  harvested and changes in  the
mutation frequency or other genetic end-points were deter-
mined  in  in vitro culture.  Since  that time, there  have
been  several  modifications  to the  procedure,  the most
significant  being the injection  of the target  organisms
into  the circulating blood of the host.  In this way, the
bacterial or yeast target cells are distributed in various
organs  and can, for example, be harvested from the liver,
lungs, and kidney.  The target cells can, in principle, be
affected  by reactive metabolites generated  from the test
chemical  in these organs.  Intra-sanguinous host-mediated
assays  using either Salmonella typhimurium  or the yeast,
 Saccharomyces  cerevisiae,  were represented in the study.

    Urine  and  other body  fluids  can be  collected from
rodents  treated with test chemicals and assayed for muta-
genic  activity.  For example, after appropriate treatment
to release conjugated metabolites, urine can be tested for
the  presence of mutagens in a conventional  in vitro  bac-
terial mutation assay.  In principle, the urine  assay  is
capable  of  detecting  mutagenic chemicals  excreted  un-
changed or after metabolic activation.

6.4  The mouse spot test

    The  mouse spot test detects mutations induced in mel-
anocyte  precurser cells in  the embryos of  specially de-
rived strains of mice.  The mouse strain carries recessive
mutations at a number of specific coat colour loci and the
embryos  are, thus, heterozygous at these loci.  Mutations
induced  at the wild-type  or normal alleles  of the  coat
colour  loci result in  the development of  clones of  the
mutant  melanocytes.   The  young mice  are examined after
birth and the mutations are expressed as patches  of  con-
trasting  coloured fur.  The mutations can result from one
of  a number of  genetic events including  gene mutations,
chromosome  deletions, mitotic crossing-over, and  loss of
whole  chromosomes. In addition to  the conventional tech-
nique of observing the appearance of coloured spots in the
fur,  a modification of  the spot test  allows the  micro-
scopic  recognition of mutant melanocytes  in preparations
of embryonic skin.

6.5  Mammalian germ cell studies

    The  classical dominant lethal  assay in male  rodents
involves the treatment of the animals with the test chemi-
cal and then mating with groups of females at intervals to
cover  the complete spermatogenic cycle.   Dominant lethal
mutations induced at any stage of spermatogenesis  can  be
detected  by dissection of  the uterine contents  of  each
female  and are  characterized by  dead fetuses  or a  re-
duction  in the numbers of  fetal implantations.  Dominant
treatment of male rats, male mice, or female mice.

    Two  other assays represented  in this group  were the
abnormal  sperm morphology assay  and the unscheduled  DNA
synthesis  (UDS)  test.  The former  assay monitors mature
spermatazoa  for irregularities in morphology at intervals
after treatment of rodents with the test  chemicals.   The
UDS  test measures DNA repair  in developing spermatocytes
and spermatids.

6.6  Drosophila assays

    Although data from drosophila tests cannot be regarded
as  appropriate alternatives to mammalian data for assess-
ing  the potential hazard to humans from exposure to geno-
toxic chemicals, drosophila are, in fact, intact, complex,
eukaryotic  organisms whose metabolic and  genetic charac-
teristics have some parallels with those of the whole mam-
mal.  Their main value in genotoxicity testing  lies  with

the  availability of assays to study the effects of chemi-
cals on both somatic and germ cells. The tests represented
in the CSSTT/2 study were:

*   the sex-linked recessive lethal mutation assay in
    germ cells;

*   tests for chromosome loss from germ cells;

*   assays for somatic mutation and recombination using
    the induction of mosaic spots in either the wings or
    the eyes.

7.  RESULTS

    The investigators met at Cap d'Agde, France,  from  15
to  21 May 1985, to assess the results of the study.  Each
group  of investigators presenting data  from a particular
type  of assay discussed their data and individual results
were  assessed and agreed.  This led to the preparation of
consensus  reports on the  response of each  assay to  the
four  test  chemicals.   These consensus  views  were then
incorporated  into  coordinators' summary  reports on each
group of assays. Summary reports were presented and criti-
cally  discussed  in  open  plenary  discussions,  thereby
allowing  the  overall  conclusions  and   recommendations
resulting  from the study  to be formulated.   Reports  of
individual   investigators,   the   coordinators   summary
reports,  and certain technical  appendices form the  main
text of the final publication together with  an  editorial
overview of the study (Ashby et al., 1988).

    The  purpose  of  this chapter  of  the  report is  to
present the results of the  in vivo studies with  the  four
test  chemicals and to construct a profile, in qualitative
terms,  of their genotoxic  activity in the  whole animal.
Quantitative  differences in response in  relation to sex,
species,  route of exposure, and  technical variations are
considered  in more detail in the next section, which also
includes  a comprehensive tabulation of the data generated
by  individual investigators (Table 4).   However, as many
of the assays were replicated in a number of laboratories,
a simplified table of results is presented here (Table 2).
It  must be emphasized that Table 2 contains the consensus
views  of  the  Working Groups  assessing  the  individual
results  and performance of each  assay and that, in  some
cases,  there were conflicting  results on the  same assay
from participating laboratories.

Table 2.  Results of the short-term  in vivo tests summarized 
          by Working Groups a
----------------------------------------------------------------------------
                                                      BP    PYR   2AAF  4AAF
1.    Cytogenetic studies

            Chromosomal aberrations
                  Mouse bone marrow                   P     N     ?     N
                  Mouse ascites cells                 P     NT    N     N
                  Rat bone marrow                     P     N     ?     N
                  Chinese hamster bone marrow         P     N     P     N

            Micronucleus tests
                  Mouse bone marrow                   P     N     P     N
                  Mouse blood - maternal              P     N     N     N
                  Mouse blood - fetal                 P     N     ?     N
                  Rat bone marrow                     P     N     P     N

            Sister chromatid exchange
                  Mouse bone marrow                   P     W     P     W
                  Mouse blood                         P     N     P     N
                  Rat bone marrow                     P     W     P     W
                  Chinese hamster bone marrow         P     N     P     N
                  Chinese hamster intestinal cells    P     N     P     N

2.    Liver-specific assays

            Altered enzyme foci - rat                 P     N     P     W
            Unscheduled DNA synthesis
                  Mouse liver                         N     N     N     N
                  Rat liver                           N     N     P     W
            S-phase hepatocytes
                  Mouse liver                         NT    NT    W     P
                  Rat liver - adult                   W     N     ?     P
                  Rat liver - weanling                P     NT    NT    NT
            Cytogenetic tests
                  Metaphase aberrations - rat         W     N     W     N
                  Micronuclei - rat                   N     N     P     N
                  SCE - rat                           W     N     ?     N
                  Ploidy - rat                        P     N     P     N
                  Ploidy - mouse                      P     N     P     N
            Primary DNA changes
                  Alkaline elution                    N     N     ?     ?
                  DNA/protein cross-links             P     N     P     NT
                  DNA unwinding                       N     N     P     N
                  with repair inhibitor               P     N     P     N

3.    Miscellaneous assays

            Carcinogenicity
                  Oral dosing - rat                   NT    NT    P     ?
                  Mouse lung adenoma                  P     N     P     N
                  Quail egg tumours                   ?     ?     ?     ?
                  UDS in rat fore-stomach             ?     N     N     N

--------------------------------------------------------------------------

Table 2.  (contd.)
----------------------------------------------------------------------------
                                                      BP    PYR   2AAF  4AAF
3.    Miscellaneous assays (contd.)

            Sebacious gland suppression - mouse       P     N     NT    NT
            Epithelial hyperplasia - mouse            P     N     NT    NT
            Peritoneal macrophages - rat              N     N     N     N
            Syrian hamster lung - mutation            P     N     ?     N
            DNA adducts
                  32P-post-labelling - rat            P     N     P     W
                  Immunochemical (ELISA)              NT    NT    ?     ?
                  Radiolabelled chemicals             NT    NT    P     P
            Immunotoxicity
                  Natural Killer (NK) cells           NT    NT    P     N
                  T-cell cytoxicity                   P     N     P     W
            Host-mediated assays - mouse
                  Salmonella typhimurium              N     N     N     N
                  Saccharomyces (liver)               P     P     P     P
                  Saccharomyces (lung)                N     N     N     N
            Urine mutagenicity
                  Rats                                P     P     P     P
                  Mice                                W     W     P     P
                  Guinea-pig                          NT    NT    P     NT

4.    Mouse spot tests

            Coat colour spots                         P     N     P     N
            Melanocyte assays                         NT    NT    P     N

5.    Mammalian germ cells

            Dominant lethal assays
                  Male mice                           P     N     N     N
                  Female mice                         P     N     N     N
                  Male rats                           NT    NT    N     N
            Sperm abnormalities
                  Mice                                P     N     P     N
                  Rats                                NT    NT    N     N
            UDS in germ cells
                  Mice                                N     N     N     N
                  Rats                                N     N     N     N

6.    Drosophila assays

            Sex-linked recessives                     N     N     N     N
            Chromosome loss                           N     N     N     N
            Somatic mutation
                  Eye spots                           P     N     P     N
                  Wing spots                          P     N     W     ?
--------------------------------------------------------------------------
a   P   =   positive
    W   =   weak positive
    N   =   negative
    ?   =   results inconclusive or not yet reported
    NT  =   not tested

7.1  Benzo [a] pyrene (BP) and pyrene (PYR)

7.1.1  Cytogenetic studies

    Assays  for  the  induction of  structural  chromosome
aberrations  or  micronuclei  in rodent  bone marrow cells
were  consistently positive with BP and negative with PYR.
Among a number of experimental variables between different
investigators,  neither the species, strain, sex, solvent,
nor route of exposure affected the qualitative result.  BP
increased  the  incidence  of sister  chromatid  exchanges
(SCE)  in mouse, rat, and  Chinese hamster bone marrow  in
every  study.  The results  with PYR, however,  were  less
clear,  and  this  presumed non-carcinogen  induced SCE at
high  dose levels in  mice after oral  or  intraperitoneal
dosing  and in male rats after oral administration, i.e. 3
of 9 SCE studies reported a weak positive result. Analysis
of  micronuclei  in  circulating blood  erythrocytes  from
maternal,  fetal, or weanling mice, and SCE in circulating
blood  lymphocytes  from adult  mice clearly discriminated
between BP and PYR.

7.1.2  Liver-specific assays

    Five  investigators provided data from observations of
altered enzyme foci in rat liver under the general heading
of  initiation/promotion  assays.   There  were,  however,
significant  protocol  variations  between  investigators.
Two protocols required administration of the rodent tumour
promotor,  phenobarbitone,  in the  drinking-water or diet
for   some  weeks  after  dosing  in  order  to  determine
initiating  activity of the test chemicals.  The promoting
activity  of the test chemicals  was studied by two  other
workers by producing initiation events in the liver before
treatment  with  the  test chemicals.   The fifth protocol
tested  the  ability  of  the  chemicals  to  induce  both
initiation  and  promotion without  discriminating between
the  two phases.  In all  cases, the evidence for  induced
pre-cancerous  changes  was  the observation  of  foci  or
clones  of cells with altered  enzyme characteristics.  BP
was shown to have significant initiating activity  in  rat
liver  in  two studies  and, in a  third study, was  shown
capable   of  promoting  nitrosamine-induced   initiation.
Using  a  fourth, essentially  experimental, protocol that
involved the transplantation of donor liver cells  to  the
host  animal, BP failed  to show promoting  activity.   No
evidence  of tumour initiating  or promoting activity  was
observed in any of the studies with PYR.

    The  rodent liver assay for  unscheduled DNA synthesis
measures  the induction of repairable lesion in the DNA of
hepatocytes  and,  theoretically,  should  be  capable  of
detecting chemicals that are metabolized in the  liver  to
produce   genotoxic  (i.e.  DNA-interactive)  metabolites.

Both  BP  and  PYR  were  reproducibly  negative  in mouse
hepatocytes  and in hepatocytes  from adult, weanling,  or
neonatal rats using either oral or intraperitoneal dosing.
The   induction   of   S-phase  DNA   synthesis  was  also
investigated and BP was shown to increase the incidence of
S-phase  cells  in  weanling  rats  and,  in  one  of  two
experiments,  a  slight  increase  in  S-phase  cells  was
observed  in adult rats.  In  mice, BP had no  significant
effect  on the  incidence of  S-phase, and  PYR showed  no
evidence   of   S-phase  synthesis   induction  in  either
species.

    Limited data were presented on the cytogenetic effects
of  the chemicals in liver-derived  cells.  In epithelial-
like cells from weanling rats, BP was observed to induce a
small  increase in the incidence  of structural chromosome
aberrations  and SCE; tests with PYR were negative.  These
findings,  however, require confirmation.  No  evidence of
micronucleus-induction  was detected in rats  treated with
BP  or PYR after partial hepatectomy.  Using a cytofluoro-
metric  method, an  increase in  the ratio  of diploid  to
tetraploid cells was observed in liver-derived cells after
treatment of rats with BP, but not after dosing with PYR.

    Three  different assays were used to study the ability
of the chemicals to induce single-strand breaks  (SSB)  in
liver cell DNA.  Both BP and PYR were  uniformly  negative
in the alkaline elution assay for SSB conducted  in  three
laboratories.  BP, however, was shown to induce crosslinks
between  DNA and protein, while  PYR was negative in  this
assay.  The third assay in this group was a measure of SSB
based  on the degree  of alkali-induced unwinding  of  DNA
molecules.   When  the  DNA-repair  inhibitor,   adenosine
arabinoside was incorporated, BP was shown to  induce  SSB
in  mouse liver  cell DNA  using this  technique.  In  the
absence  of the repair  inhibitor, SSB were  not observed.
PYR  produced negative results in the DNA-unwinding assay.
The results from this group of assays suggest that  BP  is
capable  of inducing SSB in  the DNA of mouse  liver cells
that  are effectively repaired  in the absence  of a  DNA-
repair inhibitor.  In rats, the observation of DNA/protein
crosslinks  tentatively suggests the  induction of SSB  in
rat  liver cells, but  additional studies are  required to
assess the DNA effects of BP in rat liver.

7.1.3  Miscellaneous assays

    Two investigators are producing data that are directly
related  to the induction of tumours.  In the lung adenoma
assay,  mice were injected  with BP or  PYR by the  intra-
peritoneal  route, 2-3 times each week, for up to 8 weeks.
The  mice were examined  23 weeks after  the start of  the
study  and the numbers  of adenoma on  the surface of  the
pulmonary  lobes were recorded.  BP  induced a significant
increase  in adenomas compared with  the untreated control

group  while no increase in the induction of these tumours
was  observed  in  mice  injected  with  PYR.   This assay
differentiated  very  clearly  between  the   carcinogenic
activity of BP and PYR.  The second study in this category
involved  injecting the test material into the yolk sac of
quail eggs and then observing the birds for  the  presence
of tumours at intervals after hatching.  In birds examined
at approximately 3 months, there was no evidence of tumour
induction  by either BP or  PYR.  Surviving birds will  be
maintained for up to 12 months before being  examined  for
the  presence  of  tumours and,  therefore,  a  definitive
conclusion on the carcinogenicity of BP and PYR  to  quail
is not yet possible.

    One investigator studied the induction of UDS  in  the
fore-stomach of rats after oral dosing; the data  with  BP
were  considered to be  equivocal while PYR  gave negative
results.

    Two assays involved the application of the test chemi-
cals to mouse skin followed by histological examination of
skin  sections.   BP  induced epithelial  hyperplasia  and
suppression of sebaceous glands, both of which  have  been
correlated   with   polycyclic  hydrocarbon-induced   skin
carcinogenesis.   PYR  failed  to elicit  either  of these
responses.

    The  transformation of rat peritoneal  macrophages has
been  advocated  as  a short-term   in vivo test for poten-
tially  carcinogenic chemicals.  Macrophages isolated from
rats  dosed with BP  or PYR, however,  failed to show  any
evidence  of  transformation  in experiments  conducted in
three laboratories.

    Somatic  mutation data on  cells isolated from  Syrian
hamster lung tissue, after dosing with the test chemicals,
were  presented  by  one investigator.   Cultures of cells
from  animals dosed with BP  showed a higher frequency  of
mutations  at  the  6-thioguanine locus  than  those  from
untreated  hamsters,  suggesting the  induction of somatic
mutations in lung cells from this species.   PYR  produced
negative mutation data in this assay.

    32P-labelling   of purified DNA was used to detect DNA
adducts in tissues from rats and mice treated intraperito-
neally  with the test chemicals.  Measurable levels of PYR
adducts were not detected in DNA from mouse  liver,  lung,
or  kidney, or from rat liver or lung tissues.  BP adducts
were identified in all of these tissues.

    Only one of the two assays for immunotoxicity was con-
ducted with the BP/PYR pair. Thus, the T-cell assay showed
that  BP, but not PYR, was capable of inducing a cytotoxic
response in rat T-cells.

    Two  investigators  provided  data from  host-mediated
assays  in mice dosed with  BP or PYR.  In  tests with the
yeast,  Saccharomyces  cerevisiae,  a significant  increase
in  mitotic gene conversion  was detected in  yeast  cells
isolated  from the liver of  mice dosed with either  BP or
PYR,  while yeast cells isolated from lung tissue produced
negative  data with both  chemicals.  No evidence  of  the
induction   of   mutations  was   observed  in  Salmonella
typhimurium isolated from liver tissues of mice dosed with
BP or PYR.

    Assays  for the detection  of mutagens in  urine  were
conducted with both rats and mice.  Mutagenic activity was
detected in urine samples from both species  after  dosing
with  BP or PYR and, therefore, the test failed to differ-
entiate between the two chemicals.

7.1.4  Mouse spot tests

    Only one investigator provided mouse spot test data on
BP and PYR.  Using the observation of coat colour spots in
off-spring as evidence of somatic mutation, BP was clearly
positive and PYR negative.

7.1.5  Mammalian germ cell assays

    BP  induced dominant lethal mutations in both male and
female mice after intraperitoneal injections but not after
oral dosing.  The response in the male was  highly  stage-
specific;  only the mature spermatozoa  were sensitive and
spermatid  and spermatocyte stages were  unaffected. Domi-
nant lethal assays with PYR were reproducibly negative.

    Studies  of  morphological  abnormalities  in   mature
spermatozoa   were  conducted  in   mice  after  oral   or
intraperitoneal dosing.  BP was uniformly positive in this
assay,  regardless of the  route of exposure.   Although a
weak  positive  response  was  observed  in  one  of seven
experiments  with PYR, this observation  was not confirmed
and the consensus view was that PYR did not  induce  sperm
abnormalities.  In studies of the induction of unscheduled
DNA synthesis in rat and mouse male germ  cells,  negative
results were obtained with both BP and PYR.

7.1.6  Drosophila assays

    In  all, ten studies were conducted with BP and PYR in
drosophila  and in each case  the oral route of  treatment
was   used.   Germ  cell  studies,   i.e.  the  sex-linked
recessive lethal mutation assay and the test  for  chromo-
some  loss, were uniformly  negative with both  chemicals.
In contrast, tests for somatic mutation and recombination,
based on the observation of eye or wing spots, showed that
BP  was able to induce genetic changes in somatic cells in
each of three studies.  PYR was consistently negative.

7.2  2-Acetylaminofluorene and 4-acetylaminofluorene

7.2.1  Cytogenetic studies

    Seven  investigators  provided  data from  analysis of
metaphase  chromosomes in mouse  bone marrow cells.   2AAF
induced  a  significant  increase  in  the  incidence   of
structural  chromosome  aberrations  in only  two of seven
assays,  one of which  was considered to  be inconclusive.
The  results of assays  with 4AAF were  negative with  the
exception  of two assays in which the data were inconclus-
ive.  In contrast, 2AAF was reproducibly positive and 4AAF
consistently  negative  in  15 of  16  micronucleus  tests
conducted in mouse bone marrow erythrocytes.  The route of
exposure,  i.e. oral dosing or  intraperitoneal injection,
did  not appear to influence the outcome of these studies.
Micronuclei were produced by 2AAF in rat bone marrow cells
and  a small  increase was  also recorded  in one  of  two
assays  with 4AAF.  Two workers investigated the influence
of  these  two chemicals  on  the incidence  of structural
chromosome  aberrations  in  Chinese hamster  bone marrow;
2AAF  was positive in  this species and  4AAF gave a  weak
positive result in one of the two studies.  Both chemicals
failed  to  induce  detectable chromosome  damage in mouse
ascites   tumour  cells.   Four  micronucleus  tests  were
conducted  with mouse blood erythrocytes.   An increase in
micronuclei was recorded by one worker in blood cells from
weanling  mice dosed with  2AAF, although when  samples of
maternal  or  fetal  blood  were  examined,  increases  in
micronuclei were not apparent.  However, studies conducted
in  another  laboratory showed  that  2AAF was  capable of
increasing  the incidence of  micronuclei in fetal  blood.
Thus,  2AAF is clearly  clastogenic to rodent  bone marrow
cells while 4AAF was considered by most  investigators  to
be devoid of clastogenic activity.

    Nine  assays for sister chromatid exchanges (SCE) were
conducted in bone marrow cells from mice, rats, or Chinese
hamsters.   2AAF was clearly positive in this test and the
consensus  view was that  4AAF, although negative  in four
assays,  was capable of inducing a small (but significant)
increase  in SCE.  In  studies with mouse  lymphocytes and
Chinese  hamster  intestinal  epithelial cells,  2AAF  was
positive and 4AAF was considered to be negative.

7.2.2  Liver-specific assays

    As  discussed in section 7.1.2, there were a number of
variations   between  the  five  protocols   used  in  the
initiation/promotion  assays.  Significant initiation  and
promotion  activity was demonstrated in  studies with 2AAF
based  on the observation  of foci of  cells with  altered
enzyme  characteristics in rat liver.  These assays failed
to  discriminate clearly between the  two chemicals, since

4AAF  was  shown to  be an initiator  in the hands  of one
investigator  and had weak  promoting activity in  another
study.   Both  chemicals,  therefore,  proved  capable  of
producing  these pre-cancerous changes in rat liver tissue
though with different degrees of potency.

    2AAF  and 4AAF failed to induce detectable unscheduled
DNA  synthesis  (UDS)  in  mouse  hepatocytes  after  oral
dosing.   Quite  different  results were  obtained  in rat
liver,  however, and 2AAF was  shown to induce UDS  in rat
hepatocytes  in each of  the seven laboratories  that sub-
mitted data. Three of these laboratories also reported the
detection  of a small increase in UDS in rats after dosing
with  4AAF.  The other four laboratories obtained negative
data.  Thus, 2AAF induced UDS in hepatocytes from rats but
not mice, and 4AAF elicited a weak UDS response in the rat
and was negative in mouse cells.

    The results of the analysis of S-phase  DNA  synthesis
indicated  that the presumed  non-carcinogen, 4AAF, was  a
very  potent inducer of S-phase cells in the liver of both
rats  and mice.  2AAF, on  the other hand, induced  only a
small  increase in S-phase  cells in the  mouse and  three
studies in rat liver produced one clear positive  and  two
negative results.

    In  cytogenetic experiments in cells  derived from rat
liver,  2AAF appeared to  induce a small  increase in  the
incidence  of  structural  aberrations and,  in  partially
hepatectomised  rats,  a  significant increase  in  micro-
nuclei.  4AAF gave negative  results in tests  for chromo-
somal  aberrations,  micronuclei,  and SCE  in  rat  liver
cells.   When the ratio of diploid to tetraploid cells was
investigated  in rat and mouse  liver, an increase in  the
ratio  was observed in both species after dosing with 2AAF
but not in response to 4AAF.

    The  alkaline elution assay  for single strand  breaks
(SSB)   produced conflicting results between three labora-
tories.   Two  investigators presented  negative data from
assays  with both chemicals  while a third  observed  that
both 2AAF and 4AAF produced SSB in this test.   Using  the
detection  of  DNA/DNA  or  DNA/protein  crosslinks  as  a
measure  of SSB, 2AAF was  shown to produce SSB.   No data
were  presented on 4AAF.  Another  investigator calculated
SSB from the amount of DNA unwinding induced by the chemi-
cals.  2AAF and 4AAF gave positive and  negative  results,
respectively, either in the presence or absence of  a  DNA
repair  inhibitor.  These data suggest  that, unlike 4AAF,
2AAF  is capable of producing  SSB in rat liver  DNA under
appropriate experimental conditions.

7.2.3  Miscellaneous assays

    A  2-year  oral  dosing carcinogenicity  study in rats
with  2AAF  and 4AAF  is  still in  progress.  Preliminary
findings  in a small number  of animals examined after  26
weeks of dosing showed that an increase in  liver  tumours
was already apparent in animals dosed with 2AAF; there was
no  evidence of tumour  induction in the  4AAF-dosed  ani-
mals.

    The  lung  adenoma assay,  in  which mice  were  given
intraperitoneal injections of the test chemicals 2-3 times
a week for up to 8 weeks, clearly separated 2AAF and 4AAF.
2AAF  induced a significant increase in adenomas while the
incidence in mice dosed with 4AAF was comparable with that
of untreated mice.

    There was no evidence of the induction of UDS  in  rat
fore-stomach  after oral dosing with  either chemical, and
the assay for the induction of somatic mutations in Syrian
hamster  lung cells produced  inconclusive data with  2AAF
and was negative in tests with 4AAF.  The  rat  macrophage
test was reproducibly negative with both 2AAF and 4AAF.

    Three  techniques were applied to  detect the presence
of  DNA  adducts formed  in  various tissues  after dosing
rodents  with  the test  chemicals.   Data from  the ELISA
technique, which requires the use of monoclonal antibodies
to detect specific adducts, are not yet  available.   How-
ever,  the 32P-post-labelling  method indicated  that 2AAF
formed adducts with DNA in rat liver and lung and in mouse
liver,  lung, and kidney after intraperitoneal dosing.  No
data  were presented from  mice dosed with  4AAF.  In  the
rat,  however, 4AAF formed adducts  in liver and lung  DNA
though at a much lower rate than 2AAF.  In  studies  using
radiolabelled test materials, both chemicals were shown to
form adducts with DNA in the liver and lungs of rats after
intraperitoneal  administration, although adduct formation
was considerably less with 4AAF than with 2AAF.

    2AAF  produced positive results in both immunotoxicity
assays.   However,  although  4AAF  was  negative  in  the
Natural  Killer  (NK) cell  assay,  data from  the  T-cell
cytotoxicity  test  suggested  that  it  was  capable   of
inducing a measurable cytotoxic response in rat T-Cells.

    Nine  sets of data  were considered from  mouse  host-
mediated  assays.  In eight of these, including assays for
genetic  changes in yeast  isolated from liver,  lung  and
kidney,  and  for  mutations in  salmonella  isolated from
liver  tissue, 4AAF was uniformly negative.  2AAF produced
two weak positive responses and five negative  results  in
the same eight studies.  The ninth assay,  however,  indi-
cated  that both chemicals induced mitotic gene conversion

in yeast isolated from liver tissue.  In  addition,  muta-
genic materials were detected in urine samples  from  rats
and  mice after dosing with either isomer and from guinea-
pigs  after  dosing with  2AAF.   4AAF data  on guinea-pig
urine were not available.

7.2.4  Mouse spot tests

    Six separate mouse spot tests were conducted with 2AAF
and  the results were equally divided between positive and
negative.  An increase in coat colour spots  was  detected
in  two studies while  in two others,  no evidence of  the
induction  of coat colour spots  was observed.  Similarly,
in  the  melanocyte assay,  one  positive result  and  one
negative were reported.  Four of five spot tests conducted
with  4AAF were negative and the data were inconclusive in
the fifth.

7.2.5  Mammalian germ cell assays

    Dominant  lethal mutation assays conducted  in male or
female  mice or male rats  and tests for the  induction of
UDS in rat or mouse male germ cells were  uniformly  nega-
tive with both compounds.  In the test  for  morphological
abnormalities  in mature spermatozoa, two  assays in which
2AAF  was given orally to mice or rats were also negative.
After  intraperitoneal  dosing,  however, 2AAF  induced  a
significant  increase in the  incidence of abnormal  mouse
sperm.   4AAF was negative  in both species  regardless of
the  route of exposure.  These data suggest that, although
2AAF  or its metabolites appear capable of penetrating the
testes  and  adversely  affecting sperm  morphology  after
intraperitoneal  dosing, there is no evidence for interac-
tion of 2AAF metabolites with germ cell DNA.

7.2.6  Drosophila assays

    The   mutagenic  effects  of  the   two  chemicals  on
drosophila germ cells were studied in tests for sex-linked
recessive  lethal mutations in  five laboratories and  for
chromosome  loss in two  laboratories.  Data from  six  of
these  studies  indicated  that neither  chemical produced
genetic  damage in drosophila.  In the seventh laboratory,
however,  2AAF  was  clearly mutagenic  in  the sex-linked
recessive  lethal  assay;  4AAF produced  a  weak positive
result in this study.

    2AAF induced eye or wing spots in somatic mutation and
recombination  assays in drosophila, although the response
was weak in two of the studies.  The somatic mutation data
generated  from tests with 4AAF  were ambiguous, producing
one  weak positive result, one inconclusive result and one
negative.

7.3  Summary of the  in vivo genotoxicity of the four chemicals

    A  comprehensive  in vivo genotoxicity profile has been
generated  on the four test  chemicals, each of which  had
previously  shown  evidence  of mutagenic  activity  in  in
 vitro screening  tests.  It is relevant,  in this section,
to  consider the mammalian genotoxicity in relation to the
hazard  assessment procedures recommended by many regulat-
ory bodies.  Table 3 shows the mutagenic activity  of  the
chemicals  in the  in vivo tests common to most legislative
guidelines.  Based on these results, and in the absence of
pharmacokinetic and metabolic data, BP would be considered
to  present a potential mutagenic  and carcinogenic hazard
and  2AAF  would be  regarded  as a  potential carcinogen.
These  observations  are,  of course,  corroborated by the
established carcinogenic activity of these two chemicals.

    The  abbreviated data base  shown in Table  3 suggests
that  neither 4AAF nor PYR present a significant genotoxic
hazard  and it is useful  to consider whether the  compre-
hensive  data  generated  in CSSTT/2  support this initial
prediction.   The  summarized consensus  results (Table 2)
show that conclusive data for PYR were presented  from  48
distinct   in vivo tests and PYR was considered to be nega-
tive in 43 of these.  The remaining five tests suggested a
weak induction of SCE in rodent bone marrow that  was  not
reproduced  in all laboratories,  a single positive  host-
mediated assay, and evidence of the excretion of mutagenic
metabolites in rodent urine.  None of these data seriously
question  the validity of the original prediction that PYR
is not a significant genotoxic hazard.

Table 3.  Activity of the four test chemicals in established
           in vivo mutagenicity assays
------------------------------------------------------------------------
                                                  BP    PYR   2AAF  4AAF
      Somatic cell tests

            Metaphase chromosome analysis         P     N     Pa    N
            Micronucleus test                     P     N     P     N
            Mouse spot test                       P     N     Pa    N

      Germ cell tests

            Dominant lethal assay                 P     N     N     N
            Heritable translocation test          NT    NT    NT    NT
            Mammalian germ cell cytogenetics      NT    NT    NT    NT
------------------------------------------------------------------------
a   Data not consistent between laboratories
    NT = not tested
    P  = positive    
    N  = negative

    A  similar analysis of  the 4AAF data  indicates  that
conclusive  results were obtained from  50 different types
of   in vivo tests.  Of these, 38 were regarded as negative
and  the remaining twelve assays produced positive or weak
positive  results.   Like  BP, 4AAF  produced  a  positive
result in a host-mediated assay and in tests for mutagenic
activity  in  rodent urine.   Other observations, however,
suggest  that 4AAF may  not be devoid  of  in vivo genotox-
icity.   For example, 4AAF induced  detectable unscheduled
DNA synthesis in rat liver hepatocytes that was reproduced
in three different laboratories. One investigator reported
the  induction  of micronuclei  in  rat bone  marrow cells
after  intraperitoneal dosing with 4AAF, and five of seven
studies  conducted in rat  or mouse bone  marrow  recorded
small,  but significant, increases in SCE.  There was also
data  to suggest that 4AAF was capable of adduct formation
with  liver cell DNA in  rats.  All the germ  cell data on
4AAF  were  negative.   The positive  findings  in somatic
tissues  cannot be dismissed  as inconsequential and  they
raise  serious doubts about  the prediction, based  on the
data  shown  in  Table 3,  that  4AAF  does not  present a
significant  genotoxic hazard.  The outcome  of the 2-year
rat  carcinogenicity  study with  4AAF  is crucial  to the
assessment  of  the validity  of  this prediction  and, in
fact, to the evaluation of the whole CSSTT/2 data base.

8.  ASSESSMENT OF THE PERFORMANCE OF THE ASSAYS

    The purpose of this section is to present an objective
assessment of the performance of the  in vivo tests  as  it
relates  to the carcinogenic  activity of the  test chemi-
cals. (A detailed tabulation of the qualitative results of
each assay is presented in Table 4).  Data from a study of
only  four chemicals do not, of course, provide sufficient
information  on which to judge the overall value of a par-
ticular  assay for discriminating between non-carcinogenic
and  potentially carcinogenic chemicals.  However,  when a
data  base of this magnitude  is available, in which  many
tests  were replicated in  a number of  different  labora-
tories,  then a unique insight into the utility, reproduc-
ibility, and reliability, as well as the accuracy  of  the
tests, can be obtained. Thus, data from the current study,
together  with the considerable experience of the investi-
gators  and assessors, enable  the performance of  many of
the assays to be comprehensively evaluated.

    Although  a high proportion of  test systems performed
reasonably  well with these  four chemicals, some  assays,
not  surprisingly, failed to  meet the predetermined  cri-
teria  for acceptable performance (section 4).  The rodent
host-mediated  and  urine  mutagenicity  assays  are  good
examples.   They  involve  either the  exposure  of marker
organisms  to the chemicals or  its metabolites  in vivo or
the  detection of mutagenic excretory products in urine of
treated  rodents. Both types  of tests were  conducted  in
several  laboratories and neither proved capable of ident-
ifying  the two carcinogens among the four  in vitro  muta-
gens,  i.e. the host-mediated assays  were generally nega-
tive  with the four test  chemicals while the urine  tests
were  uniformly positive.  These observations suggest that
neither  of these two classes  of tests has any  practical
value  in evaluating the  in vivo genotoxicity of chemicals
known to be genotoxic  in vitro.  The results obtained from
the  peritoneal macrophage transformation assay  suggest a
similar conclusion.  This is a form of host-mediated assay
in  which the transformation of macrophages, isolated from
the  peritoneum  of  treated rats,  is  investigated in  in
 vitro culture.   The  four  test chemicals  gave  negative
results in each of three laboratories.


Table 4.  IPCS CSSTT  in vivo study - summary of qualitative
          results from individual investigatorsa
-------------------------------------------------------------------------------------------------
        Assay                                  Chapter    BP     PYR     2AAF    4AAF   Route of
                                               numbersb                                 exposure
-------------------------------------------------------------------------------------------------
1.  CYTOGENETICS

   1.1  Chromosomal aberrations

        1.1.1   Mouse bone marrow                  8      P       N       NT      NT      o
                                                   8      NT      NT      P       ?       ip
        1.1.2   Mouse bone marrow                  9      P       N       NT      NT      o
                                                   9      NT      NT      N       N       ip
        1.1.3   Mouse bone marrow                 10      P       N       N       N       o
        1.1.4   Mouse bone marrow                 11      P       N       N       N       o
        1.1.5   Mouse bone marrow                 12      P       N       P       ?       o
        1.1.6   Mouse bone marrow                 13      P       NT      N       N       o
        1.1.7   Mouse bone marrow                 14      P       N       ?       N       ip
        1.1.8   Mouse ascites tumour cells        13      P       NT      N       N       o
        1.1.9   Rat bone marrow                   15      P       N       NT      NT      o
                                                  15      NT      NT      ?       N       ip
        1.1.10  Chinese hamster bone marrow       16      P       N       P       W       o
        1.1.11  Chinese hamster bone marrow       17      W       N       W       N       o

   1.2  Micronuclei

        1.2.1   Mouse bone marrow                 19      P       N       P       N       o
        1.2.2   Mouse bone marrow                 20      P       N       P       N       o
        1.2.3   Mouse bone marrow                 21      P       N       P       N       o
        1.2.4   Mouse bone marrow                 22      P       N       N       N       o
        1.2.5   Mouse bone marrow                 23      P       N       P       N       o
        1.2.6   Mouse bone marrow                 24      P       W       P       W       o
        1.2.7   Mouse bone marrow                 25      P       N       NT      NT      o
        1.2.8   Mouse bone marrow                 26      P       N       P       N       o
        1.2.9   Mouse bone marrow                 27      P       N       P       N       o
        1.2.10  Mouse bone marrow                 28      P       N       NT      NT      o
                                                  28      NT      NT      P       N       ip
        1.2.11  Mouse bone marrow                 29      P       N       P       N       ip
        1.2.12  Mouse bone marrow                 29      P       N       NT      NT      o
        1.2.13  Mouse bone marrow                 30      P       N       P       N       o
                                                  30      P       N       P       N       ip
        1.2.14  Mouse bone marrow                 31      P       N       P       N       ip
        1.2.15  Mouse bone marrow                 32      P       N       P       N       o
        1.2.16  Mouse bone marrow                 33      P       N       P       N       o
        1.2.17  Mouse blood - weanling            34      P       N       P       ?       o
        1.2.18  Mouse blood - fetal               34      P       N       N       N       o
        1.2.19  Mouse blood - maternal            34      P       N       N       N       o
        1.2.20  Mouse blood - fetal               32      P       N       P       N       o
        1.2.21  Rat bone marrow                   35      NT      NT      P       N       o
        1.2.22  Rat bone marrow                   36      P       N       NT      NT      o
                                                  36      NT      NT      P       W       ip
--------------------------------------------------------------------------------------------------------------

Table 4.  (contd.)
--------------------------------------------------------------------------------------------------------------
        Assay                                  Chapter    BP     PYR     2AAF    4AAF   Route of
                                               numbersb                                 exposure
--------------------------------------------------------------------------------------------------------------
   1.3  Sister chromatid exchange

        1.3.1   Mouse bone marrow                 38      P       N       P       W       ip
        1.3.2   Mouse bone marrow                 39      P       W       P       W       o
        1.3.3   Mouse bone marrow                 40      P       N       P       W       o
        1.3.4   Mouse bone marrow                 41      P       W       P       W       ip
        1.3.5   Mouse bone marrow                 42      P       N       P       N       o
        1.3.6   Mouse bone marrow                 43      P       N       W       N       o
        1.3.7   Rat bone marrow                   44      P       W       P       W       o
        1.3.8   Mouse lymphocytes                 45      P       N       P       N       o
        1.3.9   Chinese hamster bone marrow       46      P       N       P       N       ip
                Chinese hamster bone marrow       46      P       N       P       N       o
        1.3.10  Chinese hamster intestinal cells  46      P       N       P       N       o

2.  LIVER-SPECIFIC ASSAYS

   2.1  Initiation/promotion

        2.1.1   Altered enzyme foci               50      NT      NT      N       N       o
        2.1.2   GGT-positive foci                 51      P       N       P       N       o
        2.1.3   GGT-positive foci                 52      P       N       P       P       o
        2.1.4   GGT-positive foci - promotion     53      P       N       P       W       o
        2.1.5   GGT-positive foci                 54      N       N       P       N       o
        2.1.6   GGT-positive foci                 73      NT      NT      P       N       o

   2.2  Unscheduled DNA synthesis (UDS)

        2.2.1   UDS in mouse liver                56      N       N       N       N       o
        2.2.2   UDS in rat liver                  56      N       N       P       N       o
        2.2.3   UDS in rat liver                  57      NT      NT      P       W       o
        2.2.4   UDS in rat liver                  58      N       N       P       W       o
        2.2.5   UDS in mouse liver                58      NT      NT      N       N       o
        2.2.6   UDS in rat liver                  59      NT      NT      P       N       o
        2.2.7   UDS in rat liver                  60      NT      NT      P       W       o
        2.2.8   UDS in rat liver                  61      NT      NT      P       N       o
        2.2.9   UDS in rat liver                  62      NT      NT      P       N       o
        2.2.10  UDS in weanling rat liver         63      N       NT      NT      NT      o
        2.2.11  UDS - neonate rat                 61      NT      NT      W       NT      o

   2.3  S-phase hepatocytes

        2.3.1   Rat                               56      W       NT      NT      P       o
        2.3.2   Mouse                             56      NT      NT      NT      W       o
        2.3.3   Rat                               58      N       N       P       P       o
        2.3.4   Mouse                             58      NT      NT      W       P       o
        2.3.5   Rat                               59      NT      NT      NT      P       o
        2.3.6   Rat - weanling                    63      P       NT      NT      NT      o
        2.3.7   Rat                               60      NT      NT      N       P       o
        2.3.8   Rat                               62      NT      NT      N       P       o
        2.3.9   Rat                               61      NT      NT      NT      P       o
--------------------------------------------------------------------------------------------------------------

Table 4.  (contd.)
--------------------------------------------------------------------------------------------------------------
        Assay                                  Chapter    BP     PYR     2AAF    4AAF   Route of
                                               numbersb                                 exposure
--------------------------------------------------------------------------------------------------------------
   
   2.4  Cytogenetic analysis

        2.4.1   Aberrations in liver              65      W       N       W       N       o
                epitheloid cells
        2.4.2   SCE in liver epitheloid cells     65      W       N       ?       N       o
        2.4.3   Micronuclei in hepatocytes        66      N       N       P       N       o
   
   2.5  DNA strand breaks

        2.5.1   Alkaline elution                  68      N       N       N       N       o
        2.5.2   Alkaline elution                  69      N       N       N       N       o
        2.5.3   Alkaline elution                  70      N       W       P       P       o
        2.5.4   DNA-DNA/DNA-protein crosslinks    70      P       N       P       NT      ip
        2.5.5   DNA unwinding                     71      N       N       P       N       o
        2.5.6   DNA unwinding (with araA)         71      P       N       P       N       o

3.  MISCELLANEOUS ASSAYS

   3.1  Carcinogenesis

        3.1.1   2-year rat study                  73      NT      NT      P       *       o
        3.1.2   Mouse lung adenoma                74      P       N       P       N       o
        3.1.3   Quail egg                         75      *       *       *       *       

   3.2  Supplementary assay

        3.2.1   Rat fore-stomach UDS              76      ?       N       N       N       o
        3.2.2   Sebaceous gland suppression       77      P       N       NT      NT      der
        3.2.3   Epithelial hyperplasia            77      P       N       NT      NT      der
        3.2.4   Rat hepatocyte ploidy             78      P       N       P       N       o
        3.2.5   Mouse hepatocyte ploidy           78      P       N       P       N       o
        3.2.6   Syrian hamster 6-TG-resistant     79      P       N       ?       N       ip
                lung cells
        3.2.7a  32P-post labelling - rat liver    80      P       N       P       W       ip
        3.2.7b  32P-post labelling - rat lung     80      P       N       P       W       ip
        3.2.7c  32P-post labelling - mouse        80      P       N       P       NT      ip
                liver
        3.2.7d  32P-post labelling - mouse        80      P       N       P       NT      ip
                lung
        3.2.7e  32P-post labelling - mouse        80      P       N       P       NT      ip
                kidney
        3.2.8   Radiolabelled test compounds      81      NT      NT      P       P       ip
        3.2.9   ELISA rat                         82      NT      NT      *       *       o

   3.3  Immunotoxicity

        3.3.1   T-cells - rat                     83      P       N       P       W       ip
        3.3.2   Natural Killer cells - rat        84      NT      NT      P       N       o
--------------------------------------------------------------------------------------------------------------

Table 4.  (contd.)
--------------------------------------------------------------------------------------------------------------
        Assay                                  Chapter    BP     PYR     2AAF    4AAF   Route of
                                               numbersb                                 exposure
--------------------------------------------------------------------------------------------------------------
   3.4  Transformation of peritoneal macrophages

        3.4.1   Rat macrophages                   85      N       N       N       N       o
        3.4.2   Rat macrophages                   85      N       N       N       N       o
        3.4.3   Rat macrophages                   85      N       N       N       N       o

   3.5  Host-mediated assays

        3.5.1a  Mouse/yeast - mitotic             86      NT      NT      N       N       o
                recombination - liver
        3.5.1b  Mouse/yeast - mitotic             86      NT      NT      N       N       o
                recombination - lung
        3.5.1c  Mouse/yeast - mitotic             86      NT      NT      N       N       o
                recombination - kidney
        3.5.1d  Mouse/yeast - mutation -          86      NT      NT      W       N       o
                liver
        3.5.1e  Mouse/yeast - mutation -          86      NT      NT      N       N       o
                lung
        3.5.1f  Mouse/yeast - mutation -          86      NT      NT      W       N       o
                kidney
        3.5.2a  Mouse/yeast - gene conversion     87      P       P       P       P       o
                liver
        3.5.2b  Mouse/yeast - gene conversion     87      N       N       N       N       o
                lung
        3.5.3   Mouse/salmonella - liver          88      N       N       N       N       o

   3.6  Urine mutagenicity

        3.6.1   Rat urine extract                 89      P       P       P       P       ip
        3.6.2   Rat urine extract                 90      P       P       P       P       ip
        3.6.3   Mouse urine extract               91      W       W       P       P       o
        3.6.4a  Rat urine                         92      P       P       P       P       ip
                                                  92      P       P       P       P       o
        3.6.4b  Guinea-pig                        92      NT      NT      P       NT      ip
        3.6.5   Rat urine                         93      P       P       P       P       ip

4.  MOUSE SPOT TEST

        4.1.1   Coat colour                       95      NT      NT      P       N       ip
        4.1.2   Coat colour                       96      NT      NT      N       N       o
                                                  96      NT      NT      N       NT      ip
        4.1.3   Coat colour                       97      P       N       P       N       ip
        4.1.4   Melanocytes                       98      NT      NT      P       ?       ip
        4.1.5   Melanocytes                       99      NT      NT      N       N       ip
--------------------------------------------------------------------------------------------------------------

    Table 4.  (contd.)
--------------------------------------------------------------------------------------------------------------
        Assay                                  Chapter    BP     PYR     2AAF    4AAF   Route of
                                               numbersb                                 exposure
--------------------------------------------------------------------------------------------------------------

5.  MAMMALIAN GERM CELL STUDIES

   5.1  Dominant lethal

        5.1.1   Female mice                       102     P       N       N       N       ip
        5.1.2   Male mice                         103     P       N       N       N       ip
        5.1.3   Male mice                         104     P       N       N       N       ip
                                                  104     N       ?       NT      NT      o
        5.1.4   Male mice                         105     P       N       N       N       ip
                                                  105     N       NT      NT      NT      o
        5.1.5   Male rat                          106     NT      NT      N       N       o

   5.2  Sperm abnormalities

        5.2.1   Mouse                             108     P       N       P       N       ip
        5.2.2   Mouse                             109     W       N       N       N       o
        5.2.3   Mouse                             110     P       W       P       N       ip
        5.2.4   Mouse                             111     P       N       N       N       o
        5.2.5   Rat                               112     NT      NT      N       N       o

   5.3  Unscheduled DNA synthesis

        5.3.1   Rat spermatocytes                 113     N       N       N       N       o
        5.3.2   Mouse sperm                       114     N       N       N       N       ip
                                                  114     N       NT      NT      NT      o

6.  DROSPHILA ASSAYS

   6.1  Sex-linked recessive lethal

        6.1.1   SLRL assay                        116     N       N       P       W       o
        6.1.2   SLRL assay                        117     N       N       N       N       o
        6.1.3   SLRL assay                        118     N       N       N       N       o
        6.1.4   SLRL assay                        119     N       N       N       N       o
        6.1.5   SLRL assay                        120     N       N       N       N       o
--------------------------------------------------------------------------------------------------------------

Table 4.  (contd.)
--------------------------------------------------------------------------------------------------------------
        Assay                                  Chapter    BP     PYR     2AAF    4AAF   Route of
                                               numbersb                                 exposure
--------------------------------------------------------------------------------------------------------------

   6.2  Chromosome loss

        6.2.1   Germ cell                         121     N       N       N       N       o
        6.2.2   Germ cell                         116     N       NT      N       NT      o

   6.3  Somatic mutation and recombination

        6.3.1   Eye spots                         116     P       N       W       W       o
        6.3.2   Eye spots                         122     P       N       P       N       o
        6.3.3   Wing spots                        118     P       N       W       ?       o

--------------------------------------------------------------------------------------------------------------
a   From: Ashby et al. (1988)
b   Numbers refer to chapters in Ashby et al. (1988)

P   Positive        
NT  Not tested
N   Negative        
?   Inconclusive
W   Weak positive   
*   Results not available

8.1  Cytogenetic assays

    Assays that utilize target cells in rodent bone marrow
for detecting chromosomal effects, i.e., chromosomal aber-
rations,  micronuclei, or sister chromatid  exchanges, are
widely  advocated  for  investigating the  activity  of  in
 vitro genotoxins  in the intact  animal and represent  the
largest group of tests in this study.

8.1.1  Chromosomal aberrations

    Data  from metaphase chromosome studies  in mouse bone
marrow were provided by seven investigators. Each investi-
gator  successfully discriminated between BP  and PYR, and
five sets of data determined 4AAF to be negative while two
were inconclusive.  The results of chromosome studies with
2AAF, however, were conflicting.  Two laboratories identi-
fied  2AAF as a clastogen, one set of data were inconclus-
ive  and  four  investigators reported  2AAF  as negative.
There appears to be no simple explanation for  these  dis-
crepancies,  which were not associated with differences in
mouse  stain,  route  of treatment,  sampling interval, or
other  aspects of experimental  design.  The mouse  micro-
nucleus assay (section 8.1.2) showed 2AAF to be an  in vivo
clastogen   (though less potent than  BP) and it is  clear

that the dosing schedules used in the metaphase chromosome
assays  were more than adequate to induce chromosome aber-
rations,  i.e., 2AAF would be expected to be detected as a
clastogen  in the metaphase chromosome  assay.  One expla-
nation  for the negative findings in four laboratories may
be  the resolving power  of the sample  sizes used.   Most
standard  protocols  for  the bone  marrow metaphase assay
require at least 50 cells per animal from 10  animals  per
dose/time group (i.e., 500 cells) to be analysed for aber-
rations at each data point.  This recommended minimum fig-
ure  was only achieved by three of the seven investigators
and  three others  presented data  on only  250 cells  per
dose/time  group.   These observations  suggest that, with
samples  of  this  size,  almost  a  10-fold  increase  in
aberrations  over the control incidence  would be required
to  demonstrate a significant induction  of aberrations by
2AAF.

    Two investigators conducted studies in Chinese hamster
bone  marrow cells.  PYR was negative in both studies.  In
experiments  in which 500  cells per dose/time  group were
analysed,  BP and 2AAF  were clearly positive  while, when
only 300 cells were used, the results were  classified  as
weak positive with both carcinogens.  It is interesting to
note that 4AAF induced a weak positive response in Chinese
hamster bone marrow in one study.

    One set of data from rat bone marrow was presented and
clearly  separated  BP  from  PYR.   2AAF,  however,  gave
unambiguous data and 4AAF was negative.

    The  performance of the bone marrow cytogenetic assays
did  not, in general, meet the criteria for a reliable and
reproducible short-term  in vivo test.  There are, however,
serious  reservations regarding the numbers of animals and
the numbers of cells analysed in many of the  studies  and
the  results suggest that  protocols designed to  give  an
acceptable resolving power should be strictly adhered to.

8.1.2  Micronuclei

    Eighteen laboratories conducted micronucleus tests; 16
laboratories  performed assays on  bone marrow cells  from
mice  and  two investigators  used  rats.  Data  were also
presented  on the incidence of  micronuclei in circulating
blood cells from adult and fetal mice.

    Mouse  bone marrow micronucleus assays  were conducted
to a common protocol with only minor deviations.  At least
5  mice were used  for each dose/time  interval with  1000
cells being analysed from each animal. The data from these
assays  showed a high  level of agreement  between labora-
tories.  Seventeen sets of data on BP were uniformly posi-
tive  and 16  sets on  PYR were  negative.  Similarly,  14
investigators  showed 2AAF to  be capable of  inducing  an

increase in micronuclei and 4AAF was negative in 14 cases.
There were only occasional anomalous results. For example,
one laboratory failed to detect 2AAF, and another investi-
gator  recorded weak positive  results from both  PYR  and
4AAF.   These anomalies do  not detract from  the  general
accuracy  and reproducibility of mouse  micronucleus tests
with the four test chemicals.

    One  investigator used erythrocytes from mouse periph-
eral  blood to assay  the incidence of  micronuclei.   The
results  were similar to those obtained in the bone marrow
studies, i.e. BP and 2AAF were positive while PYR and 4AAF
were considered to be negative by the criteria adopted for
a positive response.  In two laboratories, fetal blood was
obtained  from  mice on  day 15 of  pregnancy, 30 or  48 h
after  oral dosing of the mother.  BP was positive and PYR
was negative in each of these studies. The fetal assay was
less  sensitive  to the  effects  of 2AAF,  one laboratory
observing  a small increase  in micronuclei and  the other
finding no increase.  4AAF was inactive in fetal cells.

    Two  micronucleus studies were  conducted in rat  bone
marrow  cells and the results suggest that the response in
the  rat  was  weaker than  that  in  the mouse.   In  one
laboratory,  2AAF was positive  and 4AAF negative;  BP and
PYR were not tested.  The second investigator  reported  a
positive  response with BP  and a negative  one with  PYR.
This  investigator's result with 2AAF is shown as positive
in  Table  4.   However,  using  criteria  decided  by the
micronucleus  assay working group,  it was concluded  that
2AAF was negative in this assay.  It was  also  considered
that the protocol used in the rat studies was not designed
for the optimum detection of micronuclei.

    The  results from the  micronucleus tests confirm  the
value of the assay in mouse bone marrow as an accurate and
reproducible  means of qualitative  discrimination between
carcinogenic and non-carcinogenic chemicals.

8.1.3  Sister chromatid exchange

    Nine  investigators presented data on the induction of
sister chromatid exchanges (SCE)  in vivo using mice, rats,
or Chinese hamsters.  The test chemicals were administered
by  gavage or intraperitoneal injection  and most investi-
gators analysed SCE in bone marrow cells.  One set of data
were  presented on SCE in mouse peripheral lymphocytes and
another  set  on  Chinese  hamster  intestinal  epithelial
cells.   The  two  established carcinogens,  BP  and 2AAF,
induced  an  increase in  SCE in each  of the six  studies
conducted  in mouse bone marrow.  In two of these studies,
PYR induced a weak positive response and 4AAF  was  weakly
positive in four of the six assays.  Weak positive results
were  also obtained in rat bone marrow assays with the two
presumed  non-carcinogens, while BP and  2AAF were clearly

positive.   In experiments conducted in  blood lymphocytes
in mice and in bone marrow cells and intestinal epithelial
cells  in  Chinese hamsters,  BP  and 2AAF  gave  positive
results  while PYR and  4AAF were negative.   None of  the
experiments  suggested that the route of administration of
the  test chemicals significantly influenced the qualitat-
ive results.

    Both  the  established  carcinogens were  consistently
positive in tests for the induction of SCE in three rodent
species,  suggesting that the SCE assay is a sensitive and
reproducible   in vivo test for genotoxic  carcinogens. PYR
or  4AAF,  however,  induced small  (but  significant) in-
creases in the incidence of SCE in 8 of 22 tests, although
the   presumed  non-carcinogens  were  much   less  potent
inducers  of SCE than their carcinogenic analogues.  These
observations  raise  a  number of  questions regarding the
significance  of small increases in  SCE in  in vivo tests.
In  the first instance,  if 4AAF is  confirmed to be  non-
carcinogenic  in the current  rodent bioassay, then  these
weak responses have to be regarded as  `false  positives'.
If,  however,  4AAF is  shown  to have  weak  carcinogenic
activity,  then  the  weak positive  responses obtained in
four  of the six mouse  bone marrow studies can  be inter-
preted  as accurately reflecting the genotoxicity of 4AAF.
In the report of the SCE Working Group (Tice et al., 1988)
the  biological  relevance  of  statistically  significant
increases  in SCE at  unrealistically high doses  was con-
sidered.   It was suggested  that multiple mechanisms  are
involved in the formation of SCE and that one of these may
be  a  response to  stress, either at  the cell or  animal
level,  caused by  a toxic  response to  high doses  of  a
chemical.   The small induction of SCE observed after high
doses  of  PYR  or 4AAF,  therefore,  may  not be  related
mechanistically  to the larger increases in SCE seen after
low  doses of BP or  2AAF.  Thus, the final  assessment of
the  performance of the   in vivo SCE assays depends,  to a
large extent, on the outcome of the 2-year  bioassay  with
the AAF analogues, i.e. a negative result with  4AAF  will
cast serious doubts on the value of SCE tests for investi-
gating the  in vivo activity of  in vitro genotoxins.

8.2  Liver assays

    The activity of certain genotoxic chemicals appears to
be  confined  to  the liver.  Such  chemicals  do not,  of
course,  induce detectable genetic changes  in bone marrow
cells or other extra-hepatic tissues and this has led to a
need  to develop techniques for investigating genotoxic or
related changes in liver cells.  The main thrust  of  this
research  has centred on the  demonstration of unscheduled
DNA  synthesis in hepatocytes but data were also presented
from a variety of other assays that used target  cells  in
the liver.

8.2.1  Initiation and promotion

    Two of the assays used in this group were specifically
aimed  at detecting initiating activity of the test chemi-
cals.  The chemicals were administered after partial hepa-
tectomy, and the mice were then treated with the promotor,
phenobarbital,  for  several  weeks before  liver sections
were  examined for the  occurrence of pre-cancerous  foci,
i.e.  groups of cells having elevated levels of the marker
enzyme,  gamma-glutamyl-transpeptidase   (GGT).  Both these
protocols  have  been  used successfully  with  many  test
chemicals  and, in the present  study, successfully separ-
ated  BP and 2AAF from PYR and 4AAF.  A third protocol was
used  to investigate the  promoting activity of  the  test
chemicals after initiation by pre-treatment with dimethyl-
nitrosamine.   This,  too, successfully  separated BP from
PYR,  but,  although 2AAF  produced  an increase  in  GGT-
positive  foci, a weak positive response was also observed
with  4AAF.   The  two  other  procedures  used  to  study
initiation/promotion  events based on altered  enzyme foci
failed  to detect BP in  one assay and 2AAF  in the second
assay  and  are  considered  to  be,  primarily,  research
techniques and not yet suitable for routine application.

    The two better-established assays showed quite clearly
that both BP and 2AAF initiated altered enzyme foci in rat
liver.   The two presumed non-carcinogens  were completely
negative and these tests, although they cannot be regarded
as "short-term" in the true sense, have a useful role in
investigating liver carcinogenesis.

8.2.2  Unscheduled DNA synthesis and S-phase analysis

    The  investigators in this group used the detection of
unscheduled DNA synthesis (UDS) as a measure of DNA repair
initiated  in response to  chemical-induced damage to  the
DNA.  In  each study,  UDS  was detected  using autoradio-
graphic techniques that also allowed simultaneous measure-
ment  of S-phase cells, i.e. those cells undergoing normal
DNA  synthesis as a  prelude to mitosis.   Seven  investi-
gators examined 2AAF and 4AAF for the induction of UDS and
S-phase  cells in  hepatocytes of  male rats,  and in  two
laboratories  all four test chemicals were investigated in
rats  and mice.  The induction of UDS and S-phase cells in
rat  fore-stomach was investigated in  another laboratory.
Although  there  were  minor protocol  variations, the UDS
working  group concluded that none  of these significantly
affected  the results and there was excellent agreement in
the observations and conclusions of the investigators.

    2AAF  was shown to be  a potent inducer of  UDS in rat
hepatocytes and also caused proliferation of hepatic cells
as shown by an increase in the number of cells in S-phase.
In mice, however, 2AAF failed to induce SCE in  either  of
two  laboratories,  though there  was  evidence of  a weak

induction of S-phase cells.  Only low doses were examined,
i.e.  up to 50mg/kg, but the results indicate an important
species difference in the response to 2AAF.

    Administration  of 4AAF to rats  produced negative UDS
results  in four laboratories and weak positive results in
three  laboratories.   Although  the three  weak  positive
results  were  initially  determined without  recourse  to
statistical analysis, a comprehensive analysis of the data
by  Margolin  and  Risko  (1988)  confirmed  the   initial
interpretation.   The  six investigators  who recorded the
number  of  S-phase cells  observed  a marked  increase in
hepatic  cell proliferation in rats dosed with 4AAF.  This
compound failed to induce UDS in mouse liver but did cause
an increase in S-phase cells.

    For  comparative purposes, two laboratories  also con-
ducted  UDS tests on hepatocytes  in vitro.   Both 2AAF and
4AAF induced UDS in rat hepatocytes but were  negative  in
mouse cells.

    BP  and PYR were tested  in rats by two  investigators
and  one  assay was  conducted  in mice.   Both  chemicals
failed  to induce UDS in either species after oral dosing.
In  contrast, BP  gave positive  results in  both rat  and
mouse hepatocytes treated  in vitro.

    In  an investigation of the  UDS activity of the  four
chemicals  in rat fore-stomach, 2AAF, 4AAF, and PYR failed
to induce UDS while BP gave equivocal results.

    The performance of the UDS assay in rodents dosed with
2AAF  or 4AAF is worthy of further consideration.  2AAF is
a  potent rat hepatocarcinogen and a potent inducer of UDS
in rat hepatocytes.  In the mouse, however, 2AAF  did  not
induce UDS and, on balance, available data suggest that it
does  not produce liver tumours.  Thus, for this chemical,
the  UDS results accurately reflect its hepatocarcinogenic
potential.  The current lack of cancer data on  4AAF  pre-
cludes this kind of comparison, but the UDS  data  suggest
that  if 4AAF is carcinogenic, it is markedly less so than
2AAF.

    Although fewer tests were conducted with BP  and  PYR,
the data indicate that neither chemical induces UDS in rat
or mouse liver, although BP was positive  in   in vitro UDS
tests.  The reason for this negative response to BP has an
important  bearing on the  validity of the  UDS assay  for
detecting potentially carcinogenic chemicals.  Although BP
is an established carcinogen, it has never been  shown  to
produce  tumours  in  rodent liver.   There  is sufficient
evidence,  however,  to suggest  that DNA-reactive adducts
are  formed in liver cells  after dosing rodents with  BP.

For example, most of the data that show BP to be genotoxic
 in   vitro are derived from tests in which metabolic acti-
vation is provided by an enzyme mixture derived  from  rat
liver.   In  addition,  BP clearly  induces altered enzyme
foci  in rat liver,  there is tentative  evidence for  the
induction  of  DNA  strand breakage  based on DNA-DNA/DNA-
protein crosslinks, and 32P-post-labelling  techniques in-
dicate  the presence of DNA  adducts of BP metabolites  in
both rat and mouse liver.  Therefore, although BP is not a
hepatocarcinogen  in  rodents,  there are  data to suggest
that (a) BP is metabolized in rodent liver to DNA-reactive
metabolites,  (b) interaction of  DNA with BP  metabolites
does occur, and (c) based on data from the  DNA  unwinding
test, some repair of the DNA lesions takes place.  On this
basis,  the  liver  UDS assay  should,  theoretically,  be
capable of detecting BP-induced lesions in the liver.  All
these  observations  indicate  that such  factors  as  the
distribution  of BP to  the liver after  oral dosing,  the
rate  of formation of  genotoxic metabolites, the  rate of
detoxification,  and the rate and nature of the DNA repair
process  yield an insufficient  number of DNA  adducts  to
produce a detectable UDS response.

    In practice, of course, BP is easily detected in other
 in   vivo tests  such as  those  conducted in  bone marrow
cells.   However, the  results with  BP and,  to a  lesser
extent,  4AAF, suggest the need  for additional evaluation
of  the performance of  the  in vivo UDS assay  with liver-
specific genotoxins.

8.2.3  DNA strand breaks

    Data  were considered from three  different assay sys-
tems  used to detect single-strand breaks in DNA.  Results
from the alkaline elution assay, in which two laboratories
found  no activity with  any of the  four test  chemicals,
indicate that this assay is of little value in investigat-
ing  in vivo genotoxicity. The test for DNA-DNA/DNA-protein
crosslinks  and the DNA  unwinding assay in  rodent  liver
cells both gave very promising results and warrant further
evaluation.

8.2.4  Cytogenetics

    Three  sets  of data  were  considered in  this group.
Micronucleus  assays  were  conducted in  rat  liver cells
following partial hepatectomy. The results were similar to
those  obtained in the rat  liver UDS assay, i.e.  a dose-
dependant  response  was  obtained with  2AAF,  while  the
remaining  three  chemicals  gave negative  results.   The
other  two  data  sets  were  generated  from  analyses of
chromosome  aberrations  and SCE  in epithelial-like cells
derived  from weanling rat liver.  Although the assays are
relatively  simple  with  clearly defined  end-points, the

results were either equivocal or negative and  the  sample
sizes, i.e. the numbers of animals/cells per  dose  group,
were  too small to  permit an effective  assessment.  Both
the micronucleus test and the chromosome assays are worthy
of further evaluation.

8.3  Miscellaneous assays

    This section contains a wide variety of  assays,  some
of which have been in use for many years while others, few
of  which were undertaken in more than one laboratory, are
relatively new or in the process of development.

8.3.1  Specific carcinogenicity assays

    Neither of the two procedures considered here  can  be
regarded as short-term tests and final results from one of
them, the assay in quail eggs, were not available  at  the
time  of the assessment.  The results of the mouse adenoma
assay,  however, were available and showed a decisive dis-
crimination between BP and PYR and a much smaller and less
convincing difference between 2AAF and 4AAF.

8.3.2  Supplementary assays

    Data from the sebaceous gland suppression test and the
epidermal  hyperplasia assay have shown a good correlation
with  skin carcinogenicity and have proved useful for com-
paring  the carcinogenic potential of polycyclic hydrocar-
bons.  Their value in this area was confirmed by  the  re-
sults  of assays with  BP and PYR  but the application  of
these assays to other classes of chemicals is of uncertain
value.

    The  hepatocytes  of  rodents include  mono- and binu-
cleated  cells and nuclei that may be diploid, tetraploid,
or octaploid.  The ratio of cells with different levels of
ploidy  in liver varies  with species and  age and can  be
altered by exposure to certain chemicals.  There have been
references  to the effects  of various chemicals  on liver
ploidy ratios over a number of years, but there appears to
be  no consistent relationship between  ploidy changes and
chemical carcinogenesis. Thus the observation that the two
carcinogens, BP and 2AAF, increase the ratio of diploid to
tetraploid  cells is of uncertain significance.  The assay
may  be of value in  studying liver cell kinetics  but its
value  in the detection of   in vivo genotoxicity is debat-
able.

    The general principles of the  in vivo/in vitro somatic
mutation assay were developed in the late 1970s and showed
initial promise as a test for investigating organ-specific
mutations    induced   by   chemical   carcinogens.    One
investigator  provided  data  from a  modification of this
technique  in  which cells  isolated  from lung  tissue of

treated  Syrian  hamsters  were cultured   in vitro and the
induction  of  mutations  was determined.   A reproducible
increase  in mutations was  observed in lung  cells  after
dosing with BP, but the results with 2AAF and the two non-
carcinogens were considered to be negative.  Although this
is a logical and potentially useful approach  to  studying
 in   vivo genotoxicity,  the  method requires  much  wider
evaluation  before it can  be considered as  an acceptable
assay.

    The   detection  and  identification  of  DNA  adducts
provides  conclusive  evidence  that a  metabolite  of the
administered  chemical  has  interacted with  one  of  the
important  target macromolecules of carcinogenesis. Theor-
etical  considerations  indicate  that, for  many chemical
classes,  the formation of  DNA adducts is  a prerequisite
for  permanent genetic changes, such as mutations, and for
the initiation stage of carcinogenesis. Research has shown
a direct relationship between the administered dose  of  a
chemical  and DNA-adduct formation, so  that precise dosi-
metry  in  specific  organs is  possible. Three approaches
were  used in the study, though results from a novel tech-
nique  using  monoclonal  antibodies to  identify specific
adducts are not yet available.  One study involved the use
of radiolabelled 2AAF and 4AAF; while binding to liver DNA
was  detected with both compounds, the covalent binding of
2AAF  was approximately seven  times that of  4AAF.  These
data correlate well with the results of the UDS  assay  in
rat liver, which showed 2AAF to be a potent inducer of UDS
while 4AAF was, at best, a weak inducer.

    The  above  assay  has the  disadvantage  of requiring
radiolabelled test chemicals, and a significant advance in
the  field  was the  introduction  of techniques  in which
animals are dosed with unlabelled test chemicals  and  the
DNA is radiolabelled after isolation and purification. The
32P-labelled    normal nucleotides and  nucleotide adducts
are separated by thin-layer chromatography and detected by
autoradiography,  and a comprehensive,  organospecific DNA
adduct  profile is obtained.   The data presented  in  the
current  study showed that  2AAF adducts were  detected at
significant  levels in the DNA  of mouse liver, lung,  and
kidney and in rat liver and lung.  4AAF adducts  were  not
detected  in mouse tissue and  at only very low  levels in
rat liver.  PYR adducts were not detected in either rat or
mouse  tissues and BP produced  adducts with DNA in  liver
and lung of both species and in mouse kidney.

    The  detection  and  identification of  DNA adducts is
potentially  an  extremely  powerful tool  in the investi-
gation of  in vivo genotoxicity.  With the establishment of
`cold'  techniques, i.e. those not requiring radiolabelled
test  chemicals, this type of assay can be applied to most

chemical  classes.   Although  further development  is re-
quired, particularly with regard to the genotoxic signifi-
cance of different types of adduct, the method shows great
promise for the future.

8.3.3  Immunotoxicity assays

    In the Natural Killer (NK) cell assay, the activity of
NK  cells was measured at intervals after dosing rats with
either  2AAF  or 4AAF.   The  carcinogen, 2AAF,  induced a
significant  change in NK  cell cytotoxicity that  was not
observed  after dosing with  4AAF.  The T-cell  assay,  in
which  immunocompetent  T-cells  are  able  to  react   to
carcinogen-induced tissue changes, responded positively to
BP and 2AAF; PYR failed to induce any change in reactivity
and  4AAF gave a weak  positive response.  The results  of
both  types  of  assay closely  reflected the carcinogenic
activity  of  the test  chemicals.   The relevance  of the
observed immunological changes to the carcinogenic process
is unclear and, thus, their potential value  in  detecting
precarcinogenic  changes  caused  by  genotoxic  and  non-
genotoxic  carcinogens  remains  to  be  fully  evaluated.
Before they can be accepted as anything more than a useful
research  tool, further investigations of  the specificity
of  the immunological reactions and a wider evaluation are
imperative.

8.3.4  Host-mediated assays and urine mutagenicity tests

    As described at the beginning of Section 8, both these
classes of test failed to meet the criteria for an accept-
able short-term  in vivo assay. The host-mediated assay was
a favoured  in vivo procedure in the early 1970s, though in
recent  years  questions  have been  raised concerning its
sensitivity.  Although the intrasanguinous modification to
the original intraperitoneal technique offered theoretical
advantages, the results of the present study confirmed the
inadequacy  of the assay.  It is concluded that both these
procedures are not appropriate for the investigation of  in
 vitro genotoxins.

8.4  Mouse spot tests

    Although data were presented from two types  of  mouse
spot test, they are, in effect, different means of observ-
ing the same genetic alterations, i.e. the melanocyte test
detects  the change at the  cellular level while the  coat
colour  test demonstrates the  expression of the  cellular
changes.

    BP  and PYR were only tested in one laboratory and the
spot test successfully identified the carcinogen. In tests
with  4AAF, one investigator's results  were considered to
be inconclusive and four other sets of data, including one

after  oral dosing and three  after intraperitoneal admin-
istration,  were negative.  Six laboratories  tested 2AAF;
there  were three positive results and two negatives after
intraperitoneal  administration.   2AAF was  also negative
after oral dosing.  Examination of the protocols indicated
that  2AAF induced detectable  genetic changes only  after
administration  of the material as a solution in dimethyl-
sulfoxide,  i.e.  negative  results  followed  the  admin-
istration  of corn oil formulations  of 2AAF, illustrating
the  critical effect of the  vehicle on the absorption  of
the test chemical.

    An overview of the mouse spot test data indicates that
intraperitoneal  administration  of an  appropriate formu-
lation of the test chemicals produces results that reflect
their  carcinogenic  activity.   The original  coat colour
procedure  appears to be more reliable than the melanocyte
technique,  which may be influenced by sporadic mutational
events  and inadequate melanization of the hair follicles.
However,  the dependence on  the intraperitoneal route  of
administration may be a disadvantage, and the  mouse  spot
test  appears to add very little to the information gained
more easily from bone marrow cell cytogenetic assays.

8.5  Assays in mammalian germ cells

    For assessment purposes the germ cell assays have been
separated  into two groups:  those that are  considered to
involve a direct interaction with DNA, and the  assay  for
sperm  abnormalities, the genetic significance of which is
uncertain.

8.5.1  Dominant lethal and unscheduled DNA synthesis assays

    Classical dominant lethal tests were conducted in male
or female mice and in male rats.  PYR, 2AAF, and 4AAF gave
unequivocally   negative  results  after   intraperitoneal
dosing. In an oral dosing study in male mice, BP was nega-
tive  and PYR produced inconclusive  data. After intraper-
itoneal  administration of BP, however, there was conclus-
ive  evidence  of dominant  lethal  induction in  male and
female mice.  In the male, dominant lethality was confined
to  the late spermatid/spermatozoa stages  of spermatogen-
esis.

    Results  of UDS assays in rat spermatocytes after oral
dosing  and in mouse spermatocytes after oral or intraper-
itoneal  administration were negative with all four chemi-
cals.  The negative data with BP are not  altogether  sur-
prising  as dominant lethality  was only observed  in late
spermatids/spermatozoa,  in which the  DNA is highly  con-
densed and in which UDS does not occur.

    Based  on the dominant  lethal test results,  BP is  a
confirmed,  though relatively weak,  germ cell mutagen  in
mice after intraperitoneal dosing. Published data indicate

that  BP does not induce heritable translocations at doses
that  produce dominant lethality (Generoso  et al., 1982),
although  this may be  due, in part,  to the fact  that BP
appears  to  induce  mainly chromatid  deletions  in  bone
marrow cell chromosomes, with very few exchange-type aber-
rations.

8.5.2  Sperm abnormality tests

    This  assay is based on the observation of morphologi-
cal  alterations in mature spermatozoa  after treatment of
animals  at all stages  of spermatogenesis with  the  test
chemicals.   BP induced an  increase in the  incidence  of
abnormal  sperm  in  mice after  oral  or  intraperitoneal
dosing and 2AAF was also positive following  oral  gavage.
Tests with 4AAF were uniformly negative and PYR produced a
weak  positive response in one of four assays.  Thus, both
carcinogens produced sperm abnormalities under appropriate
experimental conditions.

    The  important questions raised by  these observations
relate  to the relevance of morphological abnormalities in
sperm  to  germ  cell  mutations  and  to  carcinogenesis.
Although  changes in sperm  head shape may  be genetically
determined,  it is uncertain whether they are due to geno-
toxicity or to other toxic phenomena.  A  positive  result
in the test, therefore, indicates that the  test  material
or  its metabolites are  able to reach  the germ cells  in
sufficient  quantities  to  induce an  adverse  biological
effect. Although a good correlation has been shown between
the  induction  of  sperm abnormalities  and  carcinogenic
activity  (Wyrobek et al.,  1983), the relevance  of  this
correlation  to short-term testing strategies  is doubtful
as  more  appropriate   in vivo tests are  available, e.g.,
bone  marrow cytogenetic assays.  Equally  doubtful is the
relevance  of  the test  to  heritable genetic  damage, as
there is no clear-cut evidence that abnormal sperm  are  a
consequence of induced DNA damage.

8.6  Drosophila assays

    The sex-linked recessive lethal assay and the test for
chromosome  loss in drosophila germ cells failed to show a
consistent  separation of BP and  2AAF from PYR and  4AAF,
respectively.   Three tests based on  somatic cell changes
were  positive  with  BP and  negative  with  PYR but  the
results  of tests with the  acetylaminofluorenes were less
decisive.   2AAF  produced an  unambiguous positive result
and  two weak positives, while the three results with 4AAF
were  weak positive, negative and inconclusive.  Thus, the
drosophila germ cell assays were insensitive to  the  four
 in   vitro genotoxins and, although the  somatic cell pro-
cedures  appeared to be  more sensitive, it  was concluded
that the main value of drosophila tests is to provide com-
prehensive  analyses of the genetic mechanisms involved in
the induction of mutations by chemicals.

9.  SELECTION OF THE MOST EFFECTIVE  IN VIVO ASSAYS IN RELATION 
TO THEIR PERFORMANCE

    In a study of this nature it is inevitable  that  some
assays,  even  well-established  procedures, will  fail to
perform well.  Indeed, the recognition of inadequate tests
is as important a goal as the identification of those that
are  useful and acceptable.  Other assays produced results
that correlated well with the presumed carcinogenic status
of the test chemicals but are not widely used or available
or  are still in  the process of  development.  Yet  other
tests that performed adequately in the collaborative study
cannot  be regarded as practicable short-term tests either
because  of their technical  complexity or because  of the
length of time required to produce results.  Most  of  the
latter  group of tests  have important research  roles  in
carcinogenesis.

9.1  Assays that are not considered appropriate for routine
 in vivo testing of chemicals for genotoxic activity

    Host-mediated  and urine mutagenicity  assays (section
8.3.4),  the  peritoneal macrophage  transformation assay,
and  the alkaline elution  assay for single  strand breaks
(section  8.2.3)  proved incapable of  separating the car-
cinogen/non-carcinogen  pairs  and  appear to  have little
value   in   investigating   in vivo genotoxicity.   The  in
 vivo/in   vitro somatic   mutation  procedure   in  Syrian
hamsters  (section 8.3.2) also  falls into this  category,
although  this  conclusion  should not  detract  from  the
initial  promise  of  this approach  in  Chinese  hamsters
(McGregor, 1988a).

    Drosophila tests (section 8.6) have also proved gener-
ally  inadequate  in  the identification  of  in vivo geno-
toxins and it is concluded that their main value  in  gen-
etic  toxicology  is to  provide  analysis of  the genetic
mechanisms involved in the induction of mutations. Similar
reservations  apply to the initiation/promotion techniques
using  liver  foci (section  8.2.1)   and the  analysis of
ploidy levels in liver tissue (section 8.3.2). Both assays
are  more appropriate to investigation of the carcinogenic
process  than to the identification of  in vivo genotoxins.
The  results of the  immunological assays (section  8.3.3)
closely   reflected  the  carcinogenic  potential  of  the
chemicals, but they can only be regarded as research tools
pending  a greater understanding of the specificity of the
immunological  reactions  and  a wider  evaluation  of the
procedures.

    Two  assays  conducted in  mouse  skin, i.e.  the seb-
aceous  gland suppression test and the observation of epi-
dermal hyperplasia, confirmed their value in the detection
of  carcinogenic polycyclic hydrocarbons such as BP.  Both
tests  failed to respond to 2AAF when applied dermally and
are  not  considered  to be  suitable for investigating  in
 vivo genotoxicity in general.

9.2  Assays that satisfy some or all of the criteria for an 
acceptable short-term  in vivo test

9.2.1  Assays currently in general use

    The  mouse micronucleus test  (section 8.1.2) was  the
most  widely  represented single  assay  in the  study and
produced the best overall performance with only occasional
anomalous  data.   Qualitative  results were  not affected
significantly by the route of exposure, strain or  sex  of
mouse,  or  treatment  schedule.  The  mouse  bone  marrow
micronucleus assay was confirmed as a robust and sensitive
short-term  in vivo test for genotoxic chemicals.

    Both the micronucleus test and the analysis  of  meta-
phase chromosome aberrations in rodent bone marrow respond
to  similar chromosome breakage  events and they  would be
expected to produce qualitatively similar results with the
four  test compounds.  Table 4 shows that this was not the
case  and the metaphase chromosome  procedure was signifi-
cantly  less sensitive than the  micronucleus test.  Poss-
ible  explanations for this  lack of sensitivity  are dis-
cussed above (section 8.1.1) and suggest that the  use  of
metaphase chromosome analysis should not be undervalued on
the basis of these data.

    The  sister chromatid exchange (SCE)  procedure repro-
ducibly  detected the two carcinogens, but also produced a
number  of weak positive results with 4AAF and PYR.  Taken
at  face value, therefore, the  in vivo SCE results clearly
reflect  the relative potency of the two chemical pairs in
 in   vitro tests.  As discussed in section 8.1.3, however,
very  weak SCE responses  may be caused  by  non-genotoxic
mechanisms  and a final  judgement on the  significance of
the weak activity of 4AAF must be delayed until  its  true
carcinogenic status is defined.

    After considering the data from the three  classes  of
bone  marrow assay, the  conclusion of the  Steering Group
(Ashby et al., 1988) was - `the mouse  micronucleus  assay
provides  the simplest and most effective measure of geno-
toxicity  in the  bone marrow  and, as  such, it  is  rec-
ommended here as a primary  in vivo genotoxicity test'.

    Despite the successful performance of the micronucleus
test  with these four chemicals,  it is apparent from  the
literature  that certain liver  carcinogens do not  induce

detectable  chromosomal effects in the bone marrow.  In an
acceptable  testing strategy, therefore,  negative results
in  the  micronucleus  test should  be  supplemented  with
evidence  of non-genotoxicity in the  liver.  This concept
led  to the extensive evaluation  of liver-specific assays
in  the CSSTT/2 study and the main candidate in this group
of tests was the rat liver UDS assay (section 8.2.2).

    The  liver carcinogen, 2AAF,  was a potent  inducer of
UDS  in  rat liver  while  most investigators  defined its
presumed   non-carcinogenic  analogue,  4AAF,  as  a  weak
genotoxin  in this system.   Unlike the micronucleus  test
result,  BP was devoid  of activity in  liver UDS  assays.
This is consistent with its lack of carcinogenicity in rat
liver.

    Although  the  significance  of  the  weak  liver  UDS
activity  of 4AAF will  only be resolved  when  definitive
carcinogenicity  data  are  available, the  findings raise
certain  problems  of  interpretation that  are common, in
part,  to the weak bone  marrow activity of 4AAF  and PYR.
In  the UDS assays,  2AAF was a  potent inducer of  UDS at
doses  between 5 and 100 mg/kg while 4AAF showed only weak
activity  between 50 and 1000 mg/kg, and then only in some
experiments.   In general, the activity of 4AAF was within
the  range of control  variability and, as  a result,  the
UDS  Working Group questioned its  biological significance
(Mirsalis, 1988).  A detailed analysis, however, indicated
the  high  statistical  significance  of  the  4AAF   data
(Margolin  and Risko, 1988).  Thus, although the rat liver
UDS  assay  discriminated quantitatively  between 2AAF and
4AAF,  the  significance  of  the  weak  activity  of 4AAF
remains  unresolved.   These  observations  suggest   that
qualitative  determinations  in  terms of  `positive'  and
`negative'   are  too  simplistic  for  hazard  assessment
purposes and one of the most useful points raised  by  the
present  study is the  need for appropriate  positive test
criteria,  in biological and statistical terms, for widely
used  in vivo tests.

    Thus,  the mouse bone marrow micronucleus assay is the
method  of choice for primary   in vivo testing of  in vitro
genotoxins   and the rat  liver UDS assay,  providing  the
above  problems can be  resolved, appears to  be the  most
promising complementary liver-specific test.

9.2.2  Assays that show promise for future development

    Accepting  that  bone  marrow assays  are suitable for
detecting  extra-hepatic somatic cell genotoxins  and that
the  rat liver UDS  assay can, with  certain  reservations
(see  sections 8.2.2 and  9.2.1), be used  to  investigate
liver-specific  genotoxins, it is useful to consider which
other  assays evaluated in this  study may have a  role in
the assessment of  in vivo genotoxicity.

    Development  of assays to detect  single-strand breaks
in  DNA  and  to detect  and  identify  DNA adducts  will,
undoubtedly,  continue.  It will be  interesting to follow
the  progress of the assays for DNA-DNA/DNA-protein cross-
links  and  DNA unwinding,  although  it is  probable that
their  technical complexity will limit  widespread use and
acceptance.  Of the DNA adduct techniques represented, the
post-labelling  method appears to hold  most promise.  The
need  for radiolabelled test  compounds or specific  mono-
clonal  antibodies  is  eliminated and  the assay provides
information  on the direct  interaction of test  chemicals
with  DNA in  a variety  of target  organs, including  the
liver.   Providing a reliable  and robust protocol  can be
established  and pending the provision of more information
regarding  the  relationship between  DNA adduct formation
and genotoxic changes to the DNA, this assay could have an
important  role  to play  in  the assessment  of genotoxic
hazard.

    The  performance  of  the cytogenetic  assays in liver
cells  was  disappointing but,  theoretically, they could,
with  further development,  prove to  be of  value in  the
assessment  of liver-specific genotoxicity.   The reliance
of  the  current  micronucleus procedure  on partial hepa-
tectomy is a disadvantage, but the development of alterna-
tive means of stimulating hepatic mitotic activity, either
 in vivo  or in short-term culture, could be the basis of a
useful   short-term  liver-specific  assay.    Cytogenetic
assays  on  circulating  blood cells  performed reasonably
well and the theoretical and practical advantages of tests
for  SCE  or  micronuclei  in  circulating  blood   should
encourage further development of these tests.

    The  mouse spot test,  in its original  form  (section
6.4), successfully separated the carcinogen/non-carcinogen
pairs  and, providing care is taken in the selection of an
appropriate  solvent or formulation,  it appears to  be  a
useful and reproducible assay. Its availability is limited
by  the need to  maintain specific mouse  strains and  its
overall sensitivity, compared to the micronucleus test, is
suspect,  as positive results  in the present  study  were
dependant on intraperitoneal dosing.

9.3  The detection of germ cell mutagens

    The  performance  and  significance of  the three germ
cell assays represented in the study are  discussed  above
(section 8.5).  Only BP was clearly identified as  a  germ
cell mutagen in the dominant lethal assay.  The failure of
the  sperm abnormality test to discriminate between BP and
2AAF suggests that this assay is affected  by  non-genetic
toxic  effects  and is,  therefore,  of doubtful  value in
predicting  the induction of  mutations in mammalian  germ
cells.

    Although  there are theoretical reasons  why BP should
not induce UDS in germ cells (section 8.5.1),  this  nega-
tive  result raises questions  similar to those  resulting
from the failure of BP to induce UDS in rat liver (section
8.2.2),  i.e. why  does BP  not induce  detectable UDS  in
target cells where there is evidence, from  other  assays,
of genotoxic activity?

    As  concluded by Ashby  et al. (1988)  `the germ  cell
activities of BP have served to focus  several  important,
but unresolved, issues associated with the conduct of, and
the  interpretation  of  data  derived  from,  germ   cell
genotoxicity and mutagenicity assays'.

9.4  Influence of the route of administration of the test chemicals

    A  feature of the  micronucleus assays was  that their
qualitative  separation of BP and  2AAF from PYR and  4AAF
was not influenced by the route of administration  of  the
chemicals.   Dominant lethal mutations, however, were only
detected after intraperitoneal dosing with BP; oral dosing
was  ineffective.   Similarly,  the mouse  spot  test  was
dependant  on the intraperitoneal route  of administration
for  the demonstration of  mutagenic effects.  The  intro-
duction of the test material in the vicinity of the uterus
in  the spot test circumvents the natural pharmacokinetics
and  metabolic routes of  the test animal,  and it can  be
argued  that this unrealistic pattern of exposure does not
reflect  true   in vivo genotoxicity.  It  can be similarly
argued  that the purpose  of  in vivo tests is  to  achieve
maximum  exposure of the  target cells and  that intraper-
itoneal dosing can be justified for this reason.   It  may
be,  as suggested by  Shelby (1986), that  intraperitoneal
dosing should be reserved for assays used in  a  screening
mode,  i.e. for the  detection of genotoxic  activity, and
that for investigating the  in vivo activity of established
genotoxins,  a  route  of administration  should be chosen
that  reflects possible human exposure.  Arguments such as
these  must  be  resolved before  strategies  for  testing
chemicals for genotoxic activity can be fully harmonized.

10.  CONCLUSIONS

    Of  the fifty or  so separate  in vivo techniques  rep-
resented  in the CSSTT/2 study, only a small number satis-
fied  the criteria defined for an acceptable short-term  in
 vivo test (section 3.2).  These criteria, however, defined
a  very  specific  purpose, i.e.  the identification of  in
 vivo activity of established  in vitro genotoxins. In order
to  generate a comprehensive  data base on  the four  test
chemicals, assays included in the study were  not  limited
to, for example, those that were considered most likely to
meet the criteria.  As a result, some  investigators  sub-
mitted  data from assays that were not designed for detec-
ting   in vivo genotoxins but, nevertheless, provided valu-
able information on a broad spectrum of biological effects
of  the  test  chemicals. The  failure  of  such tests  to
satisfy  the  narrow  selection criteria  used  in CSSTT/2
should  not be regarded as a reflection on the validity of
the procedures for specific research purposes.  With these
considerations  in mind, and based on the concept (section
3.2)   that short-term assays are  required to investigate
both  liver-specific  and extra-hepatic  genotoxicity, the
following conclusions appear appropriate.

1.  Most of the  in vivo somatic cell assays  for  genotox-
icity were able to discriminate between the  two  carcino-
gen/non-carcinogen  pairs,  although,  in some  instances,
weak   activity  was  observed  in  tests  with  the  non-
carcinogens, particularly with 4AAF.

2.    The  weak  genotoxicity  of  4AAF  in  some  studies
suggested  that  qualitative  determinations in  terms  of
`positive'  and `negative' are  too simplistic for  hazard
assessment  purposes and highlights the need for appropri-
ate  positive test criteria, in biological and statistical
terms, for widely used  in vivo tests.

3.   The insensitivity of some  assays to one or  other of
the two pairs of compounds supports the concept that nega-
tive   in vivo data should be  produced from at  least  two
assays  in  different  tissues, i.e.  one in extra-hepatic
somatic  tissues and one in  the liver, if they  are to be
used for assessing genotoxic hazard.

4.   The mouse bone marrow micronucleus test was confirmed
as a robust and sensitive assay and is the assay of choice
for primary  in vivo testing of  in vitro genotoxins.

5.   The overall performance  of the rat  liver UDS  assay
suggests  that  it could  be  complementary to  the micro-
nucleus  test  providing  the  reservations  raised  above
(sections  8.2.2 and 9.2.1) can be satisfied.  Development
and evaluation of alternative procedures for investigating
liver genotoxicity should be actively pursued.

6.   Certain widely advocated assays,  including the host-
mediated  and  urine  mutation  assays  and  tests   using
drosophila  are concluded to be  inappropriate for hazard-
assessment purposes.

7.  BP and 2AAF showed genotoxic activity in the organs in
which they also produce tumours.  Their genotoxic activity
in  other tissues, however, precludes the use of data from
short-term  tests for making predictions of organ-specific
carcinogenicity.

8.   The majority of  the positive responses  observed  in
this  study were obtained after oral administration of the
test  chemicals  and  at doses  substantially below lethal
levels,  suggesting that short-term  in vivo assays are not
intrinsically insensitive, as is sometimes inferred. Posi-
tive  results were obtained  in the dominant  lethal assay
and the mouse spot test after intraperitoneal dosing only;
both  assays produced negative  results after oral  admin-
istration.  These  facts suggest  that the intraperitoneal
route  should be reserved  for the detection  of genotoxic
activity and that, for hazard-assessment purposes, a route
of administration should be chosen that is relevant to the
perceived route(s) of human exposure.

9.   The  germ  cell  mutagenicity  exhibited  by  BP  was
adequately  predicted  by  the somatic  cell  mutagenicity
assays,  adding  weight to  the  suggestion that  tests on
mammalian  germ  cells should  only  be conducted  to gain
further  information on the  mutagenic spectrum of  estab-
lished  in vivo somatic cell mutagens.

10.  The occurrence of chemical carcinogens that  fail  to
show  evidence  of  genotoxicity in   in vitro and   in vivo
assays   indicates the existence  of a class  of chemicals
that  induce cancer by  a mechanism that  is not a  conse-
quence of a direct interaction with DNA.   The  acceptance
of the validity of the concept of non-genotoxic mechanisms
of cancer induction is considered to be a crucial question
in the future deployment of short-term tests  in  carcino-
genesis.

11.  The overall conclusion from the CSSTT/2 study is that
short-term   in vivo tests  have a  vital  role to  play in
hazard   assessment.   This  role   is  to  define   which
chemicals,  identified  as genotoxic  from  in vitro tests,
are  active  in vivo and, thus,  are those most  likely  to
present   a  carcinogenic/mutagenic  hazard   to  mammals,
including humans.

REFERENCES

ASHBY,   J.,   DE   SERRES,   F.J.,   DRAPER,   M.,   ISHIDATE,   M.,   Jr,
MARGOLIN,  B.H.,  MATTER, B.,  & SHELBY, M.D.,  ed. (1985)    Evaluation  of
 short-term  tests  for  carcinogens, Amsterdam, Oxford,  New York, Elsevier
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ASHBY,   J.,   DE   SERRES,   F.J.,   SHELBY,   M.D.,    MARGOLIN,    B.H.,
ISHIDATE,  M., Jr, &  BECKING, G.C., ed.  (1988a)  Evaluation of  short-term
 tests  for carcinogens: Report of  the International Programme on  Chemical
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Cambridge University Press, Vol. 1.

ASHBY,  J.,  SHELBY,  M.D., & DE SERRES, F.J. (1988b)  Overview of the IPCS
collaborative  study on  in vivo assay systems and conclusions.  In:  Ashby,
J.,  de  Serres, F.J.,  Shelby, M.D., Margolin,  B.H., Ishidate, M.,  Jr, &
Becking, G.C., ed.  Evaluation of short-term tests for  carcinogens:  Report
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DE  SERRES, F.J. & ASHBY, J., ed. (1981)  Evaluation of short-term tests for
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GENEROSO,  W.M.,  CAIN,  K.T., HELLWIG,  C.S.  &  CACHEIRO,  N.L.A.  (1982)
Lack  of  association between  induction  of dominant-lethal  mutations and
induction  of heritable translocations with benzo (a) pyrene  in postmeiotic
germ cells of male mice.  Mutat.  Res., 94: 155-163.

HICKS,  M.R.,  ASHBY,  J., &  PENMAN,  M.  (1988)   Review  of  the  rodent
carcinogenicity data for the four test chemicals. In: Ashby, J., de Serres,
F.J.,  Shelby,  M.D., Margolin,  B.H., Ishidate, M.,  Jr, & Becking,  G.C.,
ed.  Evaluation   of  short-term  tests  for  carcinogens:   Report  of  the
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MCGREGOR,   D.   (1988a)   Summary  report   on  the  performance  of   the
miscellaneous   group  of   in vivo assays  (carcinogenicity,  DNA  adducts,
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MCGREGOR,  D.  (1988b)   The activities  of 2AAF,  4AAF, BP  and PYR  in  in
 vitro assays  for genetic toxicity. In: Ashby, J., de Serres, F.J., Shelby,
M.D., Margolin, B.H., Ishidate, M., Jr, & Becking, G.C., ed.  Evaluation  of
 short-term  tests for carcinogens: Report of the International Programme on
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Kingdom, Cambridge University Press, Vol. 2, pp. 345-350.

MARGOLIN,  B.H.  &  RISKO, K.J.   (1988)   The  statistical analysis  of  in
 vivo genotoxicity  data: case studies of  the rat hepatocyte UDS  and mouse
bone  marrow micronucleus assays. In:  Ashby, J., de Serres,  F.J., Shelby,
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MIRSALIS,  J.C.  (1988)  Summary report  on the performance of  the  in vivo
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PATON,  D.  &  ASHBY, J.  (1988)  Source and analytical details of the test
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SHELBY, M.D.  (1986)  A case for the continued use of  the  intraperitoneal
route of exposure.  Mutat. Res., 70: 169-171.

TICE,  R.R.   (1988)   Summary report  on  the  performance of  the  in vivo
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Kingdom, Cambridge University Press, Vol. 1, pp.  233-253.

WHO  (1985)  Environmental  Health  Criteria  47:  Summary  report  on  the
evaluation  of short-term tests for carcinogens (collaborative study on   in
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WYROBEK,    A.J.,   GORDON,   L.A.,   BURKHART,    J.G.,   FRANCIS,   M.W.,
KAPP,  R.W.,  LETZ,  G.,  MALLING,  H.V.,  TOPHAM,  J.C., &  WHORTON,  M.D.
(1983)  An evaluation of the mouse sperm morphology test and other tests in
non-human mammals: A report of the United States  Environmental  Protection
Agency Gene-Tox Program.  Mutat. Res., 115: 1-7.

RESUME

ETUDE COLLECTIVE DU PROGRAMME INTERNATIONAL SUR LA SECURITE DES
SUBSTANCES CHIMIQUES (IPCS) POUR L'EVALUATION ET LA VALIDATION
DES EPREUVES DE COURTE DUREE RELATIVES AUX CANCEROGENES

    La première partie de ce projet, qui traite des études
 in vitro,  a été publiée en 1985 (Ashby et al., 1985);  on
en trouvera un résumé dans le No 47 de la  série  Critères
d'hygiène  de  l'environnement  (OMS  1985).   La  seconde
partie, qui fait l'objet du présent rapport, a été publieé
en 1988 (Ashby et al., 1988).

    Devant  la nécessité d'évaluer l'intérêt  des épreuves
de courte durée pour la recherche des substances mutagènes
et cancérogènes, il devenait indispensable d'organiser des
études  collectives inter-laboratoires à  l'échelle inter-
nationale. Ces épreuves de courte durée devaient compléter
les épreuves classiques à long terme sur rongeurs  ou  s'y
substituer.   C'est le problème du  choix ainsi que de  la
fiabilité  et de  la sensibilité  de ces  épreuves  qui  a
conduit  à la première grande  entreprise de collaboration
au  niveau international, à savoir le Programme coopératif
international  pour  l'évaluation  des épreuves  de courte
durée  relatives aux cancérogènes  (IPESTTC) (de Serres  &
Ashby, 1981).  Les résultats de cette étude  ont  confirmé
la  fiabilité et la  faisabilité de l'épreuve  de mutation
des  salmonelles pour une première identification des sub-
stances  cancérogènes et mutagènes.   On a également  con-
staté qu'avec cette épreuve, certains produits notoirement
cancérogènes chez les rongeurs étaient impossibles ou tout
du moins très difficiles à mettre en évidence.  Un certain
nombre  d'autres  épreuves  examinées  dans  le  cadre  de
l'étude  IPESTTC se sont  révélées capables de  mettre  en
évidence   quelques-uns   des  cancérogènes   murins  pour
lesquels  l'épreuve  sur  salmonelle donnait  un  résultat
négatif.  Toutefois, la base  de données était  trop  res-
treinte  pour qu'on puisse recommander une épreuve capable
de compléter l'épreuve de mutation des salmonelles.

    Au  vu des résultats de l'étude IPESTTC, il est apparu
nécessaire  de  poursuivre  ce type  de  coopération  afin
d'établir  a) quel  est l'ensemble  d'épreuves  in vitro le
plus efficace pour un premier tri des substances chimiques
sur  la base de  leur activité génotoxique  et  b) quelles
sont  les épreuves  in vivo de courte durée les plus utiles
pour  confirmer la génotoxicité et  le pouvoir cancérogène
des  substances  chimiques  chez les  mammifères.  L'étude
collective pour l'évaluation et la validation des épreuves
de  génotoxicité  et  de cancérogénicité  de  courte durée
(CSSTT), a été proposée par le Programme international sur
la sécurité des substances chimiques (IPCS) et le National
Institute  of  Environmental  Health Sciences  (NIEHS) des
Etats-Unis   d'Amérique,   l'une   des  institutions   qui
participent  au Programme IPCS.  Devant l'ampleur de cette

entreprise et du fait des problèmes logistiques que posent
l'organisation et la gestion de ce genre  d'études  inter-
nationales,   le  projet  a  été  divisé  en  deux  études
distinctes:  l'Etude collective sur les  épreuves de géno-
toxicité  et de cancérogénicité  de courte durée   in vitro
(CSSTT/1)   et  l'Etude  collective sur  les  épreuves  de
mutagénicité et de cancérogénicité de courte durée  in vivo
(CSSTT/2).

    L'Etude  CSSTT/1 a permis de constituer une vaste base
de  données  à  partir d'épreuves   in vitro très  diverses
portant  sur dix produits chimiques organiques.  Parmi eux
figuraient  huit  substances  de cancérogénicité  reconnue
pour  les rongeurs qui s'étaient révélées soit négatives à
l'épreuve  sur salmonelle soit difficiles à reconnaître de
cette manière, ainsi que deux substances considérées comme
non  cancérogènes.  On a procédé à l'évaluation de données
tirées  de  90 groupes  d'épreuves effectuées  par quelque
60 scientifiques.   Quatre types d'épreuves ont  donné des
résultats suffisamment bons pour qu'on puisse envisager de
les utiliser en complément à l'épreuve sur salmonelle.  Il
s'agissait de la recherche des aberrations chromosomiques,
des mutations géniques et des transformations néoplasiques
en culture de cellules mammaliennes ainsi que de l'épreuve
d'aneuploïdie  des  levures.   Sauf  dans  le  cas  de  la
recherche  des  aberrations chromosomiques,  on a constaté
que les protocoles généralement utilisés pour ces épreuves
devaient  être étudiés plus à fond avant d'être considérés
comme tout à fait acceptables.

    La  principale conclusion de l'étude  CSSTT/1 relative
aux  épreuves  in vitro,  c'est que  en combinant l'épreuve
de recherche des aberrations chromosomiques à l'épreuve de
mutation  des salmonelles, on  peut procéder à  un premier
criblage efficace des substances cancérogènes.

    Lors de l'étude IPESTTC, sept substances sur  14  sup-
posées  non cancérogènes ont donné  des résultats positifs
dans  un grand nombre  d'épreuves  in vitro.   D'après  les
données   in vivo limitées  qu'a  fournies cette  étude, il
semblerait  que ces sept produits soient inactifs dans les
épreuves  de courte durée  in vivo.   Les produits non can-
cérogènes  de deux des couples cancérogène/non-cancérogène
soumis  à l'étude IPESTTC, à savoir le couple benzo [a] py-
rène/pyrène  (BP/PYR) et le couple acétyl-2 aminofluorène/
acétyl-4  aminofluorène  (2AAF/4AAF), constituent  de bons
exemples  de ces réactions différentes  et d'ailleurs, ces
couples  de produits avaient été retenus pour la partie  in
 vivo de  l'étude  collective  (CSSTT/2).  Toutefois,  il y
avait  doute quant à  la non cancérogénicité  supposée  du
4AAF  et une part  très importante de  l'étude a été  con-
sacrée  à la mise en  route d'études de cancérogéniticé  à
long terme sur le 2AAF et le 4AAF chez le rat.

    L'objectif  de l'étude CSSTT/2 était  donc de produire
un  profil de données complet  à partir d'une large  gamme
d'épreuve  in vivo de courte durée afin de voir comment les
différents paramètres génétiques des tissus-cibles se com-
portent  vis-à-vis  de  produits chimiques  classés  comme
génotoxiques  d'après les épreuves  in vitro.   La finalité
de  l'étude était de recenser les épreuves  in vivo suscep-
tibles d'être utilisées pour déterminer l'activité  in vivo
de substances notoirement génotoxiques.

    Quatre-vingt-dix-sept  chercheurs de 16 pays  ont par-
ticipé  à l'étude  in vivo,  les données présentées portant
sur  une cinquantaine de techniques distinctes. Les résul-
tats  ont été évalués lors d'une réunion de ces chercheurs
qui  s'est tenue au Cap d'Agde (France) en mai 1985.  On a
préparé une série de rapports comportant une évaluation de
chaque  groupe d'épreuves, des rapports récapitulatifs sur
les  épreuves  relatives  aux cellules  germinales  et les
épreuves  d'hépatotoxicité et des rapports  résumant toute
la base de données relative à chaque couple  de  produits.
Par  la suite,  on a  préparé un  récapitulatif  de  toute
l'étude  in vivo en vue de sa publication définitive.

    Seule  une  faible  proportion des  épreuves examinées
lors  de  l'étude  CSSTT/2  ont  satisfait  aux   critères
d'acceptabilité  comme  épreuves  in vivo de  courte durée.
Toutefois les épreuves examinées lors de cette étude ne se
limitaient pas à celles jugées a priori les plus conformes
aux  critères.   De la  sorte, on a  pu tirer des  données
provenant  d'épreuves qui, à l'origine,  n'étaient pas des
épreuves  de génotoxicité  in vivo,  des renseignements sur
une  large  gamme  d'effets biologiques  produits  par ces
quatre substances. La plupart des épreuves de génotoxicité
 in vivo  portant  sur des cellules somatiques permettaient
de distinguer les deux couples cancérogène/non-cancérogène
encore  que dans certains  cas, les produits  non cancéro-
gènes,  en particulier le  4AAF aient présenté  une faible
activité.  L'insensibilité de certaines épreuves à l'un ou
à  l'autre des deux couples de produits chimiques, corrob-
orent  l'idée selon laquelle il faut obtenir des résultats
négatifs   in vivo  dans au moins  deux épreuves pratiquées
sur  des  tissus  différents  avant  de  considérer  qu'un
produit chimique n'est pas génotoxique  in vivo.

    Il a été confirmé que l'épreuve des  micro-noyaux  sur
moëlle osseuse de souris est robuste, sensible  et  repro-
ductible et on la recommande pour une  première  recherche
de  la  génotoxicité  in vivo et   in vitro.   Les résultats
généraux  obtenus au moyen  de l'épreuve sur  foie de  rat
pour  la synthèse anarchique  de l'ADN incitent  à  penser
qu'elle  pourrait  compléter  l'épreuve des  micro-noyaux,
mais  il  faut  en étudier  encore  la  sensibilité et  la

sélectivité.   Certaines épreuves pourtant  largement pré-
conisées, notamment les épreuves par passage sur hôte, les
épreuves  de mutagénicité des  urines et les  épreuves sur
drosophile, se sont révelées impropres à une évaluation du
risque.

    Les  résultats de l'étude CSSTT/2 ont confirmé que les
épreuves   in vivo de courte durée  ont un rôle  capital  à
jouer  dans l'évaluation du risque, rôle qui consiste dans
l'identification   des  produits  chimiques   qui  s'étant
révélés  génotoxiques  in vitro,  sont  également actifs  in
 vivo et   comportent  donc vraisemblablement  un risque de
cancérogénicité  ou de mutagénicité pour les mammifères en
général et l'homme en particulier.

RESUMEN

PROGRAMA INTERNACIONAL DE SEGURIDAD DE LAS SUSTANCIAS QUIMICAS (IPCS):
ESTUDIO EN COLABORACION SOBRE EVALUACION Y COMPROBACION DE PRUEBAS
A CORTO PLAZO PARA SUSTANCIAS CARCINOGENAS

    La  primera  parte  del presente  proyecto, relativa a
estudios  in vitro, se publicó en 1985 (Ashby et al., 1985) y se
resumió en la publicación 47 de la  serie  "Environmental
Health  Criteria" (OMS, 1985).   La segunda parte  es  el
tema del presente informe y se publicó en 1988  (Ashby  et
al., 1988).

    La   necesidad  de  estudios  en   colaboración  entre
laboratorios  en  escala  internacional surgió  de que era
imprescindible  investigar el valor de las pruebas a corto
plazo  para detectar las  sustancias químicas mutágenas  y
carcinógenas.   Se propusieron los  ensayos a corto  plazo
como  métodos  que  sustituyeran o  complementaran  a  los
tradicionales   ensayos   biológicos  a   largo  plazo  en
roedores.   La preocupación por la elección de las pruebas
a corto plazo y por su fiabilidad y sensibilidad condujo a
que  se  realizara  el  primer  ejercicio  importante   de
colaboración  internacional:  el programa internacional en
colaboración sobre evaluación de las pruebas a corto plazo
para sustancias carcinógenas (IPESTTC) (de Serres y Ashby,
1981).  Los resultados de ese estudio confirmaron el valor
de  la prueba de mutación de salmonelas como ensayo fiable
y  factible para la identificación  primaria de sustancias
carcinógenas  y mutágenas.  Se  observó también que  en la
prueba de salmonelas no se detectaban o sólo se detectaban
con grandes dificultades algunas sustancias de cancerogen-
icidad  conocida en los roedores.  Otros ensayos incluidos
en   el  estudio  IPESTTC  pudieron   detectar  sustancias
carcinógenas para roedores que eran negativas en el ensayo
de salmonelas.  Sin embargo, la base de datos de apoyo era
demasiado  pequeña  para  permitir la  recomendación de un
ensayo   que  complementara  la  prueba   de  mutación  de
salmonelas.

    Los   resultados  del  estudio  IPESTTC   pusieron  de
manifiesto  que  se  necesitaría  un  nuevo  ejercicio  en
colaboración  para  determinar:   a)  la  combinación  más
eficaz  de ensayos  in vitro para la  detección primaria de
la actividad genotóxica de sustancias químicas, y  b)  las
pruebas  in vivo a corto plazo más útiles para confirmar la
posibilidad  de genotoxicidad y cancerogenicidad  en mamí-
feros.   El Programa Internacional sobre  Seguridad de las
Sustancias  Químicas  (IPCS)  y el  Instituto  Nacional de
Ciencias de Higiene del Medio (NIHS) de los Estados Unidos
de  América,  como  institución participante  en  el IPCS,
propusieron  el estudio en colaboración sobre evaluación y
comprobación  de las pruebas a  corto plazo para la  geno-
toxicidad y la cancerogenicidad (CSSTT). Dadas la magnitud
prevista  del proyecto y la logística de la organización y
administración   de  los  proyectos   internacionales,  el

proyecto  se dividió en  dos estudios independientes:   el
estudio en colaboración sobre pruebas a corto  plazo  para
la  genotoxicidad  y  la cancerogenicidad  (CSSTT/1)  y el
estudio  en  colaboración sobre  pruebas   in vivo  a corto
plazo para sustancias mutágenas y carcinógenas (CSSTT/2).

    En  el estudio CSSTT/1 se reunió una abarcante base de
datos procedentes de una amplia gama de  ensayos   in vitro
realizados   con diez productos químicos orgánicos cuidad-
osamente  seleccionados.   Incluían  ocho  sustancias   de
cancerogenicidad  probada  para  roedores, que  resultaban
negativas   o  difíciles  de  detectar  en  el  ensayo  de
salmonelas  y dos sustancias químicas consideradas como no
carcinógenas.   Se evaluaron los datos procedentes de casi
90  series de ensayos  efectuados por unos  60 científicos
participantes.   El rendimiento de cuatro tipos de ensayos
se  estimó  suficiente  para considerarlos  como  posibles
pruebas   complementarias   del   ensayo  de   salmonelas.
Incluyeron  las pruebas para  la determinación de  aberra-
ciones cromosómicas, mutaciones génicas y transformaciones
neoplásicas en células de mamífero en cultivo, y un ensayo
para  determinar  la  aneuploidia en  levaduras.   Con  la
excepción del ensayo de aberraciones cromosómicas, resultó
evidente  que  los  protocolos utilizados  en general para
esas  pruebas exigían evaluación adicional  antes de poder
considerarlos plenamente aceptables.

    La  principal conclusión del estudio CSSTT/1 sobre las
pruebas   in vitro fue  que el  empleo  de los  ensayos  de
aberraciones  cromosómicas en asociación con  la prueba de
mutación de salmonelas podía resultar un detector primario
eficaz para posibles sustancias carcinógenas nuevas.

    En el estudio IPESTTC, siete de las catorce sustancias
no  carcinógenas presuntas dieron resultados  positivos en
muchas  de las pruebas   in vitro.  Los limitados  datos de
pruebas   in vivo disponibles  procedentes  de ese  estudio
permiten  pensar que esas  siete sustancias químicas  eran
inactivas en las pruebas a corto plazo  in vivo.    En  dos
pares   de  sustancias  carcinógenas/no   carcinógenas  de
IPESTTC  (benzo [a] pireno/pireno (BP/PIR) y  2-acetilamino-
fluoreno/4-acetilamino-fluoreno  (2AAF/4AAF),  las sustan-
cias  no  carcinógenas  proporcionaron buenos  ejemplos de
tales respuestas diferentes, de modo que se seleccion-aron
esos  pares de sustancias  químicas para la  parte  in vivo
del  estudio en colaboración (CSSTT/2).  Sin embargo, hubo
una fuerte duda respecto a la presunta falta  de  cancero-
genicidad del 4AAF, y una parte crucial del estudio fue el
comienzo  de bioensayos de cancerogenicidad  a largo plazo
del 2AAF y el 4AAF en ratas.

    Por  consiguiente, el objetivo del estudio CSSTT/2 fue
producir un abarcante perfil de datos procedentes  de  una
amplia gama de pruebas  in vivo a corto plazo,  como  medio
de  conocer el mecanismo por  el que los distintos  puntos

finales  genéticos de los tejidos  destinatarios fundamen-
tales responden a sustancias químicas definidas como geno-
tóxicas  in vitro.   La meta final consistía en identificar
qué  ensayos   in vivo podrían  usarse para  determinar  la
actividad  in vivo de los productos genotóxicos probados.

    Participaron  en  el  proyecto de  pruebas   in vivo 97
investigadores de 16 países y se presentaron datos de unas
cincuenta  técnicas  in vivo distintas.  Los  resultados se
evaluaron  en una reunión  de investigadores celebrada  en
Cap  d'Agde (Francia)  en mayo  de 1985.   Se preparó  una
serie de informes que comprendían una evaluación  de  cada
grupo  de ensayos, informes resumidos sobre los ensayos en
células  germinales y las pruebas hepáticas específicas, e
informes  resumidos sobre la  base total de  datos corres-
pondiente a cada par de sustancias químicas.   Después  se
preparó un examen general de todo el estudio  in vivo listo
para la publicación final.

    Sólo  una pequeña proporción de los ensayos represent-
ados en el estudio CSSTT/2 satisficieron los criterios que
definen  una prueba  in vivo a corto plazo  aceptable.  Sin
embargo,  los  ensayos  incluidos  en  el  estudio  no  se
limitaron  a los que  podían cumplir con  más probabilidad
los criterios.  Así, aunque los datos procedían de ensayos
que  no estaban destinados fundamentalmente  a identificar
la   genotoxicidad  in vivo,    proporcionaron  información
sobre  un  amplio espectro  de  efectos biológicos  de las
cuatro  sustancias químicas.  La mayoría de los ensayos en
células sométicas  in vivo para determinar la genotoxicidad
discriminaron entre las sustancias carcinógenas/no carcin-
ógenas  de  los  dos pares,  aunque  en  algunos casos  se
detectó  una actividad débil en las pruebas con sustancias
no  carcinógenas, en particular con el 4AAF.  La insensib-
ilidad de algunos ensayos a uno u otro de los dos pares de
productos químicos apoya el concepto de que deben obtener-
se  datos  in vivo negativos en dos ensayos por lo menos en
diferentes  tejidos antes de  que pueda aceptarse  que una
sustancia química carece de genotoxicidad  in vivo.

    Se  confirmó que la  prueba de micronúcleos  de médula
ósea  de ratón  es un  ensayo potente,  sensible y  repro-
ducible,   que  se  recomienda  para  las  pruebas  in vivo
primarias   de  las sustancias  genotóxicas  in vitro.   El
rendimiento  general  del  ensayo en  células hepáticas de
rata  para la síntesis no programada de ADN permite pensar
que puede ser complementario de la prueba de micronúcleos,
aunque  requieren investigación adicional ciertos aspectos
de  sensibilidad y selectividad de ese ensayo.  Se llegó a
la  conclusión de que ciertos  ensayos ampliamente susten-
tados,  que  incluyen los  de  mutagenicidad por  orina  y
mediada por el huésped y las pruebas que utilizan la mosca
drosófila,   son   inapropiados  para   la  evaluación  de
riesgos.

    Los resultados del estudio CSSTT/2 confirmaron que las
pruebas  in vivo a  corto  plazo tienen  que desempeñar una
función  crucial en la evaluación de los riesgos y que esa
función  consiste  en  identificar los  productos químicos
que,  una  vez demostrada  la genotoxicidad  in vitro,  son
activos  in vivo y  tienen  así  mayores probabilidades  de
presentar  un  riesgo de  cancerogenicidad o mutagenicidad
para los mamíferos, incluido el hombre.


    See Also:
       Toxicological Abbreviations