COMPARATIVE GENETIC TOXICOLOGY

COMPARATIVE GENETIC TOXICOLOGY

The Second UKEMS Collaborative Study

Edited by

J.M. PARRY Department of Genetics, University College Swansea

and

C.F. ARLETT MRC Cell Mutation Unit, University of Sussex

M MACMILLAN

© United Kingdom Environmental Mutagen Society 1985 Softcover reprint of the hardcover 1st edition 1985 978-0-333-38897-6

All rights reserved. No reproduction, copy or transmission of this publication may be made without written permission.

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Any person who does any unauthorised act in relation to this publication may be liable to criminal prosecution and civil claims for damages.

First published 1985

Published by THE MACMILLAN PRESS LTD Houndmills, Basingstoke, Hampshire RG21 2XS and London Companies and representatives throughout the world

British Library Cataloguing in Publication Data

Comparative genetic toxicology : second UKEMS collaborative genotoxicity study. 1. Chemical mutagenesis 2. Toxicology I. Parry, J.M. II. Arlett, C.F. 575.2'92 QH465.C5 ISBN 978-1-349-07903-2 ISBN 978-1-349-07901-8 (eBook) DOI 10.1007/978-1-349-07901-8

Contents

Preface xi

Part 1 INTRODUCTION

1 Design of the ~tudy 'scientific rationale and chemical selection' J. Ashby (Alderley Park) 3

2 The genotoxicity of benzidine and 4-dimethylaminoazobenzene: A survey of the literature E. Waters (Swansea) 9

Part 2 BACTERIAL MUTATION (CORE STUDY)

3 Evaluation of the mutagenic activity of 4-dimethylaminoazobenzene (DAB) and 4-cyanodimethylaniline (CDA) in the Salmonella/ micro-some assay D. Anderson and S.D. Blowers (Carshalton) 41

4 Salmonella mutagenicity testing of benzidine, 4, 4'' -diarninoterphenyl, 4-dimethylaminoazobenzene and 4' -cyanodimethylaniline using three different S9 preparations R.S. U. Baker and A.M. Bonin (Sydney, Australia) 43

5 The mutagenic activity of the structurally related compounds benzidine (BZD) and 4, 4" -diaminoterphenyl (DAT) in the Salmonella/mammalian-microsome assay T.M. Brooks and L.P. Gonzalez (Sittingbourne) 49

6 Reverse mutation in Salmonella typhimurium R. Forster and R.D. Gwilliam (Medmenham) 55

7 Bacterial mutagenicity of BZD and DAT S.A. Hubbard and C.M. Hunt (Guildford) 59

8 4-Dimethylaminoazobenzene (DAB) and 4-cyanodimethylaniline (CDA): Reverse mutation tests with Salmonella typhimurium TA1538 A.Ladner and J. W. Furlong (Loughborough) 63

9 Comparative mutagenic activity on Salmonella typhimurium TA1538 of benzidine and its terphenyl analogue 4,4" -diaminoterphenyl using

vi Contents

chicken liver, Aroclor-induced and non-induced rat liver S9s E.D. Massey, J. Haley, A.E. Hill and P.M. Setterfield (Southampton)

10 A comparison of the mutagenicity of benzidine and 4,4" -diaminoter-phenyl with uninduced mouse and rat liver S9 D. Paes, D. Gatehouse and D. Tweats (Ware)

11 The testing of four UKEMS trial compounds for bacterial mutation in the Salmonella/microsome assay using (1) Aroclor-induced rat liver, (2) chicken liver S9 and (3) the York modification of the standard Ames protocol S.E. Riley and R.C. Gamer (York)

12 Bacterial mutation tests on CDA and DAB using Salmonella typhimu-rium TA1538 and three types of S9 activation. R.J. Tye (Finham)

13 A comparison of the effects of uninduced mouse, uninduced rat and Aroclor-induced rat liver S9 on reversion in Salmonella by DAB and CDA. R.B. Varley, J.D. Rae and D.J. Kirkland (Ledbury)

14 Studies on the metabolic activation of benzidine and its terphenyl derivative to mutagens in the Salmonella plate incorporation assay by various liver preparations. J.M. Walters and R.D. Combes (Portsmouth)

15 Mutagenicity of BZD, DAT, DAB and CDA in the bacterial muta-genicity test P. Watkins and C. Rickard (Alderley Park)

16 Bacterial mutagenicity assays: Co-ordinators' report. S. Venitt and R. Forster (Sutton and Rome, Italy)

Part 3 MODIFICATION OF THE BACTERIAL MUTAGENICITY ASSAYS

17 Studies on the mutagenicity of benzidine and its terphenyl analogue in the plate-incorporation test using liver microsome and cytosol

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fractions. J.M. Walters and R.D. Combes (Portsmouth) 147 18 The in vitro mutagenicity of benzidine and 4,4" -diaminoterphenyl.

D. Paes, D. Gatehouse, D. Tweats andY. Holtby (Ware) 155 19 Urinary mutagenicity in rats after administration of benzidine and its

terphenyl analogue, 4,4" -diaminoterphenyl. J. Gibson (London) 165 20 Enhanced mutagenicity of DAT to Salmonella ( -S9) when evaluated

under conditions of reduced light. J. Ashby, R.D. Callander and D.J. V. Paes (Alderley Park and Ware) 175

21 Weak mutagenicity of 4,4" -dinitroterphenyl to Salmonella in the absence of S9 mix. J. Ashby and R.D. Callander (Alderley Park) 179

22 The comparative mutagenicity of 4-dimethylaminoazobenzene and 4-cyanodimethylaniline in plate-incorporation tests and fluctuation assays. D. Gatehouse, D. Wedd and K. Wharton (Ware) 181

23 The weak mutagenicity of CDA to strain T A98 of Salmonella typhimurium. J. Ashby, R.D. Callander and D. Paton (Alderley Park) 189

Contents vii

Part 4 YEAST GENOTOXICITY ASSAYS

24 'Petite' mutagenesis by benzidine, DAT, DAB and CDA in Saccharomyces cerevisiae. L.R. Ferguson (Auckland, New Zealand) 195

25 The comparative sensitivity of repair proficient and repair deficient strains of yeast to BZD, DAT, DAB and CDA. T.A. Tippins and J.M. Pa"y (Swansea) 205

26 A study of the genotoxicity of DAB, CDA, BZD and DAT in the strain JD1 of Saccharomyces cerevisiae. D. Kelly and J.M. Parry (Swansea) 211

27 The induction of mitotic gene conversion in Saccharomyces cerevisiae by the compounds BZD, DAT, DAB and CDA. R. Calvert and J.M. Parry (Swansea) 221

28 The genotoxicity of BZD and DA T in yeast strain D7. J.M. Parry (Swansea) 231

29 Gene mutation in yeast induced by DAB, CDA, BZD and DAT. I. de G. Mitchell and P.J. Gilbert (Stock) 241

30 The induction of mitotic aneuploidy and crossing-over in yeast. F. Ali and J.M. Parry (Swansea) 253

31 Assay for forward mutation in Schizosaccharomyces pombe Pl. N. Loprieno and R. Forster (Pisa and Rome, Italy) 259

32 A summary of the effects of benzidine, 4,4" -diaminoterphenyl, 4-dimethylaminoazobenzene and 4-cyanodimethylaniline as observed by the yeast group in the Second UKEMS Collaborative Study. JM. Parry (Swansea) 267

Part 5 POINT MUTATION ASSAYS IN CULTURED MAMMALIAN CELLS

33 Mutagenicity of benzidine and 4,4" -diaminoterphenyl in Chinese hamster V79 cells. M.R. O'Donovan (Nottingham) 281

34 DNA damage in the rat liver in vivo and mutagenicity to V79 cells in vitro for BZD, DAT, DAB and CDA. S. Parodi, M. Pala, P. Russo, C. Balbi, G. Fassina, M.L. Abelmoschi and S. Carlone (Genoa, Italy) 291

35 The absence of mutagenic activity of benzidine and 4,4" -di-aminoterphenyl in Tk6 human lymphoblast cells. J. Cole, C.F. Arlett and W.J. Muriel (Brighton) 301

36 The mutagenic effects of DAB and CDA in a thymidine kinase-deficient clone of Friend mouse leukaemia cells. P.G. McKenna (Coleraine) 307

37 The mutagenicity of benzidine, 4,4" -diaminoterphenyl, N, N­dimethyl-4-aminoazobenzene and 4-cyano-N ,N-dimethylaniline in the L5178Y mouse lymphoma assay. J.C. Kennelly and R.C. Garner (York) 311

viii Contents

38 The induction of trifluorothymidine resistance in L5178Y Tk + ;­

mouse lymphoma cells by 4-dimethylaminoazobenzene and 4-cyanodimethylaniline. R.J. Tye (Finham) 319

39 Induction of trifluorothymidine resistance in mouse lymphoma L5178Y Tk + ;- cells following treatment with benzidine and 4,4"-diaminoterphenyl in vitro. D.H Edgar (Huntingdon) 327

40 Mammalian cell mutation assays with benzidine, 4,4" -diamino-terphenyl, 4-dimethylaminoazobenzene and 4-cyanodimethyl-aniline. C.F. Arlett (Brighton) 333

Part 6/N VITRO CYTOGENETICS

41 SCE tests of Butter Yellow, 4-cyanodimethylaniline, benzidine and diaminoterphenyl using Chinese hamster V79 cells in the presence of S9 mix. D. Gatehouse, M. Tiley and D.J. Tweats (Ware) 341

42 A comparison of the chromosome damaging effects, in cultured human lymphocytes, of benzidine (BZD) and 4,4" -di­aminoterphenyl (DAT) in the presence of Aroclor-induced and uninduced rat liver S9. J.C. Asquith, L.K. Hogan, J.N. Fullwood, J.D. Rae and D.J. Kirkland (Ledbury) 355

43 Induction of chromosome aberrations in Chinese hamster liver cells treated with BZD, DAT, DAB and CDA. A. Lafi (Swansea) 363

44 Chromosome analysis of cultured rat liver epithelial cells treated with BZD, DAT, DAB or CDA. G. Mala/lah and N. Danford (Swansea) 367

45 Effects of two carcinogens and two structural analogues on the frequencies of chromosome aberrations, polyploidy and sister chromatid exchanges in vitro. R.A.J. Priston, L. Reynolds and B.J. Dean (Sittingbourne) 373

46 The effects of BZD, DAT, DAB and CDA on mitotic chromosome aneuploidy and the nuclear spindle. A. Lafi, E.M. Parry and N. Danford (Swansea) 381

47 Summary and evaluation of cytogenetic assays in vitro. D. Scott (Manchester) 387

Part 7 UNSCHEDULED DNA SYNTHESIS

48 Activity of four UKEMS trial compounds in a He La cell DNA repair assay. J. Campbell and R.C. Garner (York) 399

49 Activity of CDA, DAB and 6BT in the in vivo rat hepatocyte DNA repair assay. J. Ashby and W. Keen (Alderley Park) 405

Contents

Part 8 COMPARISON OF S9 PREPARATIONS

50 Preparation and characterisation of S9 fractions. S.A. Hubbard, T.M. Brooks, L.P. Gonzalez and J. W. Bridges (Guildford and

ix

Sittingbourne) 413

Part 9 IN VIVO CYTOGENETICS

51 Chromosome studies in hepatic cells from rats exposed to genotoxic chemicals. M.G. Clare and B.J. Dean (Sittingbourne) 441

52 4-Dimethylaminoazobenzene and 4-cyanodimethylaniline: Micronucleus test in Alderley Park Wistar derived rats. P. Watkins (Alderley Park) 453

53 Evaluation of benzidine and 4,4" -diaminoterphenyl in the micronucleus test. R.J. Proudlock and J.A. Allen (Huntingdon) 461

54 BZD and DAT: Investigation in two mouse bone marrow assays, micronucleus test and chromosomal aberration test at mitosis. J.D. Adler and U. Kliesch (Neuherberg, Germany) 467

55 4-Dimethylarninoazobenzene (DAB) and 4-cyanodimethylaniline (CDA): An in vivo cytogenetic study in the Alderley Park (Wistar) rat. R. Albanese (Alderley Park) 475

56 Evaluation of the in vivo cytogenetic assays. B.J. Dean (Swansea and Sittingbourne) 483

Part 10 IN VIVO ASSAYS AND THE METABOLISM OF THE TEST COMPOUNDS

57 Sex-linked recessive lethal tests in Drosophila with benzidine, 4,4" -diaminoterphenyl, 4-dimethylaminoazobenzene and 4' -cyanodimethylaniline. D.S. Angus (Newcastle, Australia) 493

58 An evaluation of the genotoxicity of BZD, DAT, DAB and CDA in the sex-linked recessive lethal test using Drosophila melanogaster. J.M. Parry and P.B. Sinclair (Swansea) 499

59 Effects of 4-dimethylaminoazobenzene (DAB) and 4-cyanodimethyl-aniline (CDA) in the mammalian spot test. J. W. Hart (Ballerup, Denmark) and R. Fahrig (Hannover, Germany) 507

60 A comparison of the tissue distribution and covalent binding to hepatic DNA of [3 H] benzidine and [3 H] 4,4" -diarninoterphenyl in the rat. C.N. Martin, T. Watson and J.C. Kennelly (York) 515

61 Disposition of P 4 C] cyanodimethylaniline in the bone marrow of the rat. T.C. Orton and M. Osman (Alderley Park) 527

62 The binding of 4-cyano-N,N-dimethyl-P4 CJ aniline (CDA) metabolites in the rat. R. Waters, S. Edwards, D. Hesk and C.J. Logan (Swansea and Sittingbourne) 533

X Contents

63 The binding of 4-cyano-N,N-dimethyl-[14 C) aniline (CDA) metabolites to macromolecules in mice exposed to CDA. S. Edwards, D. Hesk, C.J. Logan and R. Waters (Swansea and Sittingbourne) 537

64 In vitro metabolism of 4-cyano-N,N-dimethyl-[14 C) aniline (CDA) by rat, mouse, chicken and induced rat S9 fractions. D. Hesk and C.J. Logan (Sittingbourne) 543

65 The metabolis~ of 4-cyano-N,N-dimethyl-[l4 C) aniline (CDA) by rats and mice. D. Hesk, D.H Hutson, S.K. Lakeman and C.J. Logan (Sittingbourne) 547

66 The carcinogenicity of benzidine and its analogue 4,4" -diamino-terphenyl in the F344 rat. J.J. Eldridge, K.M. Joy and R.M. Hicks (London) 563

67 The effects of BZD and DAT on nuclear ploidy in the rat liver. J.A. Styles (Alderley Park) 585

68 Co-ordinator's report: In vivo assays used in the second UKEMS collaborative study. J. Ashby (Alderley Park) 587

Part 11 GENERAL DISCUSSION

69 An overview of the results of the in vivo and in vitro test systems used to assay the genotoxicity of BZD, DAT, DAB and CDA in the Second UKEMS Collaborative Study. J.M. Parry, C.F. Arlett and J. Ashby (Swansea, Brighton and Alderley Park) 597

Index 617

Preface

The Second United Kingdom Environmental Mutagen Society (UKEMS) Collaborative Genotoxicity Study hwas, like the first (1982), set up in response to the desire of t e members to use the collective resources of the Society to make an in-depth comparison of in vitro and in vivo methods of detecting potential carcinogens and mutagens.

Members of the Society were canvassed for suggestions of suitable ideas to be received by a steering committee composed of c. Arlett, J. Ashby, M. Coombs and J. Parry. A decision was made to combine a general suggestion to investigate in vitro metabolic activation preparations with two specific pairs of chemical analogues, benzidine (BZD) with 4,4"-diaminoterphenyl (DAT) and 4-dimethylaminoazobenzene (DAB) with 4-cyanodimethylaniline (CDA). This decision was conveyed to the members at the 1982 Annual General Meeting and individuals were then asked to indicate what investi­gation they might offer in the proposed study. In this way it was possible to arrive at a critical mass before initiating the study proper.

A co-ordinating committee was then set up under the Chairman­ship of c. Arlett with J. Parry as Secretary and composed of: s. Venitt and R. Forster (prokaryotes); J. Parry and B. Kilbey (non­mammalian eukaryotes); C. Arlett (mammalian cell culture); D. Scott and B. Dean (cytogenetics); c. Garner and M. Wooder (DNA rep;iir assays); J. Ashby and M. Combs (in vivo assays); J. Bridges, S. Hubbard and T. Brooks (metabolic studies). The composition and structure of the committee was modified during the course of the study and its final structure is indicated by the format of this report.

A number of co-ordinators' meetings were held; the first at ICRF, Lincoln's Inn Fields in June 1982 marks the start of the study, We are indebted to M. Coombs and the Director of ICRF for this facility. Three further meetings were held, one in Swansea, and two at Shell Centre. We are especially grateful to Shell Inter-

xii Preface

national through M. Wooder for its hospitality.

The programme required that the basic collection of individual studies be completed by the autumn of 1983 so that appropriate further studies mi~ht be initiated to clarify any problem areas and thus place the co-~rdinators in a position to give a report to the whole Society at its April 1984 meeting. The supply and dispatch of the test chemicals were organised with great effect from the ICI Central Toxicology Laboratory and we must acknowledge the work undertaken on our behalf by D. Paton, who, in addition to synthesi­sing many of the chemicals used here, carried out extensive purifi­cation studies on the two batches of CDA. The work of synthesising the bulk of the chemicals by Lancaster Synthesis is gratefully acknowledged.

To further our aim of investigating the in vitro metabolic activity of the pairs of selected chemicals the co-ordinating committee developed the concept of using a range of S9 preparations produced from different animal sources. The preparations selected were uninduced rat liver (using rats of the same strain as the bio­assay), Aroclor-induced rat liver, uninduced mouse liver (using the same mouse strain as used in the in vivo cytogenetics assays) and uninduced chicken. The major bulk of the assays described in the study involved the use of large common batches of these S9 pre­parations. The considerable exercise of preparing the S9 was undertaken by T. Brooks and his colleagues of Shell Research, Sittingbourne. We are grateful to Shell for its support and to Sterilin for its generosity in supplying the distribution vials for the S9 preparations.

Funds for the purchase of chemicals, the synthesis of labelled compounds and for the general operation of the study were made available from the Health and Safety Executive (HSE). Without these funds and the generosity of many organisations in encouraging and supporting voluntary individual investigations we believe the whole study may never have reached fruition or, at best, have proved very insubstantial. The Health and Safety Executive has also supported an important rodent cancer bioassay of DAT by M. Hicks and col­leagues which is incorporated in the study. We are grateful to D. Gompertz of the HSE for his co-operation throughout the study.

The central secretarial activity for the study has been generated from Swansea by Glenys Bridges and Mary Owens of 'Words', who efficiently produced the present format for the study from a heterogenous collection of manuscripts. We would also like to acknowledge the secretarial assistance of Ruth Calvert and the guidance and co-operation of Peter Osborne of Macmillan.

In addition to the involvement of the many members of the Society we have also had a voluntary participation from workers overseas who provided important data for genetic activity of the study compounds. To all of these and most especially to the group

Preface X Ill

co-ordinators acknowledgement of their very considerable efforts is appropriate. We are all grateful to John Ashby for his enthusiasm and encouragement throughout the trial and for the many contri­butions he made to the conception and execution of this work.

We wish to record our sadness at the death of Don Angus, one of our overseas collaborators, during the study.

We believe that our Society has been able to generate a very substantial and worthwhile study which will stand as an important landmark in genetic toxicology

C.F. ARLETT MRC Cell Mutation Unit Brighton

J.M. PARRY Department of Genetics Swansea

November 1984