Evaluation of the in vitro micronucleus test as an alternative to the in vitro chromosomal aberration assay: position of the GUM working group on the in vitro micronucleus test

Evaluation of the in vitro micronucleus test as an alternative to the in vitro chromosomal aberration assay: position of the GUM working group on the in vitro micronucleus test

Mutation Research 410 Ž1998. 81–116 Evaluation of the in vitro micronucleus test as an alternative to the in vitro chromosomal aberration assay: posi...

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Mutation Research 410 Ž1998. 81–116

Evaluation of the in vitro micronucleus test as an alternative to the in vitro chromosomal aberration assay: position of the GUM working group on the in vitro micronucleus test Beate Miller a

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b , Franziska Potter-Locher , Angelika Seelbach c , Helga Stopper d , ¨ Dietmar Utesch e, Stephan Madle c

Regulatory Affairs Department, Vitamins and Fine Chemicals DiÕision, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland b Toxicologyr Pathology, Preclinical Safety, NoÕartis Pharma AG, CH-4002 Basel, Switzerland c Federal Institute for Health Protection of Consumers and Veterinary Medicine, D-14191 Berlin, Germany d Department of Toxicology, UniÕersity of Wurzburg, D-97078 Wurzburg, Germany ¨ ¨ e Merck KgaA, Institute for Toxicology, D-64271 Darmstadt, Germany Accepted 1 August 1997

Abstract In order to license a pharmaceutical or chemical, a compound has to be tested for several genotoxicity endpoints, including the induction of chromosomal aberrations in vitro. A working group within the GUM has evaluated published data on the in vitro micronucleus test with the aim of judging its suitability as a replacement for the in vitro chromosomal aberration test. After strict rejection criteria were applied, a database including 96 publications and 34 compounds was obtained. For 30 of these compounds, data on both tests were available. For 24 of the 30, concordant results in both test systems were obtained Ž80% correlation.. The discordant results in 6 compounds can be explained by a known or suspected aneugenic potential of these compounds. Considering that cell types and test protocols were extremely heterogeneous, this correlation is rather encouraging. Comparison of the different protocols, and experience established within the working group yielded several recommendations for the routine use of the in vitro micronucleus test. Although many cell lines are suitable, those most often used in genotoxicity testing Že.g. CHL, CHO, V79, human lymphocytes, L5178Y mouse lymphoma cells. are recommended. Cytochalasin B may be used in the case of human lymphocytes; however, the possibility of its interaction with aneugenic test compounds should be considered. For continuously dividing cell lines, cytochalasin B is not recommended by the working group. Although, there seems to be flexibility in the choice of treatment and sampling times, the average generation time of the chosen cell line of choice should be taken into account when determining sampling time, and treatment of cells for at least one cell cycle duration is recommended. The use of appropriate cytotoxicity tests is strongly recommended. Although studies on some parameters of the test protocol may be useful, the introduction of the in vitro micronucleus test into genotoxicity testing and guidelines should not be delayed. Even in its present state, the in vitro micronucleus is a reliable genotoxicity test. Compared with the chromosomal aberration test, it detects aneugens more

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Corresponding author. Tel.: q41 Ž61. 688-6796; fax: q41 Ž61. 688-1356.

1383-5742r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 4 2 Ž 9 7 . 0 0 0 3 0 - 6

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reliably, it is faster and easier to perform, and it has more statistical power and the possibility of automation. q 1998 Elsevier Science B.V. Keywords: In vitro micronucleus test ŽMNT.; In vitro chromosomal aberration test ŽCA.; Genotoxicity testing; GUM

1. Introduction The in vitro chromosomal aberration test ŽCA. has long been part of the test battery for genotoxicity testing. For the in vivo testing of the same endpoint, the detection of micronuclei ŽMN. induced, instead of the chromosomal analysis, is not only equivalent, but even preferred in routine screening. The reasons for this are, in particular, the rapidness and easiness of the micronucleus test compared with the chromosomal aberration test, the better statistical power and the possibility of automation. In addition, the micronucleus test is suitable for detection of clastogens and aneugens due to the fact that chromosome fragments and whole lagging chromosomes lead to MN formation. Although, for the testing of chromosomal aberrations in vitro the same advantages as the in vivo testing are obvious, the in vitro micronucleus test ŽMNT. is not generally accepted as an alternative to the more tedious and time-consuming CA, owing to the fact that no guidelines for the MNT exist. This is remarkable, since: Ž1. the evaluation of MN in established cell lines or human lymphocytes for the detection of chromosome damage has been shown to be feasible by many authors Že.g. Ellard et al., 1991; Matsuoka et al., 1993; Vian et al., 1993., and especially in routine screening ŽMiller et al., 1997.; and Ž2. the CA itself was not validated at the time when it was adopted by the OECD Guidelines for Genotoxicity testing Žsee also Kirkland, 1992.. With this background, in Autumn 1995, the GUM ŽGesellschaft fur ¨ Umweltmutationsforschung e.V., German speaking section of the Environmental Mutagen Society. initiated a working group on the MNT, whose aim was to systematically evaluate published data on the MNT, and compare results with those from the CA. In the evaluation, 34 compounds with at least two valid MNT published were considered. Details on selection of the compounds and criteria for validity are given in Section 2. In the evaluation, the questions listed below had to be addressed.

Ø Is the MNT a suitable alternative to the CA with regard to the detection of structural chromosomal aberrations? Ø Does the MNT have advantages over the CA, e.g. the additional detection of aneugenic activity? Ø Can we give advice on how to perform the MNT, and contribute to a standardized test protocol?

2. Methods The initial selection of chemicals for evaluation by the GUM working group was based on a literature search Žmedline. for compounds that had been tested in both the MNT and the CA. This first list consisted of 75 chemicals. For these compounds, a more detailed literature search in several databases Že.g. medline, toxall, toxline, embase. and a preliminary evaluation of the literature obtained were carried out. Following this, rejection criteria were established, and papers were not selected for final evaluation if they fell into one or more of the following categories: Ø written in a language other than English; Ø abstracts only; Ø review articles with no data; Ø tests system other than mammalian cells; cell lines established from rare diseases; repair deficient cell lines; primary cells other than human lymphocytes or Syrian hamster embryo ŽSHE. cells. Finally, the evaluation was limited to the following cells: 3T3, Swiss albino mouse fibroblasts; CHL, Chinese hamster lung fibroblasts; CHO, Chinese hamster ovary cells; DON, Chinese hamster lung cells; HULY, human Žperipheral blood. lymphocytes; L5178Y, mouse lymphoma cells; SHE, Syrian hamster embryo cells Žprimary cells.; V79, Chinese hamster lung fibroblasts; HEPG2, human hepatocellular carcinoma cells; Ø method and results not explained in detail;

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Ø compound concentration not transferable to mgrml; Ø no negative control given Žalthough a control for only one sampling time or historical control data were accepted.; a positive control was not required; Ø data given for only one concentration of the test compound; Ø number of cells analyzed lower than 100 ŽCA. or 1000 ŽMNT. or not given; Ø no information about the kind of lesion in the CA. If fewer than two acceptable MNT publications were available, the compound was eliminated from the list. CA publications were not required at this point in order to avoid exclusion of aneugens from the database. No additional systematic literature search was carried out after the end of 1995. Each individual publication was then evaluated according to the following criteria: Ø type of assay ŽMNT or CA. and cell type; Ø use of cytochalasin B Žcyto B. Žin the MNT. and of S9 mix; Ø concentration range Žmgrml. from the lowest to the highest concentration applied; Ø treatment time and sampling time; both given as hours after start of treatment; Ø cytotoxicity endpoint Žif sufficient information was provided.; Abbreviations: CE, cloning efficiency; C count, cell number; C dens, cell density; CV, crystal violet; BNC, binucleated cells; MI, mitotic index; RI, replication index; Ø highest micronucleus ŽMN. or aberration Žaberr.. frequency as percent of cells with micronuclei or aberrations Žexcluding gaps. in the most effective treatment protocol presented; Ø author’s evaluation of the result as positivernegativerinconclusive Žor the implication of a positive result by the author.; Ø evaluation by the w orking group as positivernegativerinconclusive according to the overall impression of the experimental result. A doubling over control was not necessarily considered adequate by itself. In the case of deviations from the author’s evaluation, data were discussed by the working group; Ø the lowest concentration, if considered by itself, that yielded a positive result, was given as lowest effective concentration tested ŽLOED; in mgrml.;

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Ø if there were at least two consecutive concentrations having increased aberration frequencies Žcompared with the concurrent negative control. and the effect of the higher concentration was more pronounced than that of the lower, the effect was labeled as ‘DER yes’ Ždose–effect relationship.; Ø if at least two data sets Žpossibly with modified methodology. from the same cell line were shown the result was considered to be confirmed in one publication; Ø acceptance of a publication in spite of variations from the above requirements, and further information regarded as important by the working group, yielded a remark: r1, no. of cell evaluated not given; r2, frequency in no. of MNraberr. per 100 cells Žnot % cells with MNraberr..; r3, high toxicity genotoxin; r4, frequency of chromosomal aberrations including gaps; r5, mitotic shake-off method; r6, control level subtracted; r7, high concentration of solvent Že.g. 3.3% DMSO.; r8, no concurrent control value given. The final database obtained was provided by 96 publications and covered 34 compounds. Evaluated compounds Žabbreviations. and CAS numbers: 2-Acetylaminofluorene Ž2AAF., 53-96-3; actinomycin D ŽAMD., 50-76-0; adriamycin ŽADR., 25316-40-9; aflatoxin B1 ŽAFB1., 1162-65-8; 2aminoanthracene Ž2AA., 613-13-8; m-amsacrine ŽMAC., 54301-15-4; benzoŽ a.pyrene ŽBŽ a.P., 5032-8; bleomycin sulfate ŽBLM., 11056-06-7 Žother salts were used in some publications; molecular weight could not be determined.; cadmium chloride ŽCD., 10108-64-2; chloralhydrate ŽCH., 302-17-0; colchicine ŽCOL., 64-86-8; cyclophosphamide ŽCP., 50-18-0; diazepam ŽDZ., 439-14-5; diethylstilbestrol ŽDES ., 56-53-1; 7,12-dimethylbenzanthracene ŽDMBA., 57-97-6; econazole ŽEZ., 27220-47-9; ethylmethanesulfonate ŽEMS., 62-50-0; 5-fluorouracil Ž5-FU., 51-21-8; griseofulvin ŽGF., 126-07-8; hydroquinone ŽHQ., 123-31-9; methyl-2-benzimidazole carbamate ŽMBC., 10605-21-7; 3-methylcholanthrene ŽMCA., 56-49-5; methylmethanesulfonate ŽMMS ., 66-27-3; N-methyl-N X-nitro-N-nitrosoguanidine ŽMNNG., 70-25-7; 1-methyl-1-nitrosourea ŽMNU., 684-93-5; mitomycin C ŽMMC., 50-07-7; neocarcinostatin ŽNCS., 9014-02-2; 2-nitrofluorene

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ŽNF., 607-57-8; phenol ŽPHE., 108-95-2; pyrene ŽPYR., 129-00-0; pyrimethamine ŽPY., 58-14-0; thiabendazole ŽTB., 148-79-8; thimerosal ŽTM., 54-648; vincristine sulfate ŽVCR., 5722-7 Žother salts were used in some publications.. Colcemide and vinblastine were not included, as no additional information to that obtained with colchicine and vincristine would have been gained.

3. Results and discussion In our final evaluation of data published on the MNT, 34 compounds were included Žsee Appendix, Tables.. For 30 of these 34 compounds, comparative data on the CA were included according to our evaluation criteria. When comparing lowest effective concentrations tested in both tests in different publications, a factor of up to 10 was regarded as comparable, because test protocols and cells used by different authors were very heterogeneous. 3.1. Results from compounds eÕaluated 3.1.1. 2-Acetylaminofluorene (2AAF, CAS 53-96-3) 2AAF, an animal carcinogen, was positive in the MNT in CHL ŽLi et al., 1993. and SHE ŽSchmuck et al., 1988. cells, both in the absence and presence of metabolic activation. In V79 cells ŽGlatt et al., 1990., 2AAF was tested only without S9 and was found to be negative Ža weak effect was observed in the initial experiment, which could not be confirmed in the repeat experiment.. The CA performed in CHL cells ŽIshidate, 1988. was positive, with and without metabolic activation. Possible explanations for the negative result in the MNT in V79 cells were the low concentration range used, when compared with the MNT in CHL cells Žthe cytotoxicity endpoint was not determined., the lack of metabolic activation, and the use of only one sampling time of 24 h. As the CA was performed with much higher concentrations than the MNT, the LOEDs could not be compared. 3.1.2. Actinomycin D (AMD, CAS 50-76-0) AMD, an animal carcinogen, known to be a clastogen and an intercalating drug, which is used clinically as an antineoplastic agent, was clearly positive

in the MNT and in the CA, in different cell lines. Only data without S9 were available. In general, LOEDs were comparable; however, in one publication in which the MNT and the CA using the same cell lines were directly compared, it was shown that the MNT was more sensitive ŽDoerr et al., 1989.. 3.1.3. Adriamycin (ADR, CAS 25316-40-9) ADR, an intercalating agent with antitumor activity, was clearly positive in the MNT and in the CA, in different cell lines. LOEDs in the MNT and the CA were comparable. In the literature, ADR has been shown to inhibit nucleic acid synthesis, to cause chromosomal breaks, and to produce an irreversible block of the cell cycle traverse in G2 ŽKrishan et al., 1981.. 3.1.4. Aflatoxin B1 (AFB1, CAS 1162-65-8) AFB1, a well-known hepatocarcinogen was positive in the CA. In the MNT, the overall picture of AFB1 effects was positive. The agent was positive in SHE cells ŽSchmuck et al., 1988. and HULY ŽIskandar and Vijayalaxmi, 1981., but negative in V79 cells in one publication ŽGlatt et al., 1990.. In V79 cells, lower concentrations Žthe cytotoxicity endpoint was not determined. in the absence of metabolic activation were tested, and only one treatment time was used. LOEDs in the MNT and CA were comparable. 3.1.5. 2-Aminoanthracene (2AA, CAS 613-13-8) 2AA, an animal carcinogen, was clearly positive in the MNT in different cell lines with metabolic activation. It was also positive in 3T3 cells ŽGu et al., 1992., which are known to have some metabolic activation capacity. No valid data were available on the CA. 3.1.6. m-Amsacrine (MAC, CAS 54301-15-4) MAC, a DNA-intercalating topoisomerase II inhibitor ŽAnderson and Berger, 1994., was clearly positive in the MNT and the CA. Only data without metabolic activation were available. In one publication that directly compared both assays, the LOED in the CA was lower than that in the MNT ŽDoerr et al., 1989.. However, in this low concentration range, only weak effects were induced. In the same publication ŽDoerr et al., 1989., the LOED in the CA was

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not considered to be effective at inducing mutations at the tk locus of L5178Y cells. 3.1.7. Benzo(a)pyrene (B(a)P, CAS 50-32-8) BŽ a.P is a well-known, indirectly acting mutagen and carcinogen. In the presence of S9 mix, BŽ a.P dose-dependently induced MN in Chinese hamster cell lines ŽCHL, V79. and HULY. In SHE and 3T3 cells, it was positive without S9 mix. In the CA, it was clearly positive in Chinese hamster cell lines with S9 mix. In permanent fibroblast cell lines LOEDs in both assays were comparable. 3.1.8. Bleomycin sulfate (BLM, CAS 11056-06-7) BLM, a direct S-phase-independent clastogen, is known to be positive in various genetic endpoints in vitro and in vivo. Only data without S9 mix were available for our evaluation. In the MNT, BLM was clearly positive in HULY and mouse lymphoma cells ŽL5178Y.. In HULY, the LOED seemed to depend on treatment time. Also in the CA, BLM was also positive in various cell lines ŽChinese hamster cell lines, L5178Y and HULY.. In both test systems, LOEDs were in the same range. 3.1.9. Cadmium chloride (CD, CAS 10108-64-2) CD is used in several industries, such as electroplating, insecticide and fungicide manufacturing, and dyeing. CD was positive in the CA. In the MNT, the agent was positive in V79 cells ŽGlatt et al., 1990.. In HULY, it was positive in one study ŽMigliore and Nieri, 1991. and negative in another ŽVan Hummelen and Kirsch-Volders, 1992.. The difference between the HULY studies is probably due to the different treatment times used, because CD was positive only after a 72-h treatment. LOEDs in the MNT and the CA were comparable. 3.1.10. Chloral hydrate (CH, CAS 302-17-0) CH, an anesthetic and sedative agent, is a metabolite of trichlorethylene ŽCAS 79-01-6.. No valid CA data were available, and the MNT data were inconsistent. In the mitotic shake-off method with V79 cells, a method specifically designed to detect aneugens, CH was dose-dependently positive at concentrations from 1000 mgrml upwards ŽSeelbach et al., 1993b.. In HULY Žcyto B method., contradictory findings were obtained. CD was one of the suspect

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aneugens in the EEC Aneuploidy Project ŽParry and Sors, 1993.. It was, however, also discussed to have clastogenic activity ŽRusso et al., 1984; Furnus et al., 1990.. 3.1.11. Colchicine (COL, CAS 64-86-8) For the spindle poison COL, only data without S9 mix were considered. COL was clearly positive in the MNT with HULY Žcyto B method. and SHE cells. In SHE cells, an LOED of 2.0 mgrml was found ŽSchmuck et al., 1988.. In a single CA report, a positive result at extremely high concentrations Ž160–5000 mgrml. was described ŽGalloway et al., 1987.. 3.1.12. Cyclophosphamide (CP, CAS 50-18-0) The cytostatic agent CP is known as an extremely effective indirect, S-phase-dependent clastogen in vitro and in vivo. Only findings obtained in the presence of S9 mix are considered here. In both test systems ŽMNT and CA., clearly positive results were found in Chinese hamster cell lines and HULY. Taken altogether, the LOEDs in both test systems were comparable. 3.1.13. Diazepam (DZ, CAS 439-14-5) For DZ, a tranquilizing agent of the benzodiazepine group, the data published showed positive results in the MNT in two studies ŽV79 cells, HULY., and one inconclusive study using HULY ŽMigliore and Nieri, 1991.. In the CA, DZ was reported in two studies to be negative for induction of structural aberrations. DZ was one of the suspect spindle poisons included in the EEC Aneuploidy Project ŽParry and Sors, 1993.. It was reported to inhibit centriolar separation ŽAndersson et al., 1981. and multipolar spindles and lagging chromosomes in vitro ŽBrinkley et al., 1967.. Thus, the induction of MN, but not of chromosomal aberrations, in vitro can be expected. 3.1.14. Diethylstilbestrol (DES, CAS 56-53-1) DES, a human and animal carcinogen, was clearly positive in the MNT in different cell lines and HULY, mostly in the absence of metabolic activation. In the in vitro CA, the overall picture for DES was negative. It induced chromosomal aberrations in one study, with CHO cells using much higher concentrations than those tested in the MNT, possibly

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due to high cytotoxicity ŽNatarajan and Van Kesteren-van Leeuwen, 1981.. However, it was clearly negative for the induction of structural aberrations in all other assays and cells reported Ži.e. in CHL, SHE and HULY.. The difference between the MNT and the CA can be explained by the suspected aneugenic activity of DES, which is reported to lead to spindle disturbances ŽTucker and Barrett, 1986.. Further evidence for aneugenicity is seen from various endpoints induced, e.g. the induction of aneuploidy in SHE cells and HULY ŽTsutsui et al., 1983; Banduhn and Obe, 1985.. 3.1.15. Dimethylbenzanthracene (DMBA, CAS 5797-6) The polycyclic aromatic hydrocarbon DMBA is known to be an indirect clastogen that needs metabolic activation, and to be an in vivo mouse-skin carcinogen ŽAshby et al., 1993.. DMBA was clearly positive in the MNT and the CA when metabolic activation was provided, and also in SHE cells without S9. The LOEDs were more or less the same in both test systems, except in one CA using V79 cells ŽSalassidis et al., 1990., where the LOED was about a factor of 100 lower than those in other publications. However, in the MNT, similar concentrations were not tested. 3.1.16. Econazole (EZ, CAS 27220-47-9) EZ is a broad-spectrum antimycotic agent, which was included for testing in the EEC Aneuploidy Project ŽParry and Sors, 1993.. Negative MNT assays have been described for HULY and V79 cells at concentrations up to 40 mgrml without S9. Data from CA were not available. 3.1.17. Ethylmethanesulfonate (EMS, CAS 62-50-0) EMS, a carcinogen known as a directly acting monoalkylating agent, was clearly positive in the MNT and the CA in various cell lines and HULY. In general, the LOEDs were comparable. 3.1.18. 5-Fluorouracil (5-FU, CAS 51-21-8) 5-FU, a pyrimidine analog, acts as an antineoplastic and immunosuppressant which interferes with pyrimidine-nucleotide synthesis ŽMaier and Schmid, 1976.. 5-FU has induced MN in CHL and L51784Y mouse lymphoma cells without metabolic activation.

In one publication, a positive result in CHL was only obtained with S9 ŽLi et al., 1993.. In the CA, 5-FU was clearly positive in CHO and CHL cells without metabolic activation. The LOEDs in the MNT and CA were comparable. 3.1.19. GriseofulÕin (GF, CAS 126-07-8) The antimycotic GF is known to be carcinogenic in mice and rats ŽDeCarli and Larizza, 1988.. GF was only tested without metabolic activation. It was clearly positive in the MNT. In the one CA, the compound showed a response which was classified as positive by the authors ŽNamba and Kimoto, 1976., but as inconclusive by the working group. Thus, the LOEDs were not comparable in the MNT and the CA. GF is known to be an aneugen ŽStopper et al., 1994b., and is thought to act on the mitotic spindle by inhibiting the binding of the microtubuliassociated proteins ŽDeCarli and Larizza, 1988.. 3.1.20. Hydroquinone (HQ, CAS 123-31-9) HQ, a metabolite of benzene, induced MN in V79 cells and HULY in the absence of metabolic activation. For the CA, only one publication is known in which HQ induced structural aberrations in CHO cells in the presence of S9 ŽGalloway et al., 1987., which was possibly also due to the clastogenic activity of quinone, to which HQ is metabolized ŽGlatt et al., 1989.. In the same publication, the inconclusive finding without metabolic activation may be due to the short treatment and sampling times of 10.5 h used in the study. As this study was the only CA available, LOEDs between the MNT and the CA could not be compared. 3.1.21. Methyl-2-benzimidazole carbamate (MBC, CAS 10605-21-7) MBC, a fungicide, is suspected of being an aneugen ŽBanduhn and Obe, 1985.. Only data without S9 mix are available. MNT were clearly positive in HULY and V79 cells. According to a single CA investigation, MBC did not induce chromosomal aberrations up to 0.2 mgrml ŽBanduhn and Obe, 1985.. 3.1.22. 3-Methylcholanthrene (MCA, CAS 56-49-5) The polycyclic hydrocarbon MCA, a well-known animal carcinogen, was negative in both assays with-

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out metabolic activation. With metabolic activation it was positive in two MNT, negative in one other MNT, and was considered to be positive in two CA. The LOEDs in both tests were similar. There was no obvious explanation for the contradictory results in the MNT because the compound was not found to be a high toxicity genotoxin, and none of the authors described any problems with solubility. 3.1.23. Methylmethanesulfonate (MMS, CAS 66-27-3) The alkylating agent MMS was clearly positive in both assays, with slightly lower LOEDs in the MNT, although the LOED in the CA was also the lowest concentration tested. All results were without metabolic activation. 3.1.24. N-Methyl-N X -nitro-N-nitroso-guanidine (MNNG, CAS 70-25-7) MNNG is a known nitroso-alkylating mutagen and carcinogen ŽHsie et al., 1987.. MNNG was clearly positive in the MNT and the CA, with the same LOEDs. It was always tested without metabolic activation. MNNG also induces gene mutations and SCE ŽHsie et al., 1987.. 3.1.25. 1-Methyl-1-nitrosourea (MNU, CAS 684-935) The nitroso-alkylating agent MNU was positive in the MNT and the CA with comparable LOEDs. All data were without metabolic activation. The compound is a known inducer of mammalian gene mutations and SCE ŽKaina et al., 1991.. 3.1.26. Mitomycin C (MMC, CAS 50-07-7) MMC, an antitumor agent, was clearly positive in both assays at comparable concentration ranges, with a slightly lower LOED in the MNT. MMC is a natural antibiotic, which acts as an alkylating agent able to cross-link complementary copies of DNA, and thus affecting the synthesis and function of nucleic acids ŽKrishna et al., 1986.. 3.1.27. Neocarcinostatin (NCS, CAS 9014-02-2) NCS is a polypeptide antibiotic used in tumor therapy which causes DNA single- and double-strand breaks ŽGoldberg, 1991.. NCS was clearly positive in both assays at similar LOEDs without metabolic activation. No publications with metabolic activation were available.

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3.1.28. 2-Nitrofluorene (NF, CAS 607-57-8) NF is a mutagenic and carcinogenic nitroarene, which is formed by incomplete combustion of nitrate ŽTokiwa and Ohnishi, 1986.. NF was positive in the MNT and the CA. In the CA, one of two publications gave an inconclusive result Žauthor’s judgement, Ishidate, 1988., which was considered negative by the working group. This investigation was carried out without metabolic activation. The same cell line was also used in the publication with the positive CA with and without metabolic activation ŽMatsuoka et al., 1991.. Without metabolic activation, a higher concentration was used than in the negative CA. Thus, the one negative CA result was probably due to the low concentrations used. The LOED without metabolic activation was lower in the MNT than in the CA. 3.1.29. Phenol (PHE, CAS 108-95-2) The disinfectant phenol induces various, generally weak, genetic effects in mammalian cell culture assays, and has been reported to be a weak inducer of MN in mouse bone marrow cells ŽShelby and Witt, 1995.. In CHO cells, MN were dose-dependently induced with and without S9. In HULY Žcyto B method., a weak effect was found ŽYager et al., 1990.. In a single CA, chromosomal aberrations were induced after metabolic activation ŽIvett et al., 1989.. 3.1.30. Pyrene (PYR, CAS 129-00-0) PYR is derived from coal tar and the destructive hydrogenation of hard coal. It is also an air pollutant released in hot asphalt fumes. In most mutagenicity studies it is used as a negative control with the mutagenic structural analogue benzoŽ a.pyrene. PYR was negative for MN and CA with and without S9 in various cell types tested at concentrations up to 1010 mgrml ŽMNT. and 3300 mgrml ŽCA.. 3.1.31. Pyrimethamine (PY, CAS 58-14-0) PY is a folic acid antagonist, which inhibits dihydrofolate reductase and, thus, nucleic acid biosynthesis. Apart from one article using CHL cells, in which PY induced MN ŽOno and Yoshimura, 1996., it was reported to be negative in the MNT in V79 cells and HULY without metabolic activation, and in HULY

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with metabolic activation. In the CA, a clastogenic effect of PY in CHL and HULY in the absence of S9 was reported. It remains to be clarified, whether the discrepancy between the CA and the MNT was due to the cytochalasin B method and the different treatment. However, from Egeli and Erdogan Ž1991., Ono et al. Ž1994. and Ono and Yoshimura Ž1996., it is obvious that chromosomal damage after treatment with PY is much more expressed, or only seen, after long-term treatment of about 48–78 h. 3.1.32. Thiabendazole (TB, CAS 148-79-8) The fungicide TB, a suspected aneugen, was tested within the EEC Aneuploidy Project ŽParry and Sors, 1993.. Only data without S9 mix were considered because for CA no data with S9 mix were available. In the MNT in HULY and V79 cells, controversial findings were described. Three studies with HULY Žcyto B method. were negative. In two of them, lower concentrations than reported elsewhere were tested. In V79 cells, a dose-dependent effect was found with the mitotic shake-off method ŽSeelbach et al., 1993a.. A single CA was negative at concentrations up to 45 mgrml ŽIshidate, 1988.. 3.1.33. Thimerosal (TM, CAS 54-64-8) TM, a bactericidal and fungicidal drug, was one of the 10 suspected aneugens in the EEC Aneuploidy Project ŽParry and Sors, 1993.. Only data from MNT without S9 mix were available. TM was positive for

MN induction in V79 cells at 1.0 mgrml Žmitotic shake-off method. ŽSeelbach et al., 1993b.. An inconclusive result, as judged by the working group, was obtained with HULY Žcyto B method. up to 2.6 mgrml ŽMigliore and Nieri, 1991.. 3.1.34. Vincristine (VCR, CAS 57-22-7) VCR is a well-known spindle poison from Vinca ŽVinca alkaloid., which is used in tumor therapy. VCR is known to crystallize tubulin and, thus, to lead to colchicine-like mitotic arrest. Only data without S9 mix were available. In the MNT, VCR was positive in Chinese hamster cell lines, L5178Y and HULY, the effect was dose-dependent. In one report, chromosomal aberrations were induced at concentrations from 0.05 mgrml upwards ŽMatsuoka et al., 1993.. 3.2. ComparatiÕe eÕaluation of results from the MNT and the CA 3.2.1. OÕerall comparison For 4 compounds Ž2AA, CH, EZ, TM. data were only available for the MNT. For 30 compounds, data from both the MNT and the CA could be considered. With 24 of the 30 compounds, concordant results were obtained in both tests Ž80% correlation, see Table 1.. Twenty-three compounds were positive in both assays Ž2AAF, AMD, ADR, AFB1, MAC,

Table 1 Concordance of results from 30 compounds tested in the MNT and the CA

Concordant results

MNT

CA

No. of compounds

q y

q y

23 1 24

76.7 3.3 80.0

q q i i

i y q y

1a 3b 1c 1d 6

3.3 10.0 3.3 3.3 20.0

Total Discordant results

Total Overall total a

GF. DZ, DES, MBC. c PY. d TB. b

30

%

100

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BŽ a.P, BLM, CD, COL, CP, DMBA, EMS, 5-FU, HQ, MCA, MMS, MNNG, MNU, MMC, NCS, NF, PHE, VCR.; one compound ŽPYR. was negative in both assays. The 6 remaining compounds gave discordant results. Four compounds were positive in the MNT, but inconclusive ŽGF. or negative ŽDZ, DES, MBC. in the CA, and two compounds were inconclusive in the MNT, but positive ŽPY. or negative ŽTB. in the CA. Thus, omitting these three compounds that were inconclusive in one of the two assays, controversial findings were obtained for three compounds ŽDZ, DES, MBC.. These three compounds are suspected aneugens, and are discussed further in the Section 3.3. If the known or suspected aneugenic activity of some of the above compounds is considered, a discrepancy such as that more of compounds showing an effect only in the MNT is not surprising, as the MNT is reported to detect not only clastogens, but also aneugens. However, this does not exclude the fact that there may be single compounds which are only positive in the CA. Possible explanations for this could be different concentration ranges tested, different cytotoxicity in both assays, or induction of unstable aberrations in the CA that are not be detected in the MNT. Considering the fact that, in our evaluation of published data, cell types, test protocols and evaluation criteria were extremely inhomogeneous in the different publications, the overall correlation of 80%, in our opinion, is rather encouraging. For example, in a comparison of results from the in vivo micronucleus test and the in vivo chromosomal aberration test using the same species and tissue Žmouse bone marrow., the two assays agreed in about 53 of the 65 chemicals which corresponded to about 80% ŽShelby and Witt, 1995.. A handicap of the current database is that the compounds are those which are mostly expected to give positive answers, or which are even used as positive controls. Hence, our database is not representative of routine testing, in which most of the compounds are expected to be negative. However, in a comparative evaluation based on industrial routine testing, a similar correlation of nearly 80% Žin a database of 57 compounds. between results of the MNT and the CA was shown ŽMiller et al., 1997.. This result is very promising with regard to replacing the conventional CA by the MNT.

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On the basis of the 34 compounds included in this evaluation of published data, and the 57 compounds from routine genotoxicity testing ŽMiller et al., 1997., we have, in our opinion, a fairly good database for the MNT as an alternative to the CA. 3.2.2. Lowest effectiÕe concentrations tested (LOEDs) As already mentioned, heterogeneity of protocols, concentrations tested, and of evaluation criteria play a big role when comparing both test systems. It is even more important when comparing LOEDs. Therefore, in our evaluation, absolute values for LOEDs should be interpreted very cautiously. Moreover, LOEDs were often identical to the lowest concentrations tested. As direct comparisons between two different publications are very difficult, not too much weight should be put on the absolute values. In our evaluation, a difference of about a factor of 10 was still regarded as comparable when different cell lines and different publications were compared. Moreover, more weight must be put on publications in which direct comparisons between the two assays, using the same cell type, were reported. Where a comparison between LOEDs in the MNT and the CA was possible, LOEDs were in the same range for most compounds. This was also the case for those compounds in which a direct comparison in the same publication could be made Že.g. ADR, BLM, CP, DMBA, MCA; Doerr et al., 1989; Sorsa et al., 1992; Matsuoka et al., 1993.. There was only one compound ŽMAC; Doerr et al., 1989., in which in the same publication, the LOED in the CA was lower than in the MNT. Moreover, for AMD, BŽ a.P, MAC, MMC and MNNG, the LOEDs in the MNT were, in the same publication, by a factor of two to ten lower than in the CA. Moreover, there were some compounds in which, in the overall picture, the MNT seemed to be more sensitive than the CA Že.g. 5-FU, MMS, MMC.. A greater sensitivity of the MNT was already shown in an evaluation of industrial compounds being tested in the MNT and the CA in routine screening ŽMiller et al., 1997.. 3.3. The MNT as a tool for detection of aneugens In principle, it is assumed that the MNT is able to cover both genetic endpoints, structural chromosomal aberration and aneuploidy, as MN can occur as a

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consequence of both structural and numerical chromosomal aberrations. In the following, the MNT and the CA are compared with regard to their potential to detect aneugens. It must be noted that only the standard CA assay for the detection of structural chromosomal aberrations is considered. Induction of hyper- and hypoploidy have not been taken into consideration. Results of the MNT and CA are taken from the present analysis by the GUM working group. Evaluation of the aneugenic potential is taken from the ECETOC report ‘Aneuploidy: its significance in human disease and methods for screening chemicals’ ŽECETOC, 1997.. According to the ECETOC, report 13 of the 34 substances evaluated by the GUM working group are proven or suspected aneugens Žsee Table 2.. Six compounds under discussion are ‘aneugenic in somatic cells in vivo’ ŽCOL, VCR, DES, MBC, GF, HQ.; three compounds are in vitro aneugens ŽDZ, TB, CH. and four compounds are suspected of being aneugens ŽCD, PY, TM, EZ.. Unfortunately, suspected aneugens have not so far been tested in detail in the CA, and some of them have not been tested at all, so that it is difficult, or even impossible, to draw a comparison between the

MNT and the CA. Nevertheless, a tendency may be seen.

3.3.1. In ÕiÕo aneugens COL and VCR were clearly positive in the MNT in different cell types. These substances were also positive in the CA; however, LOEDs differed greatly Žin the case of COL, even more than 100 000-fold.. This means that, for the two substances, the MNT has been far more specific in detecting an effect than the CA. However, for each compound, there was only one CA report published. DES, MBC and GF were clearly positive in the MNT, and negative ŽDES, MBC. or inconclusive ŽGF. in a CA. HQ has been classified as being aneugenic in vitro and in vivo by the ECETOC Ž1997.. In our evaluation, HQ was clearly positive in the MNT. However, in contrast to the other in vivo aneugens, HQ also has a clastogenic potential: in our evaluation, HQ was positive in a single CA. Furthermore, it induced chromosomal aberrations in vivo ŽXu and Adler, 1990; Marrazzini et al., 1994.. In our evaluation, the LOED in the MNT was much lower than that in the CA.

Table 2 Comparison of aneugenicity data and results for the MNT and the CA

In vivo aneugens COL VCR DES MBC GF HQ In vitro aneugens DZ TB CH Suspected aneugens CD PY TM EZ

Aneugenicity ŽECETOC.

GUM evaluation

In vitro

In vivo Žsomatic.

MNT

CA

q q q q q q

q q q q q q

q Ž0.001. q Ž2. q Ž1.3. q Ž0.2. q Ž1.5. q Ž1.

q) Ž160. q) Ž0.05. y y) i) q) Ž450.

q q q

y i i

q Ž30. i i

y y) n.d.

i i i y

i y y i

q Ž0.1. i q Ž1.0. y

q Ž0.2. q Ž0.4. n.d. n.d.

Values in parentheses are LOED in mgrml. ) One report.

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3.3.2. In Õitro aneugens According to the ECETOC, DZ, TB and CH are clearly in vitro aneugens, with in vivo results having been inconclusive or negative. In our evaluation, DZ was positive in the MNT and negative in the CA. TB was only positive in the mitotic shake-off method, which is a method specifically designed to detect aneugens; however, it was negative in the MNT using HULY in 3 studies. The overall picture for TB in the MNT was inconclusive. For CH, positive and negative results were obtained in the MNT, hence, the overall picture was inconclusive. In the CA, for TB and CH, no data are available. 3.3.3. Suspected aneugens The aneugenicity data for CD, PY, TM and EZ were inconclusive or negative ŽECETOC, 1997.. According to our evaluation, CD was positive in the MNT and the CA. For PY, TM and EZ, a comparison of findings in the MNT and the CA was not possible due to inconclusive or missing data. In summary, the data evaluated here clearly show that the MNT is more reliable than the CA in detecting effects induced by aneugenic activity. It is sensitive to all the in vivo aneugens considered by the ECETOC, irrespective of their potential clastogenic effects. In the MNT, the LOEDs are consistently lower than in the CA. In view of the discussion about aneugenic effects and their routine detection in the implementation of regulation on substances, the lower LOED is certainly a great advantage of the MNT over the CA. Recently, the endpoint of non-disjunction has received much attention ŽKirsch-Volders et al., 1996; Zijno et al., 1996.. However, we are as yet unaware of any compound, detectable in the non-disjunction assay using chromosome painting, that could not be found positive in the MNT. 3.4. EÕaluation of different protocols for the MNT 3.4.1. Different cell lines CHL cells were used in five publications Žsee Appendix, tables.. In several cases ŽAAF, NF, PHE., they showed positive results without metabolic activation. The capacity of CHL cells to specifically activate the substrates is, so far, not known. CHO cells are not yet very common in the MNT. They

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appeared 7 times in the final evaluation list, and were used in 5 publications ŽOshiro et al., 1991; Majone et al., 1992; Darroudi and Natarajan, 1993; Salvadori, 1994; Miller et al., 1995., revealing their widespread use in the CA. CHL and CHO are usually used with exogenous systems if metabolic activation is required. HULY were the most common cells in the MNT Žused 56 times in 21 publications.. They seem to have some small metabolic capacity as AFB1 ŽIskandar and Vijayalaxmi, 1981., BŽ a.P ŽElhajouji et al., 1994., and CP ŽVian et al., 1993. were weakly positive in these cells without addition of an exogenous metabolizing system, although, in its presence, the effects were much greater. HULY are usually used with cytochalasin B in order to limit the MN analysis to the proliferating cell fraction which may vary between donors. A theoretical disadvantage of this test system is that the typical HULY protocol limits the observation to one cell cycle duration. L5178Y mouse cells are not very suitable for the analysis of chromosomal aberrations and, probably, were therefore used only rarely in the MNT Ž15 times in 5 publications; Doerr et al., 1989; Cole et al., 1990; Stopper et al., 1994a,b; Zhang et al., 1995.. These cells require the use of an exogenous activation system. Because of its selection for the loss of function of the thymidine kinase gene, this cell line is one of the two most often used mammalian mutation assay systems, a large genotoxicity database exists, and the cells are routinely cultured in many laboratories. V79 cells were used in 35 investigations Ž13 publications. to study the induction of MN. Their capacity to metabolically activate compounds is limited and, thus, the use of an exogenous activation system is required. They are one of the two most often used mammalian mutation assay systems Žbecause of the inactivation of the hprt gene.. Therefore, a large genotoxicity database exists, and the cells are routinely cultured in many laboratories. SHE cells were used in two studies Ž10 substances. in the MNT ŽSchmuck et al., 1988; Schnitzler et al., 1994.. They have the metabolic capacity to activate at least some indirect genotoxins Ž2AAF, AFB1, BŽ a.P, DMBA.. An evaluation of SHE cells in the MNT has been published ŽFritzenschaf et al., 1993. in which of 48 carcinogens tested, 85% yielded a positive result, and of 17 non-carcinogens, all proved negative. A large degree

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of concordance with data from the in vivo micronucleus test was found Ž89%., and the accordance with results from morphological SHE cell transformation was 95%. HepG2 were used in one MNT ŽPYR; Natarajan and Darroudi, 1991.. These cells are usually used with the assumption that they are capable of metabolically activating genotoxins via cytochrome P450-dependent pathways. However, in the investigation, PYR, was presumed to be nonmutagenic, even in the presence of activating enzymes. Thus, no information on the suitability of HepG2 in the MNT could be obtained. 3T3 cells were used in one of the publications evaluated in the MNT Ž4 compounds tested; Gu et al., 1992.. They are not supposed to metabolize pro-mutagens to their ultimate mutagenic species, although they seem to have limited metabolic activities which vary between subclones ŽGu et al., 1992.. DON cells were only used in one study analyzing chromosomal aberrations ŽAbe and Sasaki, 1977.. Thus, their suitability for the MNT cannot be judged. The number of different cell lines used is only one of the many variations in the MNT-protocol that made comparisons difficult. Overall, all the cell types gave a similar quality of result. In an effort to simplify comparison in future and to accelerate acceptance of the MNT in guidelines, the working group recommends that the choice of cell lines for routine use in the MNT is limited to those cell lines which are most easily available, and which are most often used in genotoxicity testing, i.e. Chinese hamster cells ŽCHO, CHL, or V79., L5178Y and HULY.

3.4.2. Use of cytochalasin B (cyto B) With cyto B, the microscopic evaluation can be limited to binucleate cells, which have undergone mitosis since test substance exposure. This is considered to be necessary for HULY, because the percentage of proliferating cells varies considerably between donors. In other cell types, cyto B is sometimes used because of the assumption that the evaluation is limited to the dividing cell fraction, sensitivity or MN frequency increase. However, this assumption is not supported by the data in our evaluation. In otherwise comparable experiments, the lowest LOEDs and the highest maximal frequencies were obtained sometimes with and sometimes without the

use of cyto B, and the differences were generally small. In the case of aneugens, cyto B has been found to interfere with MN formation or mitosis by members of the working group and others ŽAntoccia et al., 1993; Eckert and Stopper, 1996; Ramirez et al., 1996; Kalweit et al., 1997; Miller, unpublished results.. Among the effects seen were the formation of MN in mononucleate cells, or failure to induce MN in binucleate cells with compounds that have induced MN without cyto B. A synergism of vinblastine and cyto B in the induction of chromosome number changes Žhyperploidy and polyploidy. has also been observed, and there have been indications that cyto B might change the choice of chromosomes that malsegregate ŽMarcon et al., 1996.. Mitotic disturbances and MN can, under certain conditions, be induced by cyto B itself ŽLindholm et al., 1991; Norppa et al., 1993.. A further complication in the use of cyto B is that the cytotoxicity of cyto B itself varies to a large degree between cell lines and sometimes even between subtypes of the same cell line. However, the possibility of analyzing percentages of mono- and binucleate cells as a measure of cytotoxicity Ži.e. proliferation inhibition. is one advantage of the use of cyto B. In conclusion, although it may be necessary for HULY, the working group does not recommend the use of cyto B for other cell lines. 3.4.3. Treatment and sampling times In the papers evaluated, a variety of treatment times Žmost of them between 1 and 24 h. and sampling times Žusually in the range between 24 and 72 h. were used. All methods yielded comparable results. The so-called mitotic shake-off method is a specific method, in which, after an appropriate time, mitotic cells are selected by shake-off and cultured for an additional 3.5 h until being sampled ŽSeelbach et al., 1993a,b.. An advantage of this method is that after short treatment and sampling times, S-phase-independent MN induction can be detected, whereas under these conditions, the standard MN is quite insensitive. S-phase-independent MN induction after short treatment times can be assumed, according to current knowledge, to be due to aneugenic effects. The ideas concerning different treatment and sampling times are briefly discussed here. A short treat-

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ment time followed by a recovery period before sampling may be less likely to lead to an inhibition of cell cycle progression that may impair MN formation, than a continuous treatment. On the other hand, if the mechanism of action of a compound is limited to a specific cell cycle phase Žsuch as G2rM., the treatment of an unsynchronized culture affects only very few cells in their sensitive phase, and, if the treatment time is too short, the induction of MN may be too small to be detected. If, after substance exposure or after the addition of the compound Žunstable compounds., the sampling time is too short or too long, the effect may also be missed, because MN may not yet have been formed at a detectable frequency Žmany clastogens. or because unaffected cells may have overgrown the MN-containing population Žmany aneugens or treatment with unstable compounds.. These theoretical considerations are not directly implied by the data here as mutagenic model compounds were mainly tested. Nevertheless, these problems may well arise in the routine testing of chemicals in the toxic and subtoxic range. Although treatment schedules seem to be flexible, data show that cells should be treated for at least 0.5 time of the cell cycle duration, as a minimum, and the average generation time of the cell line used should be considered. In general, the working group recommends to treat the cells at least for about one cell cycle duration. Furthermore, in view of a replacement of the CA, short-term Ž3 to 6 h. and long-term treatment is recommended.

only at very high cytotoxicity Ž‘high toxicity genotoxin’.. Therefore, the working group strongly recommends that an appropriate cytotoxicity test is performed with every MNT, and also suggests that in publications, the data are shown or, at least, clearly described. For the choice of the cytotoxicity test it must be kept in mind that cytotoxicity assays in most cases measure one specific type of cytotoxicity, whereas another type might still be induced by the test compound. As culture and treatment conditions affect cytotoxicity, they should be adjusted, as closely as possible, to those used in the MNT. Hence, in conclusion, hamster cell lines and HULY are suitable for the MNT. Cyto B may be used in the case of HULY; however, the possibility of interactions with aneugenic test compounds should be considered. Cyto B is not recommended for all other cell lines. Although, there is flexibility in the choice of treatment and sampling times, it is recommended to treat the cells at least for about one cell cycle duration, and to take the average generation time of the cell line into consideration. The use of appropriate cytotoxicity tests is strongly recommended. Even though it may be useful to do evaluation studies on some parameters of the MNT protocol, this should not delay the introduction of the MNT into genotoxicity testing and guidelines. Even in its present state, the MNT is a reliable genotoxicity test.

3.4.4. Cytotoxicity tests The more commonly used cytotoxicity assays were cloning efficiency, cell number, the number of binucleate cells, mitotic index, and those which were less often used are cell density, replication index, or crystal violet permeation. Very often, no cytotoxicity test is mentioned in a publication. However, in the case of negative results, it must be sure that the compound was tested up to toxicity, insolubility or up to a concentration of 5 mlrml, 5 mgrml, 10 mM Žwhichever is lowest; OECD Guidelines.. It must also be sure, that the cells did not suffer from cell cycle inhibition impairing proliferation through mitosis and expression of MN. In the case of positive results, cytotoxicity data still need to be obtained in order to show that the compound was not genotoxic

Together with the fact that the MNT is able to detect clastogens with the same and sometimes with an even greater sensitivity, and has the advantage of detecting most, if not all aneugens, the MNT has several further advantages over the CA. Ø The MNT assay is much quicker than the conventional CA, in particular, because scoring is much easier than in the CA. Ø In the MNT, genetic effects are only considered in cells which go through mitotic cell division, i.e. they are viable. Ø The MNT has more statistical power because of the fact that many more cells can be analyzed. Ø Without too much effort, in the event of a positive answer in the MNT, mechanistic information can be obtained using antikinetochore staining or

3.5. AdÕantages of the MNT oÕer the CA

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in situ hybridization with various DNA probes. Ø Automation of MN scoring by means of image analysis and flow cytometry is much easier than in the CA. We think that the existing database on the MNT together with the advantages given above, more than justify implementation of the MNT in routine screening.

4. Conclusion Ž1. In this evaluation of published data, the MNT has been shown to detect all clastogens with the same accuracy as the CA, the MNT being sometimes even more sensitive. By omitting suspected aneugens, a 100% correlation in results between both assays has been obtained. With regard to a replacement of the CA, these results are very promising. However, we are aware that the database used for the evaluation consists predominantly of well-known clastogens. Ž2. The MNT has several advantages over the CA, such as the additional detection of aneugens, methodological advantages, e.g. easiness of scoring, possibility of automation, statistical power, and the fact, that it is less prone to false-positive results because MN can only occur in dividing cells are

considered in the estimation of the percentage of cells with chromosomal aberrations. Ž3. Overall, the MNT has been shown to be a robust test system and, although the cell cycle of the cell system used has to be carefully considered, with great flexibility with regard to treatment times. A variety of different cell types have been shown to be feasible. However, the use of the most common cell lines Žsuch as CHL, CHO, V79, HULY, L5178Y. is recommended. Cytochalasin B block is normally used for HULY; however, for continuously dividing cells, it is not recommended by the working group. The use of appropriate cytotoxicity tests is strongly recommended within the assay.

Acknowledgements The manuscript has been approved by the German-speaking Society for Environmental Mutation Research ŽGesellschaft fur ¨ Umwelt-Mutationsforschung, GUM: President F.E. Wurgler, Vice Pres¨ ident S. Madle, Seceretary S. Albertini, Treasurer R. Creutziger, Former President R. Fahrig.. The authors would like to thank the GUM for support in this evaluation and Veronique Thybaud for critically reading the manuscript.

B. Miller et al.r Mutation Research 410 (1998) 81–116 Appendix: GUM evaluation of data published on the MNT and the CA

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List of abbreviations and terms used in the tables. 3T3 BNC CA C count C dens CE CHL CHO confirmation

CV Cyto B

Swiss albino mouse fibroblast binucleated cells in vitro chromosomal aberration test cell number cell density cloning efficiency Chinese hamster lung fibroblasts Chinese hamster ovary cells at least two data sets Žpossibly with modified methodology. from the same cell line shown crystal violet cytochalsin B

DER DON HEPG2 high. MN raberr. fr.

HULY L5178Y LOED MI MNT MW nd r1

dose–effect relationship Chinese hamster lung cells human hepatocellular carcinoma cells highest micronucleus or aberration frequency referred to control values Žin brackets. human peripheral blood lymphocytes mouse lymphoma cells lowest effective concentration tested mitotic index in vitro micronucleus test molecular weight no data no. of cell evaluated not

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r2

r3 r4 r5 r6 r7 r8 RI Ref. S9 SHE V79 WG

given frequency in no. of MN raberr. per 100 cells Žnot % cells with MNraberr.. high toxicity genotoxin frequency of chromosomal aberrations including gaps mitotic shake-off method control level subtracted high concentration of solvent Že.g. 3.3% DMSO. no concurrent control value given replication index references metabolic activation system Syrian hamster embryo cells Chinese hamster lung fibroblasts GUM working group

References to the Appendix Ž1. J. Li, Y. Suzuki, H. Shimizu, M. Fukumoto, H. Okonogi, T. Nagashima, T. Ishikawa, In vitro micronucleus assay of 30 chemicals in CHL cells, Jikeikai Med. J. 40 Ž1993. 69–83. Ž2. G. Schmuck, G. Lieb, D. Wild, D. Schiffmann, D. Henschler, Characterization of an in vitro micronucleus assay with Syrian hamster embryo fibroblasts, Mutation Res. 203 Ž1988. 397–404. Ž3. C.L. Doerr, K. Harrington-Brock, M.M. Moore, Micronucleus, chromosome aberration, and small-colony TK mutant analysis to quantitate chromosomal damage in L5178Y mouse lymphoma cells, Mutation Res. 222 Ž1989. 191–203. Ž4. L. Vian, N. Bichet, D. Gouy, The in vitro micronucleus test on isolated human lymphocytes, Mutation Res. 291 Ž1993. 93–102. Ž5. M. Ishidate, Data Book of Chromosomal Aberration Test In Vitro Žrevised edition., Elsevier, Amsterdam, 1988. Ž6. U. Egeli, G. Erdogan, The clastogenic effect of pyrimethamine ŽDaraprim. on human chromosomes in lymphocyte cultures, Cell Biol. Toxicol. 7 Ž1991. 347–356. Ž7. T. Tsutsui, H. Maizumi, J.A. McLachlan, J.C.

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Barrett, Aneuploidy induction and cell transformation by diethylstilbestrol: a possible chromosomal mechanism in carcinogenesis, Cancer Res. 43 Ž1983. 3814–3821. Ž8. A. Matsuoka, N. Yamazaki, T. Suzuki, M. Hayashi, T. Sofuni, Evaluation of the micronucleus test using a Chinese hamster cell line as an alternative to the conventional in vitro chromosomal aberration test, Mutation Res. 272 Ž1993. 223–236. Ž9. Y. Oshiro, C.E. Piper, P.S. Balwierz, S.G. Soelter, Chinese hamster ovary cell assays for mutation and chromosome damage: data from noncarcinogens, J. Appl. Toxicol. 11 Ž1991. 167–177. Ž10. L. Vian, P. Van Hummelen, N. Bichet, D. Gouy, M. Kirsch Volders, Evaluation of hydroquinone and chloral hydrate on the in vitro micronucleus test on isolated lymphocytes, Mutation Res. 334 Ž1995. 1–7. Ž12. H. Glatt, I. Gemperlein, F. Setiabudi, K.L. Platt, F. Oesch, Expression of xenobiotic-metabolizing enzymes in propagatable cell cultures and induction of micronuclei by 13 compounds, Mutagenesis 5, Suppl. 3 Ž1990. 241–249. Ž13. K. Salassidis, U. Kulka, D. Paul, M. Bauchinger, Induction of chromosome aberrations and sister-chromatid exchange by indirectly acting mutagens in immortal mouse and rat hepatocyte lines, Mutagenesis 6 Ž1991. 59–63. Ž14. A. Seelbach, B. Fissler, S. Madle, Further evaluation of a modified micronucleus assay with V79 cells for detection of aneugenic effects, Mutation Res. 303 Ž1993. 163–169. Ž15. A. Seelbach, B. Fissler, A. Strohbusch, S. Madle, Development of a modified micronucleus assay in vitro for detection of aneugenic effects, Toxicol. Vitro 7 Ž1993. 185–193. Ž16. L. Migliore, M. Nieri, Evaluation of twelve potential aneuploidogenic chemicals by the in vitro human lymphocyte micronucleus assay, Toxicol. Vitro, 5 Ž1991. 325–336. Ž17. L. Migliore, R. Barale, D. Belluomini, A.G. Cognetti, N. Loprieno, Cytogenetic damage induced in HULY by adriamycin and vincristine: a comparison between micronucleus and chromosomal aberration assays, Toxicol. Vitro 1 Ž1987. 247–254. Ž18. L. Migliore, M. Nieri, S. Amodio, N. Loprieno, The human lymphocyte micronucleus assay: a comparison between whole blood and separated-

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