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Series: Current issues in mutagenesis and carcinogenesis, no. 41 Can a ‘relatively simple’ screening procedure for the detection of chemicals with aneugenic potential be recommended at the moment?
Mutation Research, 304 (1994) 303-307 © 1994 Elsevier Science B.V. All rights reserved 0165-1218/94/$07.00
303
MUT 01978
Series: Current Issues in Mutagenesis and Carcinogenesis, No. 41 Can a 'relatively simple' screening procedure for the detection of chemicals with aneugenic potential be recommended at the moment? B . M . M i l l e r a,,, S. M a d l e b a n d S. A l b e r t i n i a a
Pharma Division, Preclinical Research, Department of Toxicology, F. Hoffmann-La-Roche Ltd, CH-4002 Basel, Switzerland and b Federal Health Office, Max yon Pettenkofer-Institute, D-14195 Berlin, Germany (Received 1 September 1993) (Accepted 1 September 1993)
Keywords: Screening procedure; Aneugenic potential chemicals
Aneuploidy has a considerable impact upon human health as it leads to genetic disorders among neonates (Hoffmann et al., 1986; Hook, 1986) having consequences such as infertility or mental retardation, and as it is linked to malignancies and development of cancer (Tsutsui et al., 1983; Heim and Mitelman, 1986; Oshimura and Barrett, 1986). Nevertheless, the strategies for mutagenicity testing used as part of risk assessment decisions generally focus on gene mutations and structural chromosome aberrations and do not address numerical changes in depth (COM, 1989; Kramers et al., 1991; EEC DG XI, 1993; see also Madle, 1993; Fahrig et al., 1991). This is mainly due to the lack of appropriate information about the relevance of chemically induced aneuploidy in humans and the fact that no adequate validated test systems are available at the moment. Furthermore, despite the fact that several human diseases and congenital conditions are associated specifically with aneuploidy, regarding risk assessment we do not know at the moment how many of the observed 'spontaneous' aneuploidies are attributable to environmental causes/chemical exposures. To fill this gap, in 1987 a coordinated programme was initiated by the CEC. The pro-
* Corresponding author.
SSDI 0027-5107(93)E0109-2
gramme in which several European laboratories were involved, aimed at (1) evaluating the significance of aneuploidy to the human population induced by chemicals which had previously been shown to produce positive results in fungi, (2) developing and validating assay systems for the detection of chemicals capable of inducing chromosome number changes and (3) providing mechanistic understanding of the modes of action of aneugenic chemicals (Parry and Sors, 1993). Within this programme 10 chemicals were selected as model compounds. The selection was mainly based on published data of the chemicals, including positive and negative results in different test systems and because for these chemicals a variety of specific targets was predicted for their aneugenic action. In May 1993 a Special Issue of Mutation Research (Vol. 287) was dedicated to original papers and reviews of the work of the participants of the EC Aneuploidy Project. Although the results obtained for the 10 model compounds were rather heterogeneous comparing different experimental approaches or various laboratories, a 'relatively simple' screening procedure for detecting aneugenic potential was presented (Natarajan and Parry, 1993): (1) Analysis of the induction of mitotic cell division aberration in mammalian fibroblast cultures,
304 (2) Analysis of mitotic arrest and c-mitoses in rodent bone marrow, (3) Determination of meiotic delay in rodent spermatocytes. Recently, it was pointed out in detail in an article by J. Ashby that new tests need adequate validation before being implemented in or proposed for regulatory schemes (Ashby, 1993). Some general crucial points were discussed and the following remarks were made (for details see Ashby, 1993): (1) " F r o m the beginning, genetic toxicology has been faced with this basic dilemma whether to base testing strategies on confirmed precedents or on theoretically possible test outcomes." (2) "It is confusing to test a compound in a novel cell line, perhaps also with a novel genetic parameter, and to then report a positive response in isolation of other data and without adequate discussion of its significance." As one example to show the problems when developing new test strategies, Ashby mentioned the induction of aneugenic events. The aim of this comment is to critically focus on the proposed 'relatively simple' screening procedure from the practical viewpoint of genetic toxicology. Such a scheme was proposed despite the fact that Parry and Sors (1993) commented in their review: "It is clear that we are not able to recommend any one specific methodology for routine use".
the assay as compared to other systems. In general, we would agree that for initial hazard identification, test systems of high sensitivity should be used. However, can we conclude from these data that the analysis of mitotic division aberrations is really the most sensitive (reliable and predictive) assay or is it prone to lead to false-positive resuits? The question is still open whether an aneuploidy-inducing property of the 10 chemicals regarded as model compounds can be taken as a fact. Chemicals used for test development should have unequivocal evidence of hyperdiploidy induction in mammals in vivo. More crucial, we are faced with the dilemma that we do not have information about the specifity and selectivity of the test systems as long as we cannot answer the question with which test system we should compare the obtained results ('gold standard' for test selection; Albertini, 1993). Compared to the mutagenicity/carcinogenicity database we have no human data available (IARC data for carcinogenicity) and no widely accepted in vivo system for aneuploidy induction (mouse and rat database for carcinogenicity) to compare with. Thus, from our viewpoint it seems to be premature to recommend such a system for routine screening without validated data.
In vitro systems
For detection of aneugenic potential in vivo one somatic approach and one approach in germ cells were recommended. Focusing on somatic cells in vivo within the EEC programme, the following endpoints were investigated: mitotic arrest, c-mitoses, micronucleus (MN) induction and chromosome counting in bone marrow cells. A highly diverse set of positive and negative findings was obtained. The reasons for the different responses were attributed mainly to different mouse strains used, resulting in sometimes different toxicity of the chemicals, different sampling sizes or different experimental protocols including sampling intervals (see Adler, 1993; Leopardi et al., 1993). The analysis of mitotic arrest and c-mitoses in bone marrow was recommended as the second screening step. The analysis of c-mitoses in bone
Within the EC project different in vitro tests were employed. Three endpoints were evaluated: chromosome number, division aberration and micronuclei. For the majority of compounds quite variable responses were obtained in different test systems and cell types. In the studies for analysis of division aberrations in mammalian fibroblasts, all 10 chemicals were regarded as positive in the immortal Chinese hamster cell line D O N : W g 3 h . When tested in the low passage Chinese hamster cell line LUC2, nine out of the 10 chemicals were regarded as positive (Warr et al., 1993). The recommendation to investigate division aberrations in a first step of screening for aneugenic effects might be based on the high sensitivity of
In vivo systems
305 marrow, which was elaborated in one laboratory only, is mainly based on three criteria: changes of the mitotic index, chromatid contraction and spreading and decrease in anaphase frequencies (Miller and Adler, 1989). A good correlation between cell cycle delay (mitotic arrest) and aneuploidy induction has been shown for several chemicals (Manca et al., 1990; Pacchierotti et al., 1991). On the other hand, signs of spindle impairment are highly suggestive of a possible induction of aneuploidy (Hsu et al., 1983; Liang and Satya-Prakash, 1985; ()nfelt, 1987; Miller and Adler, 1989). Although the criteria for c-mitoses in bone marrow cells listed above are in general highly predictive for aneuploidy induction, scoring criteria would have to be standardised and preparational influences minimised (Miller and Adler, 1989). In the trials for induction of aneuploidy in germ cells in vivo two laboratories were involved and two main approaches have been followed the induction of hyperploid metaphases meiosis II of spermatocytes (within both laboratories) and the induction of meiotic delay in spermatocytes (additionally within one laboratory). Counting of chromosomes in metaphases meiosis II of spermatocytes is very tedious and requires more expertise than the analysis of meiotic delay. The analysis of meiotic delay gave a quite good correlation with hyperploidy induction in metaphases meiosis II (Miller and Adler, 1992). This assay was included in the identified strategy as a third step. For both the proposed in vivo system as well as the in vitro system so far no standardised descriptions for test performance and no validated databases exist. As already mentioned in the introduction, and as discussed extensively by Ashby (1993), it is - in our view - rather questionable whether we can implement new test strategies into regulatory schemes at this stage of development. Do we need germ cell tests in a primary screening procedure at the moment?
In a review of six germ-cell-specific mutagens (Auletta and Ashby, 1988) revised by Adler and
Ashby in 1989; it was concluded that there are no relevant data for the existence of germ-cellspecific mutagens. In the case of aneuploidy-inducing chemicals, different responses between somatic cells and germ cells are theoretically imaginable or even expected. However, within the EC Aneuploidy Project, no reasonably clear indication for a germ-cell-specific aneugen was apparent. At the moment there seems to be no justification for germ cell tests in screening programmes on the basis of the presently available data. Future directions
In our view it is surprising that micronucleus assays (in vitro a n d / o r in vivo) were not taken into consideration for a screening for aneugenic effects. Since the establishment of the in vivo micronucleus assay, it is known that micronuclei may be induced by clastogens as well as aneugens (Schmid, 1975; Yamamoto and Kikuchi, 1980). It seems to us that still today micronucleus assays are most suitable for detection of aneugenic effects in routine testing. Problems may arise when chemicals act on membranes as targets, as this might hinder formation of the MN membrane like it is supposed for econazole nitrate (Liang and Brinkley, 1985). However, these effects can be expected to be fairly rare. Several advantages of the MNT over the proposed tests should not be disregarded. The MNT can be combined with measurements for detection of whole lagging chromosomes (like fluorescence in situ hybridization with centromeric DNA probes, CREST staining or C-banding). Micronuclei are fast and easy to score. Furthermore, there are various automation analysis systems available or under development (e.g. image analysis, flow cytometry) that allow evaluation of large sampling sizes. Regarding future aspects, we agree with Parry and Sors (1993) and with Natarajan (1993) that the in vitro MNT has potential for further development and may provide a useful assay. So far, the MNT in vitro is not routinely performed, however, there are some promising developments (Surrall6s et al., 1992; Ellard and Parry, 1993; Matsuoka et al., 1993; Seelbach et al., 1993). The discrepancy between the results from the in vivo
306 M N a n d t h e in vitro M N test in t h e p r e s e n t e d CEC Aneuploidy Programme data underlines the necessity o f f u r t h e r extensive r e s e a r c h to clarify t h e issue o f p r e d i c t a b i l i t y . W i t h in vitro tests, t h e d o s e is only l i m i t e d by t h e c h e m i c a l ' s solubility o r its cytotoxicity. Thus, cells can b e e x p o s e d to very high doses, as long as t h e y survive a n d divide mitotically. D a t a d e r i v e d f r o m e x p e r i m e n t s c o n d u c t e d in vitro p r o v i d e an a l e r t o f p o t e n t i a l for a n e u p l o i d y i n d u c t i o n in vivo. In t h e i n t e r p r e t a t i o n o f positive findings it s h o u l d b e c o n s i d e r e d w h e t h e r t h e effects a r e r e s t r i c t e d to e x t r e m e l y high c o n c e n t r a t i o n s a n d w h a t t y p e o f d o s e - e f f e c t r e l a t i o n s h i p is o b s e r v e d . U s e of in vivo m i c r o n u c l e u s tests m a y h e l p to e v a l u a t e t h e significance o f in vitro m i c r o n u c l e u s data. I n in vivo tests t h e d o s e d e l i v e r e d to t h e t a r g e t cells is d e p e n d e n t on the uptake, m e t a b o l i s m a n d t r a n s p o r t o f t h e chemicals. F u r t h e r m o r e , t h e d o s e is l i m i t e d by t h e toxicity o f t h e test c h e m i c a l to t h e w h o l e animal. T h e in vivo M N T m a y b e c o n s i d e r e d relatively insensitive. In o r d e r to avoid u n r e l i a b l e in vivo d a t a studies s h o u l d n o t b e c o n d u c t e d unless it can b e r e a s o n ably e x p e c t e d , t h a t t h e t a r g e t tissue will be a d e q u a t e l y exposed. Since in g e n e r a l in vitro assays a r e p r e f e r a b l e for s c r e e n i n g p u r p o s e s , p r e f e r e n c e s h o u l d b e given to t h e f u r t h e r d e v e l o p m e n t a n d v a l i d a t i o n of in vitro m i c r o n u c l e u s assays. H o w e v e r , it s h o u l d b e k e p t in m i n d t h a t in in vitro systems cells m a y b e e x p o s e d to high c o n c e n t r a t i o n s which a r e sufficient for a n e u p l o i d y i n d u c t i o n while in vivo such c o n c e n t r a t i o n s d o n o t occur. C o n c e r n i n g t h e ext r a p o l a t i o n f r o m high to low doses, for a n e u g e n i c effects a n o n - t h r e s h o l d e d d o s e - r e s p o n s e relat i o n s h i p is a p r i o r i n o t e x p e c t e d on t h e o r e t i c a l grounds. O n t h e o t h e r h a n d , the p l a u s i b l e d e m o n s t r a t i o n of a t h r e s h o l d will in p r a c t i c e b e very difficult in t h e i n d i v i d u a l cases. T h e C E C A n e u p l o i d y P r o g r a m m e can b e cons i d e r e d as an i m p o r t a n t initiative to fill t h e gap t h a t exists in t h e field o f c h e m i c a l l y - i n d u c e d ane u p l o i d y . A s P a r r y a n d Sors (1993) s t a t e d , " t h e r e a r e still m a n y m e c h a n i s t i c q u e s t i o n s to b e ans w e r e d b e f o r e we a r e a b l e to d e f i n e r e l i a b l e a n d p r e d i c t i v e assays". U l t i m a t e l y t h e s o l u t i o n to t h e d i l e m m a o f p r e d i c t i v e tests for a n e u p l o i d y m u s t b e s o u g h t at t h e m o l e c u l a r level. A c o m p r e h e n -
sive k n o w l e d g e o f m e c h a n i s m s t h a t c a u s e a n e u p l o i d y will p e r m i t t h e d e v e l o p m e n t o f systems t a r g e t e d specifically for t h e critical m o l e c u l a r events.
Acknowledgements W e a r e i n d e b t e d to Dr. P. A r n i a n d Dr. E. G o c k e for constructive discussions a n d suggestions d u r i n g t h e p r e p a r a t i o n o f this c o m m e n t .
References Adler, I.-D. (1993) Synopsis of the in vivo results obtained with the 10 known or suspected aneugens tested in the CEC collaborative study, Mutation Res., 287, 131-137. Adler, I.-D., and J. Ashby (1989) The present lack for unique rodent germ-cell mutagens, Mutation Res., 212, 55-56. Adler, I.-D., U. Kliesch, P. van Hummelen and M. KirschVoiders (1991) Mouse micronucleus test with known and suspect spindle poisons: results from two laboratories, Mutagenesis, 6, 47-53. Albertini, S. (1993) Aneuploidy assays in routine screening?, in: S. Madle and L. Miiller (Eds.), Current Issues in Genetic Toxicology, BGA-Schrift 1/93, MMV, Munich, pp. 64-69. Ashby, J. (1993) Precedents or possibilities: which should guide the harmonization of mutagenicity test protocols and carcinogen prediction strategies? Mutation Res., 298, 291-295. Auletta, A., and J. Ashby (1988) Workshop on the relationship between short-term test information and carcinogenicity: Williamsburg, Virginia, Jan. 20-23, 1987, Environ. Mol. Mutagen., 11, 135-145. COM (Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment) (1989) Guidelines for the Testing of Chemicals for Mutagenicity, Department of Health Report 35, Her Majesty's Stationary Office, London. EEC DG XI (1993) Guidance Document 'A testing strategy for the assessment of the mutagenic potential of new substances'. Ellard, S., and E.M. Parry (1993) A modified protocol for the cytochalasin B in vitro micronucleus assay using whole human blood or separated lymphocyte cultures, Mutagenesis, 8, 317-320. Fahrig, R., R. Lang and S. Madle (1991) General strategy for the assessment of genotoxicity, Mutation Res., 252, 161163. Heim, S., and F. Mitelman (1986) Numerical chromosome aberrations in human neoplasia, Cancer Genet. Cytogenet., 22, 99-108. Hoffman, G.R., V.L. Dellarco and P.E. Voytek (1986) A review of the symposium on aneuploidy: etiology and mechanims, Environ. Mutagen., 8, 643-651. Hook, E.B. (1986) The impact of aneuploidy upon public health: mortality and morbidity associated with human
307 chromosome abnormalities, in: V.L. Dellarco, P.E. Voytek and A. Hollaender (Eds.), Etiology and Mechanisms, Basic Life Sciences, Vol. 36, Plenum, New York, pp. 7-33. Hsu, T.C., J.C. Liang and L.R. Shirley (1983) Aneuploidy induction by mitotic arrestants. Effects of diazepam on diploid Chinese hamster cells, Mutation Res., 122, 201209. Kramers, P.G.N., A.G.A.C. Knaap, C.A. van der Hejden, R.D.F.M. Taalman and G.R. Mohn (1991) Role of genotoxicity assays in the regulation of chemicals in the The Netherlands, Mutagenesis, 6, 487-493. Leopardi, P., A. Zijno, B. Bassani and F. Pacchierotti (1991) In vivo studies on chemically induced aneuploidy in mouse somatic and germ cells, Mutation Res., 287, 119-130. Liang, J.C., and B.R. Brinidey (1985) Chemical probes and possible targets for the induction of aneuploidy, in: V.L. Dellarco, P.E. Voytek and A. Hollaender (Eds.), Etiology and Mechanisms, Basic Life Sciences, Vol. 36, Plenum, New York, pp. 491-505. Liang, J.C., and K.L. Satya-Prakash (1985) Induction of aneuploidy by mitotic arrestants in mouse bone marrow, Mutation Res., 155, 61-70. Madle, S. (1993) Principles of a modified strategy for genotoxicity testing of new chemicals in the EC, in: S. Madle and L. Miiller (Eds.), Current Issues in Genetic Toxicology, BGA-Schrift 1/93, MMV, Munich, pp. 90-94. Manca, A., B. Bassani, A. Russo and F. Pacchierotti (1990) Origin of aneuploidy in relation to disturbances of cellcycle progression. I. Effects of vinblastine on mouse bone marrow cells, Mutation Res., 229, 29-36. Matsuoka, A., N. Yamazaki, T. Suzuki, M. Hayashi and T. Sofuni (1993) Evaluation of the micronucleus test using Chinese hamster cell line as an alternative to the conventional in vitro chromosomal aberration test, Mutation Res., 272, 223-236. Miller, B.M., and I.-D. Adler (1989) Suspect spindle poisons: Induction of c-mitotic effects in mouse bone marrow cells, Mutagenesis, 4, 208-215. Miller, B.M., and I.-D. Adler (1992) Aneuploidy induction in mouse spermatocytes, Mutagenesis, 4, 208-215. Natarajan, A.T. (1993) An overview of the results of testing
known or suspected aneugens using mammalian cells in vitro, Mutation Res., 287, 113-118. Natarajan, A.T., and J.M. Parry (1993) The detection of aneugenic chemicals, Mutation Res., 287, 1. Onfelt, A. (1987) Spindle disturbances in mammalian cells. III. Toxicity, c-mitoses and anenploidy with 22 different compounds. Specific and unspecific mechanisms, Mutation Res., 182, 135-154. Oshimura, M., and J.C. Barrett (1986) Chemically-induced aneuploidy in mammalian cells: mechanisms and biological significance in cancer, Environ. Mntagen., 8, 129-159. Pacchierotti, F., B. Bassani, P. Leopardi and A. Zijno (1991) Origin of aneuploidy in relation to disturbances of cell cycle progression. II. Cytogenetic analysis of various parameters in mouse bone marrow cells after colchicine and hydroquinone treatment, Mutagenesis, 6, 307-312. Parry, J.M., and A. Sots (1993) The detection and assessment of the aneugenic potential of environmental chemicals: the European Community Aneupioidy Project, Mutation Res., 287, 3-15. Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-15. Seelbach, A., B. Fissler, A. Strohbush and S. Madle (1993) Development of a modified micronucleus assay in vitro for the detection of aneugenic effects, Toxicol. in Vitro 7, 185-193. Surrall6s. J., E. Carbonell, R. Marcos, F. Degrassi, A. Antoccia and C. Tanzarella (1992) A collaborative study on the improvement of the micronucleus test in cultured human lymphocytes, Mutagenesis, 7, 407-410. Tsutsui, T., H. Maizuni, J.A. McLachlan and J.C. Barrett (1983) Aneuploidy induction and cell transformation by diethylstilbestrol: a possible chromosomal mechanism in carcinogenesis, Cancer Res., 43, 3814-3821. Warr, T.J., E.M. Parry and J.M. Parry (1993) A comparison of two in vitro mammalian cell cytogenetic assays for the detection of mitotic aneuploidy using 10 known or suspected aneugens, Mutation Res., 287, 29-46. Yamamoto, K.I., and Y. Kikuchi (1980) A comparison of diameters of micronuclei induced by clastogens and by the spindle poisons, Mutation Res., 71, 127-131.
303
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Series: Current Issues in Mutagenesis and Carcinogenesis, No. 41 Can a 'relatively simple' screening procedure for the detection of chemicals with aneugenic potential be recommended at the moment? B . M . M i l l e r a,,, S. M a d l e b a n d S. A l b e r t i n i a a
Pharma Division, Preclinical Research, Department of Toxicology, F. Hoffmann-La-Roche Ltd, CH-4002 Basel, Switzerland and b Federal Health Office, Max yon Pettenkofer-Institute, D-14195 Berlin, Germany (Received 1 September 1993) (Accepted 1 September 1993)
Keywords: Screening procedure; Aneugenic potential chemicals
Aneuploidy has a considerable impact upon human health as it leads to genetic disorders among neonates (Hoffmann et al., 1986; Hook, 1986) having consequences such as infertility or mental retardation, and as it is linked to malignancies and development of cancer (Tsutsui et al., 1983; Heim and Mitelman, 1986; Oshimura and Barrett, 1986). Nevertheless, the strategies for mutagenicity testing used as part of risk assessment decisions generally focus on gene mutations and structural chromosome aberrations and do not address numerical changes in depth (COM, 1989; Kramers et al., 1991; EEC DG XI, 1993; see also Madle, 1993; Fahrig et al., 1991). This is mainly due to the lack of appropriate information about the relevance of chemically induced aneuploidy in humans and the fact that no adequate validated test systems are available at the moment. Furthermore, despite the fact that several human diseases and congenital conditions are associated specifically with aneuploidy, regarding risk assessment we do not know at the moment how many of the observed 'spontaneous' aneuploidies are attributable to environmental causes/chemical exposures. To fill this gap, in 1987 a coordinated programme was initiated by the CEC. The pro-
* Corresponding author.
SSDI 0027-5107(93)E0109-2
gramme in which several European laboratories were involved, aimed at (1) evaluating the significance of aneuploidy to the human population induced by chemicals which had previously been shown to produce positive results in fungi, (2) developing and validating assay systems for the detection of chemicals capable of inducing chromosome number changes and (3) providing mechanistic understanding of the modes of action of aneugenic chemicals (Parry and Sors, 1993). Within this programme 10 chemicals were selected as model compounds. The selection was mainly based on published data of the chemicals, including positive and negative results in different test systems and because for these chemicals a variety of specific targets was predicted for their aneugenic action. In May 1993 a Special Issue of Mutation Research (Vol. 287) was dedicated to original papers and reviews of the work of the participants of the EC Aneuploidy Project. Although the results obtained for the 10 model compounds were rather heterogeneous comparing different experimental approaches or various laboratories, a 'relatively simple' screening procedure for detecting aneugenic potential was presented (Natarajan and Parry, 1993): (1) Analysis of the induction of mitotic cell division aberration in mammalian fibroblast cultures,
304 (2) Analysis of mitotic arrest and c-mitoses in rodent bone marrow, (3) Determination of meiotic delay in rodent spermatocytes. Recently, it was pointed out in detail in an article by J. Ashby that new tests need adequate validation before being implemented in or proposed for regulatory schemes (Ashby, 1993). Some general crucial points were discussed and the following remarks were made (for details see Ashby, 1993): (1) " F r o m the beginning, genetic toxicology has been faced with this basic dilemma whether to base testing strategies on confirmed precedents or on theoretically possible test outcomes." (2) "It is confusing to test a compound in a novel cell line, perhaps also with a novel genetic parameter, and to then report a positive response in isolation of other data and without adequate discussion of its significance." As one example to show the problems when developing new test strategies, Ashby mentioned the induction of aneugenic events. The aim of this comment is to critically focus on the proposed 'relatively simple' screening procedure from the practical viewpoint of genetic toxicology. Such a scheme was proposed despite the fact that Parry and Sors (1993) commented in their review: "It is clear that we are not able to recommend any one specific methodology for routine use".
the assay as compared to other systems. In general, we would agree that for initial hazard identification, test systems of high sensitivity should be used. However, can we conclude from these data that the analysis of mitotic division aberrations is really the most sensitive (reliable and predictive) assay or is it prone to lead to false-positive resuits? The question is still open whether an aneuploidy-inducing property of the 10 chemicals regarded as model compounds can be taken as a fact. Chemicals used for test development should have unequivocal evidence of hyperdiploidy induction in mammals in vivo. More crucial, we are faced with the dilemma that we do not have information about the specifity and selectivity of the test systems as long as we cannot answer the question with which test system we should compare the obtained results ('gold standard' for test selection; Albertini, 1993). Compared to the mutagenicity/carcinogenicity database we have no human data available (IARC data for carcinogenicity) and no widely accepted in vivo system for aneuploidy induction (mouse and rat database for carcinogenicity) to compare with. Thus, from our viewpoint it seems to be premature to recommend such a system for routine screening without validated data.
In vitro systems
For detection of aneugenic potential in vivo one somatic approach and one approach in germ cells were recommended. Focusing on somatic cells in vivo within the EEC programme, the following endpoints were investigated: mitotic arrest, c-mitoses, micronucleus (MN) induction and chromosome counting in bone marrow cells. A highly diverse set of positive and negative findings was obtained. The reasons for the different responses were attributed mainly to different mouse strains used, resulting in sometimes different toxicity of the chemicals, different sampling sizes or different experimental protocols including sampling intervals (see Adler, 1993; Leopardi et al., 1993). The analysis of mitotic arrest and c-mitoses in bone marrow was recommended as the second screening step. The analysis of c-mitoses in bone
Within the EC project different in vitro tests were employed. Three endpoints were evaluated: chromosome number, division aberration and micronuclei. For the majority of compounds quite variable responses were obtained in different test systems and cell types. In the studies for analysis of division aberrations in mammalian fibroblasts, all 10 chemicals were regarded as positive in the immortal Chinese hamster cell line D O N : W g 3 h . When tested in the low passage Chinese hamster cell line LUC2, nine out of the 10 chemicals were regarded as positive (Warr et al., 1993). The recommendation to investigate division aberrations in a first step of screening for aneugenic effects might be based on the high sensitivity of
In vivo systems
305 marrow, which was elaborated in one laboratory only, is mainly based on three criteria: changes of the mitotic index, chromatid contraction and spreading and decrease in anaphase frequencies (Miller and Adler, 1989). A good correlation between cell cycle delay (mitotic arrest) and aneuploidy induction has been shown for several chemicals (Manca et al., 1990; Pacchierotti et al., 1991). On the other hand, signs of spindle impairment are highly suggestive of a possible induction of aneuploidy (Hsu et al., 1983; Liang and Satya-Prakash, 1985; ()nfelt, 1987; Miller and Adler, 1989). Although the criteria for c-mitoses in bone marrow cells listed above are in general highly predictive for aneuploidy induction, scoring criteria would have to be standardised and preparational influences minimised (Miller and Adler, 1989). In the trials for induction of aneuploidy in germ cells in vivo two laboratories were involved and two main approaches have been followed the induction of hyperploid metaphases meiosis II of spermatocytes (within both laboratories) and the induction of meiotic delay in spermatocytes (additionally within one laboratory). Counting of chromosomes in metaphases meiosis II of spermatocytes is very tedious and requires more expertise than the analysis of meiotic delay. The analysis of meiotic delay gave a quite good correlation with hyperploidy induction in metaphases meiosis II (Miller and Adler, 1992). This assay was included in the identified strategy as a third step. For both the proposed in vivo system as well as the in vitro system so far no standardised descriptions for test performance and no validated databases exist. As already mentioned in the introduction, and as discussed extensively by Ashby (1993), it is - in our view - rather questionable whether we can implement new test strategies into regulatory schemes at this stage of development. Do we need germ cell tests in a primary screening procedure at the moment?
In a review of six germ-cell-specific mutagens (Auletta and Ashby, 1988) revised by Adler and
Ashby in 1989; it was concluded that there are no relevant data for the existence of germ-cellspecific mutagens. In the case of aneuploidy-inducing chemicals, different responses between somatic cells and germ cells are theoretically imaginable or even expected. However, within the EC Aneuploidy Project, no reasonably clear indication for a germ-cell-specific aneugen was apparent. At the moment there seems to be no justification for germ cell tests in screening programmes on the basis of the presently available data. Future directions
In our view it is surprising that micronucleus assays (in vitro a n d / o r in vivo) were not taken into consideration for a screening for aneugenic effects. Since the establishment of the in vivo micronucleus assay, it is known that micronuclei may be induced by clastogens as well as aneugens (Schmid, 1975; Yamamoto and Kikuchi, 1980). It seems to us that still today micronucleus assays are most suitable for detection of aneugenic effects in routine testing. Problems may arise when chemicals act on membranes as targets, as this might hinder formation of the MN membrane like it is supposed for econazole nitrate (Liang and Brinkley, 1985). However, these effects can be expected to be fairly rare. Several advantages of the MNT over the proposed tests should not be disregarded. The MNT can be combined with measurements for detection of whole lagging chromosomes (like fluorescence in situ hybridization with centromeric DNA probes, CREST staining or C-banding). Micronuclei are fast and easy to score. Furthermore, there are various automation analysis systems available or under development (e.g. image analysis, flow cytometry) that allow evaluation of large sampling sizes. Regarding future aspects, we agree with Parry and Sors (1993) and with Natarajan (1993) that the in vitro MNT has potential for further development and may provide a useful assay. So far, the MNT in vitro is not routinely performed, however, there are some promising developments (Surrall6s et al., 1992; Ellard and Parry, 1993; Matsuoka et al., 1993; Seelbach et al., 1993). The discrepancy between the results from the in vivo
306 M N a n d t h e in vitro M N test in t h e p r e s e n t e d CEC Aneuploidy Programme data underlines the necessity o f f u r t h e r extensive r e s e a r c h to clarify t h e issue o f p r e d i c t a b i l i t y . W i t h in vitro tests, t h e d o s e is only l i m i t e d by t h e c h e m i c a l ' s solubility o r its cytotoxicity. Thus, cells can b e e x p o s e d to very high doses, as long as t h e y survive a n d divide mitotically. D a t a d e r i v e d f r o m e x p e r i m e n t s c o n d u c t e d in vitro p r o v i d e an a l e r t o f p o t e n t i a l for a n e u p l o i d y i n d u c t i o n in vivo. In t h e i n t e r p r e t a t i o n o f positive findings it s h o u l d b e c o n s i d e r e d w h e t h e r t h e effects a r e r e s t r i c t e d to e x t r e m e l y high c o n c e n t r a t i o n s a n d w h a t t y p e o f d o s e - e f f e c t r e l a t i o n s h i p is o b s e r v e d . U s e of in vivo m i c r o n u c l e u s tests m a y h e l p to e v a l u a t e t h e significance o f in vitro m i c r o n u c l e u s data. I n in vivo tests t h e d o s e d e l i v e r e d to t h e t a r g e t cells is d e p e n d e n t on the uptake, m e t a b o l i s m a n d t r a n s p o r t o f t h e chemicals. F u r t h e r m o r e , t h e d o s e is l i m i t e d by t h e toxicity o f t h e test c h e m i c a l to t h e w h o l e animal. T h e in vivo M N T m a y b e c o n s i d e r e d relatively insensitive. In o r d e r to avoid u n r e l i a b l e in vivo d a t a studies s h o u l d n o t b e c o n d u c t e d unless it can b e r e a s o n ably e x p e c t e d , t h a t t h e t a r g e t tissue will be a d e q u a t e l y exposed. Since in g e n e r a l in vitro assays a r e p r e f e r a b l e for s c r e e n i n g p u r p o s e s , p r e f e r e n c e s h o u l d b e given to t h e f u r t h e r d e v e l o p m e n t a n d v a l i d a t i o n of in vitro m i c r o n u c l e u s assays. H o w e v e r , it s h o u l d b e k e p t in m i n d t h a t in in vitro systems cells m a y b e e x p o s e d to high c o n c e n t r a t i o n s which a r e sufficient for a n e u p l o i d y i n d u c t i o n while in vivo such c o n c e n t r a t i o n s d o n o t occur. C o n c e r n i n g t h e ext r a p o l a t i o n f r o m high to low doses, for a n e u g e n i c effects a n o n - t h r e s h o l d e d d o s e - r e s p o n s e relat i o n s h i p is a p r i o r i n o t e x p e c t e d on t h e o r e t i c a l grounds. O n t h e o t h e r h a n d , the p l a u s i b l e d e m o n s t r a t i o n of a t h r e s h o l d will in p r a c t i c e b e very difficult in t h e i n d i v i d u a l cases. T h e C E C A n e u p l o i d y P r o g r a m m e can b e cons i d e r e d as an i m p o r t a n t initiative to fill t h e gap t h a t exists in t h e field o f c h e m i c a l l y - i n d u c e d ane u p l o i d y . A s P a r r y a n d Sors (1993) s t a t e d , " t h e r e a r e still m a n y m e c h a n i s t i c q u e s t i o n s to b e ans w e r e d b e f o r e we a r e a b l e to d e f i n e r e l i a b l e a n d p r e d i c t i v e assays". U l t i m a t e l y t h e s o l u t i o n to t h e d i l e m m a o f p r e d i c t i v e tests for a n e u p l o i d y m u s t b e s o u g h t at t h e m o l e c u l a r level. A c o m p r e h e n -
sive k n o w l e d g e o f m e c h a n i s m s t h a t c a u s e a n e u p l o i d y will p e r m i t t h e d e v e l o p m e n t o f systems t a r g e t e d specifically for t h e critical m o l e c u l a r events.
Acknowledgements W e a r e i n d e b t e d to Dr. P. A r n i a n d Dr. E. G o c k e for constructive discussions a n d suggestions d u r i n g t h e p r e p a r a t i o n o f this c o m m e n t .
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