Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (11)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Honma, M.
Right arrow Articles by Sofuni, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Honma, M.
Right arrow Articles by Sofuni, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Mutagenesis, Vol. 14, No. 1, 23-29, January 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press

The need for long-term treatment in the mouse lymphoma assay

Masamitsu Honma1, Li-Shi Zhang2, Hiroko Sakamoto, Masayasu Ozaki, Kenji Takeshita, Maki Momose, Makoto Hayashi and Toshio Sofuni

Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The L5178Y tk+/– mouse lymphoma assay (MLA) has been widely used as a genotoxicity test for the detection of mutagens and clastogens. The standard MLA, as well as other mammalian cell gene mutation assays, usually employs a short treatment period (3–6 h). Our previous report, however, suggested that such short treatments may be insufficient for detecting some clastogens and spindle poisons. For the present study, we introduced and evaluated a longer treatment (24 h) in the MLA. We examined 15 chemicals which were evaluated as negative or inconclusive in the short-term study. Cells were exposed to the chemical for 24 h without S9 mix, cultured for 2 days and then thymidine kinase-deficient mutants were selected in 96-well microtiter plates under trifluorothymidine. Eleven chemicals yielded positive responses in the 24 h treatment MLA. They included nucleoside analogs (2'-deoxycoformycin and dideoxycytidine), a base analog (1,3-dimethylxanthine) and spindle poisons (colchicine and vinblastine sulfate), all of which do not directly affect DNA, but bring about mutations and chromosome alterations through nucleoside metabolism and chromosome segregation. Because the mutagenicities of these non-DNA targeting chemicals appear to be cell cycle dependent, treatment extending over more than one cell cycle may be required for their effect. Combining results from the present and previous studies, 31 of 34 (91%) chromosome aberration-positive chemicals exhibited positive responses in the MLA, suggesting that the sensitivity of the MLA with 24 h treatment periods approaches that of the chromosome aberration test.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
In an effort to harmonize genotoxicity testing practice internationally, the Expert Working Group on genotoxicity of the International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH) is developing a standard battery of genotoxicity tests (ICH, 1994Go, 1996Go, 1997Go). A pivotal issue is whether the mouse lymphoma assay (MLA) is needed in the battery. The MLA, which quantifies genetic alterations affecting the expression of the thymidine kinase (TK) gene (tk) in mouse lymphoma L5178Y tk+/– cells, was first developed by Clive and co-workers (Clive and Spector, 1975Go; Clive et al., 1979Go; Moore-Brown et al., 1981Go) and its protocol has been optimized (Moore and Clive, 1982Go; Moore and Howard, 1982Go; Cole et al., 1983Go; Turner et al., 1984Go; Majeska and Matheson, 1990Go; Clive et al., 1995Go; Sofuni et al., 1997Go). Subsequently adopted by many laboratories, it has been widely used for evaluating the genotoxic potential of various agents (Oberly et al., 1984Go; McGregor et al., 1988aGo, 1988bGo, 1991Go; Myhr and Caspary, 1988Go, 1991Go; Myhr et al., 1990Go; Mitchell et al., 1997Go). The MLA is capable of detecting a wide range of genetic alterations, including point mutations, large scale chromosomal changes, recombination, aneuploidy and others (Moore et al., 1985Go; Blazak et al., 1989Go; Clive et al., 1990Go; Combes et al., 1995Go; Zhang et al., 1996Go) and most of those changes occur in human carcinogenesis. The MLA is probably one of the most sensitive mammalian cell assays for predicting the carcinogenicity of chemicals. The US and EU regulatory authorities, therefore, support inclusion of the MLA in a standard genotoxicity test battery (Tennant et al., 1987Go; CEC/EU, 1989Go; DHSS UK, 1989Go; Federal Register, 1993Go; Garriot et al., 1995Go). In the Japanese guidelines for genotoxicity testing for pharmaceuticals, on the other hand, an in vitro chromosomal aberration test (CA) instead of the MLA or other mammalian cell gene mutation assay has been employed in the standard battery for a long time (Ishidate, 1988Go; MHW Japan, 1990Go).

Following the advice from the ICH, we conducted a collaborative study of the MLA under the auspices of the Ministry of Health and Welfare of Japan and the Japanese Pharmaceutical Manufacturer's Association. The major aim of the study was to validate the MLA as an alternative to the CA. We tested 34 CA-positive chemicals, mostly clastogens and spindle poisons, and investigated what proportion of them was positive in the MLA. We obtained nine negative and five marginal responses, indicating that at least 26% (9/34) of clastogens were not detected by the MLA (Sofuni et al., 1996Go; Honma et al., 1998). We therefore concluded that the MLA is not equivalent to the CA and could not be regarded as an alternative to the CA (Honma et al., 1998).

The discordant results between the MLA and the CA, however, might have been due to differing treatment periods (Sofuni et al., 1997Go; Honma et al., 1998). The standard MLA employs only short-term (3 or 4 h) treatments, while the CA employs long-term (24 or 48 h) as well as short-term (4 or 6 h) treatments (Galloway et al., 1994Go). The short-term treatment MLA may be unable to detect some clastogens and spindle poisons. In the present study, we used the long-term treatment MLA and re-examined 15 clastogens and spindle poisons that were judged to be negative or inconclusive in the MLA collaborative study


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cells and culture
L5178Y tk+/– clone 3.7.2C mouse lymphoma cells were kindly provided by Dr D.Clive (Raleigh, NC). The cells were grown in RPMI 1640 medium (catalogue no. 31800; Gibco BRL Life Technologies Inc., Grand Island, NY) supplemented with 10% heat-inactivated horse serum (BioCell Laboratories Inc., Rancho Dominguez, CA). The cultures were incubated at 37°C in an atmosphere of 5% CO2 and 100% humidity and maintained at densities ranging from 105 to 106 cells/ml.

Testing chemicals
In total, 15 chemicals were tested by the long-term (24 h) treatment MLA, all of them except urethane were ranked as CA-positive by the National Toxicology Program (NTP) report (Zeiger et al., 1990Go) and/or by the Japanese re-evaluation study using CHL/IU cells (Matsuoka et al., 1996Go) (Table IGo). These 15 chemicals showed negative, inconclusive and equivocal responses in the previous MLA collaborative study (Honma et al., 1998). All test chemicals except 2'-deoxycoformycin were supplied by Wako Pure Chemical Co. Ltd (Osaka, Japan). 2'-Deoxycoformycin was a gift from Dr T.Shigaki (Chemosero Therapeutic Research Institute, Japan).


View this table:
[in this window]
[in a new window]
  		Table I. Chemicals used and their evaluation in the 24 h treatment mouse lymphoma assay (MLA) and from published chromosome aberration test (CA) and MLA data

 
tk gene mutation assay
The tk gene mutation assay was conducted by the microwell method as described previously (Honma et al., 1998) except for treatments, which were carried out as follows. Cultures of 50 ml at a density of 2x105 cells/ml in RPMI 1640 medium supplemented with 10% horse serum were treated in flasks with a series of diluted test chemicals for 24 h in a 37°C, 5% CO2 humidified incubator. The cells were then centrifuged, washed once and resuspended in fresh medium. They were transferred to new flasks and adjusted to 50 ml cultures at a density of 2x105 cells/ml for growth through the expression period (45 h) or diluted to be plated to estimate survival.

Each experiment was performed with a single culture per treatment without S9 mix. The mutagenic potential of each chemical was investigated up to a sufficient cytotoxic condition [<20% relative survival (RS) as a rule]. We perceived no need to test concentrations >5 mg/ml.

Mutation frequencies were calculated based on the Poisson distribution as previously described (Honma et al., 1988). All results were statistically analyzed by a newly developed procedure (Hayashi et al., in preparation). The procedure consists of Dunnett's multiple comparison with the concurrent control data and a trend test to assess dose dependency after evaluation of any downturn phenomenon by Simpson and Margolin's method (Simpson and Margolin, 1986Go, 1990Go).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The results of all experiments and the mutation frequency caused by each chemical are shown in the Appendix. The mutagenic responses to the chemicals in the 24 h MLA are shown in Table IIGo. The cytotoxic (relative survival) and mutagenic responses to the chemicals in the 24 h MLA as well as the results of 3 h treatments from the previous MLA collaborative study (Honma et al., 1998) are shown in Figure 1Go. Isophorone and 1,1,1,2-tetrachloroethane were tested twice, because the first experiment yielded negative results under insufficient cytotoxicity (>20% RS). Other chemicals were tested once. Each experiment was conducted until <20% RS or a concentration of 5 mg/ml was obtained. Bromodichloromethane did not decrease RS to <20% even at the highest concentration used, but it did decrease relative total growth (RTG) to <20%. Eleven of the 15 chemicals tested were statistically positive in the 24 h treatment MLA and four were negative.


View this table:
[in this window]
[in a new window]
  		Table II. Mutagenicity in the 24 h treatment MLA

 







View larger version (150K):
[in this window]
[in a new window]
  		Fig. 1. Relative survival values and mutation frequencies in the MLA after treatments with each chemical. Thick lines with closed symbols are experiments from the 24 h treatments in the present study. Thin lines with open symbols are from the 3 h treatment experiments in the collaborative studies published previously (Honma et al., 1998).

 
All raw data are available in the Mutagenesis database. A copy can be obtained upon request to the Editor.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In a previous collaborative study, we used the MLA to examine 34 chemicals that were positive in the CA but negative in the bacterial reverse mutation assay (BRM) (Sofuni et al., 1996Go; Honma et al., 1999Go). Fourteen of the chemicals (41%) could not be judged to be positive in the MLA. This collaborative study, however, suggested that the MLA may not have detected some clastogens and spindle poisons because the treatment period (3 h) may have been too short. Among the 14 chemicals, for example, four (2'-deoxycoformycin, dideoxycytidine, 1,3-demethylxanthine and noscapine) did not produce the targeted cytotoxicity even at the maximum dose tested (5 mg/ml). Furthermore, 2'-deoxycoformycin and thiabendazole were positive in the CA with 24 h treatment, but not with pulse treatment (6 h) (Ishidate, 1987Go; Otsuka et al., 1991Go), indicating that longer exposure was needed for expression of their genotoxicity.

The updated OECD (1997) guidelines require long-term (24 or 48 h) as well as short-term treatments in the absence of S9 mix for the CA. The guidelines mention that some chemicals interacting with a mammalian replication system, such as base and nucleoside analogs, often need to be present in the culture medium for one or more complete rounds of replication to express a detectable level of cytotoxicity and clastogenicity. The idea is also expressed in the ICH guidelines for the in vitro CA (ICH, 1996Go). On the other hand, the MLA has generally employed only a short-term treatment of 3 or 4 h. Extended treatment times (>2 cell cycles) were considered inappropriate in mammalian cell gene mutation assays (MCGM), including the MLA (Aaron et al., 1994Go), because prolonged toxicity delays the cell cycle, which in turn prolongs the time for suitable mutation fixation and thus results in fluctuations in mutation frequencies. Long-term treatment was, however, sometimes employed in other MCGM systems, such as CHO/HPRT and TK6/TK (Hsie et al., 1978Go; Liber and Denault, 1991Go; Cochrane and Skopek, 1994Go; Honma and Little, 1995Go), and was effective for detecting the mutagenicity of base and nucleoside analogs, which seems to be cell cycle dependent (Hoppe et al., 1991Go; Liber and Denault, 1991Go).

Even with long-term treatment, we would not expect complete concordance between the CA and MLA; the endpoints are different and different biological processes might affect the results. In order to compare the interchangability of the CA and the MLA properly, however, the effectiveness of long-term treatment in the MLA needs to be evaluated.

The present findings that 11 out of 14 CA-positive chemicals gave positive responses with the 24 h treatment MLA demonstrated that long-term treatment can detect some genotoxic chemicals that are negative in the short-term MLA. The 11 positive chemicals included three spindle poisons (colchicine, vinblastine sulfate and thiabendazole), two nucleoside analogs (2'-deoxycoformycin and dideoxycytidine) and one base analog (1,3-dimethylxanthine). These chemicals yield mutations indirectly during chromosome segregation and DNA replication; they do not directly damage, intercalate in or form adducts with DNA. The mutagenic potential of the non-DNA targeting mutagens may be entirely cell cycle dependent, so their effective expression would require long-term treatment, just as it does in the CA.

Some chemicals that did not bring about the targeted cytotoxicity at maximum dose in the short-term MLA did so in the long-term MLA and induced statistically significant mutational responses (dideoxycytidine, 1,3-dimethylxanthine, phenacetine and thiabendazole). Other chemicals (p-tert-butylphenol, 2'-deoxycoformycin, isophorone and zearalenone) showed similar cytotoxic responses following 3 and 24 h treatments, but only the 24 h treatment revealed their mutagenic potential. These results indicate that the longer treatment allows not only expression of the cytotoxicity of the test chemicals but also provides the potential for expression of their mutagenicity.

Although 24 h treatments enhance the capacity of the MLA for detecting mutagens, clastogens and spindle poisons, this procedure may decrease the specificity of the assay, resulting in a non-mutagen being falsely judged as a MLA-positive chemical. To clarify this matter, another collaborative study was organized by the JPMA, PhRMA and EFPIA. Non-mutagens that are negative in both the BRM and the CA were investigated using the 24 h treatment MLA with the microwell and soft agar methods. In preliminary experiments almost all the non-mutagens were negative (Müller et al., in preparation; Shimada et al., in preparation).

Table IIIGo summarizes the results of the CA and the MLA, including the 24 h experiments on 40 chemicals which were tested in the MLA collaborative study (Honma et al., 1998). Among 34 CA-positive chemicals, 31 were finally judged to be positive and two to be negative by the MLA. Even though the 24 h result for bromodichloromethane was negative, it was judged equivocal, because of the marginal response in the presence of S9 mix in the short-term MLA. Thus, the MLA detected 91% (31/34) of clastogens and spindle poisons detected by the CA. We conclude that the MLA is almost equivalent to the CA for the detection of genotoxic chemicals when the 24 h treatment protocol is used. A long-term treatment trial using the MLA as well as the CA should be conducted in the absence of S9 mix if an experiment with short-term treatment was negative.


View this table:
[in this window]
[in a new window]
  		Table III. Comparison from results of the chromosome aberration test with the MLA with 24 h-treatment

 


   Acknowledgments
 
We thank Dr D.Clive for providing the L5178Y tk+/– cell line and Dr T.Shigaki (Chemosero Therapeutic Research Institute) for his kind gift of 2'-deoxycoformycin. We also thank Dr I.Yoshimura, T.Ohmori and Y.Honda for their invaluable contribution on statistical data evaluation. This study was supported by a grant from the Ministry of Health and Welfare of Japan.


   Notes
 
1 To whom correspondence should be addressed. Tel: +81 3 3700 1141; Fax: +81 3 3700 2348; Email: honma{at}nihs.go.jp Back

2 Present address: School of Public Health, West China University of Medical Sciences, Chengdu 610041, China Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

    Aaron,C.S., Bolcsfoldi,G., Glatt,H.R., Moore,M., Nishi,Y., Stankowski,L.,Jr, Theiss,J. and Thompson,E. (1994) Mammalian cell gene mutation assays working group report. Mutat. Res., 312, 235–239.[Web of Science][Medline]

    Amacher,D.E. and Turner,G.N. (1982) Mutagenic evaluation of carcinogens in the L5178Y/TK assay utilizing postmitochondrial fractions (S9) from normal rat liver. Mutat. Res., 97, 49–65.[Web of Science][Medline]

    Blazak,B.F., Los,F.J., Rudd,C.J. and Caspary,W.J. (1989) Chromosome analysis of small and large L5178Y mouse lymphoma cell colonies: comparison of trifluorothymidine-resistant and unselected cell colonies from mutagen-treated and control culture. Mutat. Res., 224, 197–208.[Web of Science][Medline]

    Caspary,W., Langenbach,R., Penman,B.W., Crespi,C., Myhr,B.C. and Mitchell,A.D. (1988) The mutagenic activity of selected compounds at the TK locus: rodent vs. human cells. Mutat. Res., 196, 61–81.[Web of Science][Medline]

    CEC/EU (1989) The Rules Governing Medicinal Products in the European Community, Vol. III. Guidelines on the Quality, Safety and Efficacy of Medical Products for Human Use. Commission of the European Communities, Brussels, Belgium, p. 103.

    Chetelat,A. (1987) Hoffman-La Roche Research Report no. B-153'750. Hoffman-La Roche, Basel, Switzerland.

    Clive,D. and Spector,J.F.S. (1975) Laboratory procedure for assessing specific locus mutations at the tk locus in cultured L5178Y mouse lymphoma cells. Mutat. Res., 31, 17–29.[Web of Science][Medline]

    Clive,D., Johnson,K.O., Spector,J.F.S., Batson, A.G. and Brown,M.M.M. (1979) Validation and characterization of the L5178Y/TK+/– mouse lymphoma mutagen assay system. Mutat. Res., 59, 61–108.[Web of Science][Medline]

    Clive,D., Turner,N.T., Krehl,R. and Eyre,J. (1985) The mouse lymphoma assay may also be used as a chromosome aberration assay. Environ. Mutagen., 7 (suppl. 3), 33.

    Clive,D., Glover,P., Applegate,M. and Hozier,J. (1990) Molecular aspects of chemical mutagenesis in L5178Y/tk+/– mouse lymphoma cells. Mutagenesis, 5, 191–197.[Abstract/Free Full Text]

    Clive,D., Bolcsfoldi,G., Clements,J., Cole,J., Honma,M., Majeska,J., Moore,M., Muller,L., Myhr,B., Oberly,T., Odelhkim,M.C., Rudd,C., Shimada,H., Sofuni,T., Thybaud,V. and Wilcox,P. (1995) Consensus agreement regarding protocol issues discussed during the mouse lymphoma workshop: Portland, Oregon, May 7, 1994. Environ. Mol. Mutagen., 25, 165–168.[Web of Science][Medline]

    Cochrane,J.E. and Skopek,T.R. (1994) Mutagenicity of butadiene and its epoxide metabolites: I. Mutagenic potential of 1,2-epoxybutene, 1,2,3,4,-diepoxybutane and 3,4-epoxy-1,2-butanediol in cultured human lymphoblasts. Carcinogenesis, 15, 713–717.[Abstract/Free Full Text]

    Cole,J., Arlett,C.F, Green,M.H.L., Lowe,J. and Muriel,W. (1983) A comparison of the agar cloning and microtitration techniques for assaying cell survival and mutation frequency in L5178Y mouse lymphoma cells. Mutat. Res., 111, 371–386.[Web of Science][Medline]

    Combes,R.D., Stopper,H. and Caspary,W.J. (1995) The use of L5178Y mouse lymphoma cells to assess the mutagenic, clastogenic and aneugenic properties of chemicals. Mutagenesis, 10, 403–408.[Abstract/Free Full Text]

    DHSS UK (1989) Guidelines for Testing Chemicals for Mutagenicity. DHSS Report on Health and Social Subjects, No. 35. HMSO, London, UK.

    Federal Register (1993) Draft Revised Toxicological Principles for the Safety Assessment of Direct Food and Color Additives Used in Food. FDA/CFSAN, Washington DC, USA.

    Galloway,S.M., Armstrong,M.J., Reuben,C., Colman,S., Brown,B.C.C., Bloom,A.D., Nakamura,F., Ahmed,M., Duk,S., Rimpo,J., Margolin,B.H., Resnick,M.A., Anderson,B. and Zeiger,E. (1987) Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: evaluation of 108 chemicals. Environ. Mol. Mutagen., 10, 1–175.

    Galloway,S.M., Aardema,M.J., Ishidate,M.,Jr, Ivett,J.L., Kirkland,D.J., Morita,T., Mosesso,P. and Sofuni,T. (1994) Report from working group on in vitro tests for chromosomal aberrations. Mutat. Res., 312, 241–261.[Web of Science][Medline]

    Garriot,M.L., Casciano,D.A., Schechtman,L.M. and Probst,G.S. (1995) International workshop on mouse lymphoma assay testing practices and data interpretations: Portland, Oregon, May 7, 1994. Environ. Mol. Mutagen., 25, 162–164.[Web of Science][Medline]

    Hoppe,H., Call,K.M., Leong,P.-M. and Thilly,W.G. (1991) Cell-cycle dependent mutation of human lymphoblasts: bromodeoxyuridine and butyl methanesulfonate. Mutat. Res., 250, 411–421.[Web of Science][Medline]

    Honma,M. and Little,J.B. (1995) Recombinogenic activity of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in human lymphoblastoid cells. Carcinogenesis, 16, 1717–1722.[Abstract/Free Full Text]

    Honma,M., Hayashi,M., Shimada,H., Tanaka,N., Wakuri,S., Awogi,T., Yamamoto,K.I., Ushio-Kodani,N., Nishi,Y., Nakadate,M. and Sofuni,T. (1999) Evaluation of the mouse lymphoma tk assay (microwell method) as an alternative to the in vitro chromosomal aberration test. Mutagenesis, 14, 5–22.[Abstract/Free Full Text]

    Hsie,A.W., O'Neill,J.P., Couch,D.B., San Sebastian,J.R., Brimer,P.A., Machanoff,R., Fuscoe,J.C., Riddle,J.C., Li,A.P. and Hsie,M.H. (1978) Quantitative analysis of radiation-and chemical-induced lethality and mutagenesis in Chinese hamster ovary cells. Radiat. Res., 76, 471–492.[Web of Science][Medline]

    ICH (1994) Technical symposium: Safety, Session 1: Harmonization of genotoxicity testing requirements. In D'Arcy,P.F. and Harron,D.W.G. (eds), Proceedings of the Second International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, Orlando 1993. The Queen's University, Belfast, UK, pp. 221–257.

    ICH (1996) Technical symposium: Safety, Session 4: Genotoxicity Testing. In D'Arcy,P.F. and Harron,D.W.G. (eds), Proceedings of the Third International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, Yokohama, 1995. The Queen's University, Belfast, UK, pp. 303–329.

    ICH (1997) Technical workshop III: Non-clinical safety testing for new medical products, Session 1: Genotoxicity. In D'Arcy,P.F. and Harron,D.W.G. (eds), Proceedings of the Fourth International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, Brussels, 1997. The Queen's University, Belfast, UK, pp. 236–261.

    Ishidate,M.,Jr (1987) Data Book of Chromosome Aberration Tests In Vitro. LIC/Elsevier, Tokyo, Japan.

    Ishidate,M.,Jr (1988) A proposed battery of tests for the initial evaluation of the mutagenic potential of medicinal and industrial chemicals. Mutat. Res., 205, 397–407.[Web of Science][Medline]

    Jotz,M.M. and Mitchell,A.D. (1981) Effect of 20 coded chemicals on the forward mutation frequency at the thymidine kinase locus in L5178Y mouse lymphoma cells. Prog. Mutat. Res., 1, 580–593.

    Liber,H.L. and Denault,C.M. (1991) Mutagenicity of 2-amino-N6-hydroxyadenine to TK6 human lymphoblast cells. Mutat. Res., 253, 91–95.[Web of Science][Medline]

    Majeska,J.B. and Matheson,D.W. (1990) Development of an optimal S9 activation mixture for the L5178YTK+/– mouse lymphoma mutation assay. Environ. Mol. Mutagen., 16, 311–319.[Web of Science][Medline]

    Matsuoka,A., Yamakage,K., Kusakabe,H., Wakuri,S., Asakura,M., Noguchi,T., Sugiyama,T., Shimada,H., Nakayama,S., Kasahara,Y., Takahashi,Y., Miura,K.F., Hatanaka,M., Ishidate Jr.,M., Morita,T., Watanabe,K., Hara,M., Odawara,K., Tanaka,N., Hayashi,M. and Sofuni,T. (1996) Re-evaluation of chromosomal aberration induction on nine mouse lymphoma assay `unique positive' NTP carcinogens. Mutat. Res., 369, 243–252.[Web of Science][Medline]

    McGregor,D.B., Brown,A., Cattanach,P., Edwards,I., McBride,D. and Caspary,W.J. (1988a) Responses of the L5178Y tk+/tk– mouse lymphoma cell forward mutation assay II: 18 coded chemicals. Environ. Mol. Mutagen., 11, 91–118.[Web of Science][Medline]

    McGregor,D.B., Brown,A.G, Cattanach,P., Edwards,I., McBridge,D., Riach,C. and Caspary,W.J. (1988b) Responses of the L5178Y tk+/tk– mouse lymphoma cell forward mutation assay: III. 72 coded chemicals. Environ. Mol. Mutagen., 12, 85–154.[Web of Science][Medline]

    McGregor,D.B., Brown,A.G, Howgate,S., McBridge,D., Riach,C. and Caspary,W.J. (1991) Responses of the L5178Y mouse lymphoma cell forward mutation assay: V. 27 coded chemicals. Environ. Mol. Mutagen., 17, 196–219.[Web of Science][Medline]

    MHW Japan (1990) Guideline for Toxicity Studies of Drugs Manual. Ministry of Health and Welfare, Tokyo, Japan.

    Mitchell,A.D., Auletta,A.E., Clive,D., Kirby,P.E., Moore,M.M. and Myhr,B.C. (1997) The L5178Y/tk+/– mouse lymphoma specific gene and chromosomal mutation assay; a phase III report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat. Res., 394, 177–303.[Web of Science][Medline]

    Moore,M.M. and Clive,D. (1982) The quantitation of TK–/– and HGPRT– mutants of L5178Y/TK+/– mouse lymphoma cells at varying times post-treatment. Environ. Mutagen., 4, 499–519.[Web of Science][Medline]

    Moore,M.M. and Howard,B.E. (1982) Quantitation of small colony trifluorothymidine-resistant mutants of L5178Y/TK+/– mouse lymphoma cells in RPMI-1640 medium. Mutat. Res., 104, 287–294.[Web of Science][Medline]

    Moore,M.M., Clive,D., Hozier,J.C., Howard,B.E., Batson,A.G., Turner,N.T. and Sawyer,J. (1985) Analysis of trifluorothymidine-resistant (TFTr) mutants of L5178Y/TK+/– mouse lymphoma cells. Mutat. Res., 151, 161–174.[Web of Science][Medline]

    Moore-Brown,M.M., Clive,D., Howard,B.E., Batson,A.G. and Johnson,K.O. (1981) The utilization of trifluorothymidine (TFT) to select for thymidine kinase-deficient (TK–/–) mutants from L5178Y/TK+/– mouse lymphoma cells. Mutat. Res., 85, 363–378.[Web of Science][Medline]

    Myhr,B.C. and Caspary,W.J. (1988) Evaluation of L5178Y mouse lymphoma cell mutagenesis assay: intralaboratory results for sixty-three chemicals tested at Litton Bionetics, Inc. Environ. Mol. Mutagen., 12, 103–194.

    Myhr,B.C. and Caspary,W.J. (1991) Chemical mutagenesis at the thymidine kinase locus in L5178Y mouse lymphoma cells: results for 31 coded compounds in the National Toxicology Program. Environ. Mol. Mutagen., 18, 51–83.[Web of Science][Medline]

    Myhr,B.C., McGregor,D., Bowers,L., Riach,C., Brown,A.G., Edward,I., McBride,D., Martin,R. and Caspary,W.J. (1990) L5178Y mouse lymphoma cell mutation assay results with 41 compounds. Environ. Mol. Mutagen., 18, 138–167.

    Oberly,T.J., Bewsey,B.J. and Probst,G.S. (1984) An evaluation of the L5178Y TK+/– mouse lymphoma forward mutation assay using 42 chemicals. Mutat. Res., 125, 291–306.[Web of Science][Medline]

    O'Donoghue,J.L., Haworth,S.R., Curren,R.D., Kirby,P.E., Lawlor,T., Moran,E.J., Phillips,R.D., Putnam,D.L., Rogers-Back,A.M., Slesinski,R.S. and Thilagar,A. (1988) Mutagenicity studies on ketone solvents: methyl ethyl ketone, methyl isobutyl ketone, and isophorone. Mutat. Res., 206, 149–161.[Web of Science][Medline]

    OECD (1997) Updated Genotoxicity Test Guidelines. OECD, Paris, France.

    Otsuka,M., Ogura,M., Okada,M., Inai,T., Kajiwara,Y., Anzinin,S., Kubo,K., Kikuno,T., Shiraishi,H., Kakimoto,K., Shigaki,T., Kuwata,S. and Umehashi,S. (1991) Mutagenicity studies of YK-176. Kiso To Rinsyo, 25, 1311–1320.

    Segawa,M., Nadamitsu,S., Kondo,K. and Yoshizaki,I. (1979) Chromosomal aberration of Don lung cells of Chinese hamster after exposure to vinblastine in vitro. Mutat. Res., 66, 99–102.[Web of Science][Medline]

    Simpson,D.G. and Margolin,B.H. (1986) Recursive nonparametric testing for dose–response relationships subject to downturns at high doses. Biometrika, 73, 589–596.[Abstract/Free Full Text]

    Simpson,D.G. and Margolin,B.H. (1990) Nonparametric testing for dose–response curves subject to down turns: asymptotic power considerations. Ann. Statist., 18, 373–390.

    Sofuni,T., Honma,M., Hayashi,M., Shimada,H., Tanaka,N., Wakuri,S., Awogi,T., Yamamoto,K.I., Nishi,Y. and Nakadate,M. (1996) Detection of in vitro clastogens and spindle poisons by the mouse lymphoma assay using the microwell method: interim report of an international collaborative study. Mutagenesis, 11, 349–355.[Abstract/Free Full Text]

    Sofuni,T., Wilcox,P., Shimada,H., Clements,J., Honma,M., Clive,D., Green,M., Thybaud,V., San,R.H.C., Elliot,B.M. and Muller,L. (1997) Meeting report on protocol issues regarding the use of the microwell method discussed in the mouse lymphoma workshop: Victoria BC, Canada, March 27, 1996. Environ. Mol. Mutagen., 29, 434–438.[Web of Science][Medline]

    Tennant,R.W., Margolin,B.H., Shelby,M.D., Zeiger,E., Haseman,J.K., Spalding,J., Caspary,W., Resnick,M., Stasiewics,S., Anderson,B. and Minor,R. (1987) Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays. Science, 236, 933–941.[Abstract/Free Full Text]

    Turner,N.T., Batson,A.J. and Clive,D. (1984) Procedure for the L5178Y/TK+/– to TK–/– mouse lymphoma cell mutagenicity assay. In Kilbey,B.J., Legator,M., Nichols,W. and Ramel,C. (eds), Handbook of Mutagenicity Test Procedure. Elsevier, Amsterdam, The Netherlands, pp. 239–268.

    Zeiger,E., Haseman,J.K., Shelby,M.D., Margolin,B.H. and Tennant,R.W. (1990) Evaluation of four in vitro genetic toxicity tests for predicting rodent carcinogenicity: confirmation of earlier results with 41 additional chemicals. Environ. Mol. Mutagen., 16, 1–14.[Web of Science][Medline]

    Zhang,L.-S., Honma,M., Matsuoka,A., Suzuki,T., Sofuni,T. and Hayashi,M. (1996) Chromosome painting analysis of spontaneous and methylmethanesulfonate-induced trifluorothymidine-resistant L5178Y cell colonies. Mutat. Res., 370, 181–190.[Web of Science][Medline]

Received on July 1, 1998; accepted on August 25, 1998.


   Abbreviations
 
PE0, plating efficiency (%) for survival; PE2, plating efficiency (%) for viability; RS, relative survival (%); RSG, relative suspension growth (%); RTG, relative total growth (%); MC, total or average number of wells containing mutant colonies per 192 wells; %SC, percent of small colony mutants; MF, mutation frequency (x10–6); ST, statistical analysis indicating a significant (+) or non-significant (–) response; nc (–), negative result obtained at concentrations up to the limit dose (5 mg/ml) with >20% RS; TX, data was excluded from evaluation due to high cytotoxicity (<10% RS); #, precipitation or separation of the test chemical was observed during treatment.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 MutagenesisHome page
M. Honma, M. Hayashi, H. Shimada, N. Tanaka, S. Wakuri, T. Awogi, K. I. Yamamoto, N.-U. Kodani, Y. Nishi, M. Nakadate, et al.
Evaluation of the mouse lymphoma tk assay (microwell method) as an alternative to the in vitro chromosomal aberration test
Mutagenesis, January 1, 1999; 14(1): 5 - 22.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (11)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Honma, M.
Right arrow Articles by Sofuni, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Honma, M.
Right arrow Articles by Sofuni, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?