Environ. Mutagen Res., 27: 145-151 (2005) Original Article

The optimized conditions for the in vitro (MN) test procedures using chamber slides

Mika Yamamoto＊, Akira Motegi, Jiro Seki and Youichi Miyamae

Toxicology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd. 1-6, Kashima 2-chome, Yodogawa-ku, Osaka 532-8514, Japan

Summary

Optimized conditions for an in vitro micronucleus (MN) test procedure were defined using a chamber slide that enabled the preparation of fine specimens without undergoing complicating pro- cedures using culture dishes. The issues investigated are 1) the effect of slide materials on the adhesion of cells, 2) the number of seeding cells necessary to obtain an adequate number of cells for observation and 3) effects of hypotonic treatment and fixation on the cytoplasmic: nuclear area ratio. In addition, we determined cell viability in each chamber using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay. The results of the investigation were as follows: 1) cell adhesion was best using plastic slides, 2) the optimum number of cells for seeding was 6.6× 103 cells/cm2, 3) the best condition for hypotonic treatment was incubation in 75 mM KCl at 37 ℃ for 5 min, and 4) the best condition for fixation was treatment of cells twice for about 2 min in ice- cold methanol containing 6％ acetic acid. Finally, the result of the MTT assay correlated with the number of viable cells in chamber as determined by the trypan blue dye exclusion assay. An in vitro MN test was conducted under these conditions using the known clastogens, Mitomycin C and dimethylnitrosamine. These clastogens dose-dependently induced a significant increase in the number of micronucleated cells with positive responses at concentrations approxi- mately 10 times lower than those of the chromosomal aberration test. On the other hand, the fre- quency of micronucleated cells in the solvent control was stable and low (0.4 ─ 1.7％). These results indicated that the in vitro MN test has a high level of sensitivity to clastogens. It was concluded that the in vitro MN test using chamber slides is a rapid, simple and sensitive method to detect clastogens.

Keywords: in vitro micronucleus test, chamber slide

short-term test is required. Introduction Recently, the in vitro micronucleus (MN) test has been The in vitro chromosomal aberration (CA) test is a well- used as a short-term test alternative to the CA test to established, frequently used short-term test to estimate detect the clastogenicity of chemicals (Matsuoka et al., the clastogenicity of chemicals (OECD, 1997). However, it 1993; Miller et al., 1995; Matsushima et al., 1999). The in is not applicable to high throughput screening because vitro MN test is rapid and simple for evaluation, because the slide preparation and metaphase analysis in the CA the measurement of micronucleus is less subjective than test requires considerable skill, experience and time metaphase analysis. Furthermore, the concordance (Matsuoka et al., 1993; Kirsch-Volders, 1997; Fenech, between the result of the in vitro MN and CA tests has 2000). Therefore, the development of a rapid and sensitive been reported to be approximately 90％ (Miller et al., 1997; Matsushima et al., 1999). Therefore, the in vitro MN test is capable of screening larger numbers of drug candi- ＊ E-mail: [email protected] dates (Matsuoka et al., 1993; Miller et al., 1995; Kirsch- Received: March 22, 2005, revised: July 14, 2005, Volders, 1997; Garriott et al., 2002; Matsushima et al., accepted: July 16, 2005 © Japanese Environmental Mutagen Society 1999). However, the slide preparation in the in vitro MN

145 test is usually the same as that of the in vitro CA test, and ferent concentrations. For experiments with metabolic this method still requires complicated, time consuming activation, the fresh medium also contained 16.7％ S9 mix procedures such as the peeling off cells from dishes, cen- (5％ S9 fraction). S9 was prepared by centrifugation trifugation of cell suspensions, and dropping cells onto (9000×g) of liver homogenate from male Sprague-Dawley slides. If complicated slide preparation procedures were rats (Slc:SD) (7 weeks of age) given phenobarbital and not required, the in vitro MN test would be more rapid 5,6-benzoflavone intraperitoneally. The ingredients in and simple. 1 mL of S9 mix were 0.3 mL of S9, 0.2 mL of 40 mM

Chamber slides have one to eight small separate areas HEPES buffer solution, 0.1 mL of 50 mM MgCl2, 330 mM (chambers) which can accommodate all procedures for KCl, 50 mM glucose 6-phosphate, 40 mM NADP and 0.1 from the seeding to the fixation on the slide. mL of distilled water. Cells were treated for 6 h or 24 h by Therefore, if the number of cells obtained by culturing in the method without metabolic activation system and for 6 chamber slides is adequate for the MN assay, the test h by the metabolic activation method. After treatment of 6 capacity for screening of drug candidates would be h or 24 h, the cells were rinsed twice with Hanks’ increased. To obtain evaluative cells which possess clear Balanced Salt Solution and incubated further in 0.8 mL of nuclei surrounded by large cytoplasmic area, it is impor- fresh medium until 48 h after the start of treatment. At the tant to optimize the slide material, the number of seeding end of this incubation, the culture medium was replaced cells, the condition of hypotonic treatment, and the condi- by 75 mM KCl solution for hypotonic treatment. Then tion of fixation in the test procedure. These conditions for cells were fixed every two minutes by two changes of ice- in vitro MN test procedures using chamber slides have cold methanol containing acetic acid and, air-dried, and not been reported. The purpose of this study was to define the chambers were removed from the slides. Each slide the optimized conditions for in vitro MN test procedures was stained with 100 µL acridine orange (3 µg/mL; AO: using chamber slides. Nacalai Tesque, Inc., Kyoto, Japan) and coverslipped. All slides were analyzed at 1,000 magnification using a fluo- Materials and Methods rescence microscope equipped with an excitation filter of 1. Chemicals and solvents 420-490 nm and a long pass barrier filter of 520 nm. The To investigate the test condition for practical treatment, number of micronucleated cells among 1,000 intact inter- cells were treated by Mitomycin C (MMC, CAS No. 50 -07- phase cells was counted. Micronucleus was identified 7, Kyowa Hakko Kogyo Ltd., Tokyo, Japan) or dimethylni- according to the following criteria based on that of P.I. trosamine (DMN, CAS No. 62-75 -9, Nacalai Tesque, Inc., Countryman and J.A. Heddle (Countryman and Heddle, Kyoto, Japan). Both chemicals were dissolved in physio- 1976). logical saline. 1) Micronucleus is surrounded by a nuclear membrane. 2) The diameter of micronucleus varies less than 1/2nd 2. Cell and medium of the diameter of the main nucleus. Chinese hamster lung (CHL/IU) cells obtained from 3) Micronucleus is lying within the cytoplasm, not the National Institute of Health Sciences, Tokyo (Japan) linked or connected to the main nuclei and with were grown in Eagle’s Minimum Essential Medium (E- staining similar to the main nucleus. MEM, GIBCO BRL, Life Technologies, Inc.) supplement- The number of cells with micronuclei in the treated ed with 10％ heat-inactivated (56℃, for 30 min) calf group was compared to that of the solvent control group serum. Cells were grown in a humidified atmosphere of using the Chi-square test. Test chemicals were consid-

95％ air 5％ CO2 at 37℃. The modal num- ered positive if the number of micronucleated cells ber was 25, and the average generation time was about 15 increased dose-dependently and differed significantly h. from the solvent control in at least 1 treated group (p＜ 0.05). 3. In vitro micronucleus test CHL/IU cells were cultivated in 4-chamber slides made 4. Cell viability determination using the 3-(4,5- of either glass (Lab-Tek, Nunc Inc., Naperville, IL), colla- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro- gen-coated glass (glass slide: Lab-Tek, Nunc Inc., mide (MTT) assay and the trypan blue dye exclusion Naperville, IL; Cellmatrix Type1-A: 3.0 mg/mL, Nitta zer- assay achin Inc., Niigata, Japan), or plastic (permanox: Lab-Tek, After cultivation, the chamber slides were divided into Nunc Inc., Naperville, IL). CHL/IU cells were diluted in two groups for measurement of the number of viable cells culture medium, and 0.8 mL of suspension was seeded in in chamber using two assay methods. For the trypan blue each chamber (1.8 cm2/chamber). After preculture (about dye exclusion assay, cells were rinsed with phosphate 18 h), the culture medium was exchanged with 0.8 mL of buffered solution, detached from each chamber by treat- fresh medium containing solvent or test chemicals at dif- ment with trypsin-EDTA, and suspended in culture medi-

146 Fig. 1 Influence of slide materials on cell morphology (a): glass slide, (b): collagen-coated glass slide, (c): plastic slide, magnification: ×50

um. The cell suspension was mixed with an equal volume optimized the number of seeding cells for observation of of 0.5％ Trypan Blue dye solution, and cells that were not micronuclei using the plastic chamber slide (data not stained were counted as viable cells using a hemacytome- shown). When CHL/IU cells were incubated in chamber ter. For the MTT assay, culture medium was exchanged slides at a density of 2.2×103 ─ 8.8×103 cells/cm2, it was with fresh medium (0.8 mL/chamber) containing 0.25 concluded that a seeding density of 6.6×103 cells/cm2 mg/mL MTT, and the cells were incubated at 37℃ for 4 was optimal for the observation of micronuclei because of h. After incubation, the medium was removed, and DMSO both the cell density and the cytoplasm: nuclear area ratio. (dimethyl sulfoxide)(0.2 mL/chamber) was added to all chambers and mixed thoroughly to dissolve the dark blue 2. Influence on the cytoplasmic area crystals. After a few minutes at room temperature, all 1) Effect of acetic acid concentration in fixative crystals were dissolved and the MTT formazan in each The cytoplasmic area of the cells was evaluated follow- chamber was transfered to a 96-well plate. The optical ing treatment with methanol containing 0, 2, 6 and 18％ densities of the wells were read on a microplate reader, at acetic acid at the end of hypotonic treatment at room tem- a test wavelength of 540 nm and a reference wavelength of perature for 10 min (Fig. 2). Other conditions used to fix 620 nm. the cells are described above in section 2.3. (In vitro micronucleus test). The higher concentration of acetic Results acid induced the more swelling of the cytoplasm. Cells 1. Influence on cell morphology and the area of fixed with 18％ acetic acid had the cytoplasm that was par- the cytoplasm tially dispersed and/or the border of each cell was Time-dependent cell proliferation on glass, on collagen- obscure. Methanol containing graded concentrations of coated glass, and on plastic is shown in Fig. 1. Cell prolif- acetic acid (3, 6, 9 and 12％) was used to establish the eration on each material was similar at 24 h incubation. best concentration and to confirm reproducibility with However, after 42 and 66 h incubation, the cells on glass respect to the cellular cytoplasmic area (Fig. 3). It was slides changed into globular or cuboidal shapes morpho- confirmed that the cytoplasmic area varied with increas- logically and aggregated time-dependently. The collagen- ing concentration of acetic acid. Concentrations of 9 and coated glass slide prevented cell morphological changes 12％ acetic acid partially dispersed the cytoplasm and/or but did not completely prevent the cellular aggregation. obscured the border of each cell. Therefore, it was con- On the other hand, plastic slides prevented both cell mor- cluded that methanol containing 6％ acetic acid was the phological changes and aggregation through the entire 66 optimal concentration to maintain the cytoplasm around h incubation period. Consequently, we concluded that the nucleus in fixative. plastic slides are best for the in vitro micronucleus test in 2) Effect of the time and temperature in hypotonic treat- that the cells proliferated as a uniform monolayer. We also ment

147 Fig. 2 Effect of acetic acid concentration in fixative on the area of the cytoplasm ─ 1 (a): 0％, (b): 2％, (c): 6％, (d): 18％, magnification: ×625

Fig. 4 Effect of time in hypotonic treatment on the area of the cytoplasm (a): 0 min, (b): 5 min, (c): 10 min, (d): 15 min, (e): 20 min, (f): 30 min, magnification: ×625

Fig. 3 Effect of acetic acid concentration in fixative on the area of the cytoplasm ─ 2 Fig. 5 Effect of temperature in hypotonic treatment on the area of (a): 3％, (b): 6％, (c): 9％, (d): 12％, magnification:×625 the cytoplasm (a): room temperature, (b): 37℃, magnification: ×625

The cytoplasmic area was assessed following hypotonic 103cells/cm2; the best condition for hypotonic treatment is treatment at room temperature for 0, 5, 10, 15, 20 and 30 incubation in 75 mM KCl at 37℃ for 5 min; and the best min (Fig. 4). After hypotonic treatment, the cells were condition for fixation is treatment two times with ice-cold fixed with methanol containing 6％ acetic acid. Other con- methanol containing 6％ acetic acid for about 2 min. ditions used to fix the cells are described above in Materials and Methods section 3 (In vitro micronucleus 3. Observation of positive response test). Without hypotonic treatment (0 min), the cells main- The number of micronucleated cells among 1,000 intact tained fibroblastic shaped. After the treatment of 20 or 30 interphase cells was counted for each group. The results min, several parts of cytoplasm were dispersed. From 5 to of MMC and DMN treatment under the optimized condi- 15 min treatment, there was no difference in the cytoplas- tion established previously are shown in Figs. 6 and 7. In mic area. the 6 h treatment/42 h recovery group and the 24 h treat- Next, the effect of hypotonic treatment at room tempera- ment/24 h recovery group without metabolic activation, ture or 37℃ on cytoplasmic area was assessed (Fig. 5). the frequencies of micronucleated cells in the solvent con- Hypotonic treatment at 37℃ increased the cytoplasmic trol were 0.4 ─ 1.2％ and 0.5 ─ 1.7％, respectively. In the 6 h area compared with treatment at room temperature. treatment/42 h recovery group with metabolic activation, These results indicated that hypotonic treatment at 37℃ the frequency of micronucleated cells in the solvent con- for 5 min was the most appropriate to swell the cytoplasm. trol was 0.6 ─ 1.2％. The frequencies of micronucleated The optimized conditions established in the in vitro cells in the solvent control in any series were consistent micronucleus test with chamber slides are as follows; the indicating reproducibility. MMC induced a dose-depen- plastic (permanox) slide is the best for cell adhesion; the dent increase in the frequency of micronucleated cells in most adequate number of cells for seeding is 6.6× the 6 h treatment/42 h recovery group and 24 h treat-

148 40 2.5

) r = 0.9944 ％ 30 ** 2

20 ** ** 1.5 10 ** Frequencies of ** ** 1 micronucleated cells ( ** ** 0 ** Optical Density (620 nm) 0

0.2 0.5 0.01 0.02 0.04 0.08 0.12 0.16 0.005 0.0025 MMC (mg/mL) 0 Fig. 6 Result of in vitro micronucleus test with Mitomycin C 0 50000 100000 150000 200000 250000 300000 (MMC) ●: 24 h treatment/24 h recovery group, ○: 6 h treat- Number of viable cells / chamber ment/42 h recovery group Fig. 8 The relationship between the number of viable cells per ＊＊ : Significantly different from solvent control (p＜0.01; chamber and the optical density that corresponded to the Chi-square test) amount of MTT formazan generated by cells in each cham- The values represent the means±S.D. of at least three inde- ber. r: the correlation coefficient pendent experiments.

sity that corresponded to the amount of MTT formazan 40 was directly proportional to the number of viable cells determined by trypan blue dye exclusion assay over a 30 range of 16,000 to 270,000 cells. The correlation coeffi- cient was calculated to be 0.9944. 20 Discussion ** There are some essential issues that need to be defined 10 ** Frequencies of ** ** ** in order to establish to an in vitro MN test procedures micronucleated cells (%) ** using a chamber slide. First, the effect of slide material on 0

0 adhesion of cells was investigated. The plastic chamber 125 250 500 31.3 62.5 1000 2000 slide provided consistent cell proliferation and good adhe- DMN (mg/mL) sion of cells compared to that of the glass or the collagen- Fig. 7 Result of in vitro micronucleus test with dimethylni- coated glass slide. These results indicated that plastic trosamine (DMN) dishes have a good record as a cell culture material in the ●: 6 h treatment/42 h recovery group in vitro micronucleus assay. The in vitro micronucleus ＊＊: Significantly different from solvent control (p 0.01; ＜ method using plastic chamber slides is also reported by Chi-square test) other investigators (Miller et al., 1995; Garriott et al., The values represent the means±S.D. of at least three inde- pendent experiments. 2002; Erexson et al., 2001). Therefore, the plastic chamber slide is considered as the best slide material for this type of study. ment/24 h recovery group without metabolic activation Although consistent cell proliferation and good adhesion method. DMN also increased the frequency of micronu- are essential for this study, the cytoplasmic area is also cleated cells dose-dependently in the 6 h treatment/42 h important in order to observe the micronucleus precisely recovery group with metabolic activation method. Both and easily (Kirsch-Volders et al., 2000). The cytoplasmic MMC and DMN treatments significantly increased the area is affected by the cell density, time and temperature frequency of micronucleated cells compared to their of hypotonic treatment, as well as by the concentration of respective solvent control group. These results were high- acetic acid in the fixative. Time and temperature of hypo- ly reproducible and showed little deviation. tonic treatment and the concentration of acetic acid in the fixative are especially important factors. Based on our 4. MTT test results, the optimal condition for maximizing cytoplasmic The relationship between the number of viable cells per area without cell lysis was to fix cells with methanol con- chamber and the amount of MTT formazan generated by taining 6％ acetic acid followed by 5 min incubation with cells in each chamber is shown in Fig. 8. The optical den- 75 mM KCl at 37℃.

149 However, there are many variations of the conditions of cells (ICHS2A, 1996; Bradley et al., 1987). Therefore, the hypotonic treatment and fixation. CHL/IU cells have been in vitro genotoxic test should be conducted with concur- cultured on plastic dishes, fixed with 1:3 acetic alcohol, rent measurement of the cytotoxicity. Based on the CA and suspended in methanol containing 1％ acetic acid test, the viable cell count is the most common method to (Matsuoka et al., 1993; Matsushima et al., 1999); V79 cells estimate the cytotoxicity; however, the viable cell count is have been cultured on glass slides in QuadriPERM dishes time consuming because this method requires the prepa- and fixed with ethanol-acetic acid (3:1) containing 0.74％ ration of cell suspension. It was reported that the number formaldehyde at the end of mild hypotonic treatment and the activity of viable cells could be measured using a (1.5％ trisodium citrate-2-hydrate for 5 min) (Kalweit et 96-well plate based on a colorimetric assay, and the MTT al.,1999; Von der Hude et al., 2000); CHO cells have been assay makes it possible to estimate the number of viable cultured on plastic chamber slides and fixed with cells based on this correlation without the preparation of methanol (Erexson et al., 2001) or methanol containing cell suspension (Mosmann, 1983). In this study, the 1％ acetic acid (Garriott et al., 2002) at the end of mild results of the MTT assay were well correlated with the hypotonic treatment (1％ trisodium citrate-2-hydrate for 5 results of trypan blue dye exclusion assay (correlation min), or fixed with methanol containing 1％ acetic acid coefficient r＝0.9944). Consequently, it was concluded without hypotonic treatment (Miller et al., 1995). These that MTT assay with chamber slides would be useful for variations may depend on cell strains and/or culture con- estimation of cytotoxicity in the in vitro MN test. ditions and suggest that the conditions of hypotonic treat- Based on our results, the in vitro MN test using cham- ment and fixation should be defined carefully in each labo- ber slides that we optimized shortened operation time to ratory with each cell type. This may be one of the reasons one fourth compared to that using culture dishes since it why the in vitro MN test has not been adapted internation- was not required complicated, time consuming proce- ally as an experimental protocol until recently (Marzin, dures. In addition, the amount of test sample for this assay 1997; Kirsch-Volders et al., 2000; 2003; Matsushima et al., using chamber slides was cut down to one fifth that for 1999). using culture dishes. Consequently, the throughput of the To evaluate the assay results, it is a prerequisite that in vitro MN test using chamber slides up four times com- background data in solvent control is reliable and stable pared to that using culture dishes. (Miller et al., 1995; Marzin, 1997; Kalweit et al., 1999). In In conclusion, the in vitro MN test using chamber slides this study, the frequency of micronucleated cells in the was a rapid and simple method for screening the genotox- solvent control ranged from 0.4 to 1.7％. The frequency of icity of drug candidates. Furthermore, cytotoxicity also micronucleated cells in solvent controls has been reported could be assessed rapidly by the MTT test with chamber to range as follows: 0.1 ─ 2.0％ (Matsushima et al., 1999), slides. 0.746±0.408％ (Matsuoka et al., 1993). Therefore, the results obtained in this assay system are in agreement Acknowledgements with the standard micronucleus assay protocol. To esti- mate the sensitivity of the assay system, we also tested The authors are grateful to Dr. Makoto Hayashi, Dr. the induction of micronuclei by two chemicals that were Herman Lilja and Dr. Marlowe Schneidkraut for their crit- routinely used in the CA test as standard positive controls ical reading of the manuscript. (MMC in tests without S9 mix and DMN in tests with S9 mix). These chemicals dose-dependently induced a signif- References icant increase in the number of micronucleated cells and Bradley, M.O., V.I. Taylor, M.J. Armstrong and S.M. Galloway these positive responses (MMC 24 h treatment/24 h (1987) Relationships among cytotoxicity, lysosomal breakdown, recovery group; 0.005 g/mL, DMN 6 h treatment/42 h ≥ µ chromosome aberrations, and DNA double-strand breaks, Mutat. recovery group; ≥ 62.5 µg/mL) were observed at concen- Res., 189, 69-79. trations of about 10 times lower than those of the CA test Countryman, P.I. and J.A. Heddle (1976) The production of micronu- 〔MMC 24 h treatment; ≥ 0.05 µg/mL, DMN 6 h treat- clei from chromosome aberrations in irradiated cultures of human ment/18 h recovery; ≥ 1000 µg/mL (Ishidate, 1998)〕. lymphocytes, Mutat. Res., 41, 321-332. While this study contains the results for only two chemi- Erexson, G.L., M.V. Periago and C.S. Spicer (2001) Differential sen- cals, these results suggest that the in vitro MN test may sitivity of Chinese hamster V79 and Chinese hamster ovary (CHO) cells in the in vitro micronucleus screening assay, Mutat. possess the higher sensitivity than the CA test. Res., 495, 75-80. In the in vitro genotoxic test, if the chemicals induce a Fenech, M. (2000) The in vitro micronucleus technique, Mutat. Res., positive response only under cytotoxic conditions, the 455, 81-95. response may not be due to the direct effect of the chemi- Garriott, M.L., J.B. Phelps and W.P. Hoffman (2002) A protocol for cal but may be due to a secondary effect via or the in vitro micronucleus test I. Contributions to the development release of endonucleases from lysosomes in damaged of a protocol suitable for regulatory submissions from an examina-

150 tion of 16 chemicals with different mechanisms of action and dif- somal aberration test, Mutat. Res., 272, 223-236. ferent levels of activity, Mutat. Res., 517, 123-134. Matsushima, T., M. Hayashi, A. Matsuoka, M. Ishidate Jr., K.F. ICHS2A (1996) Guidance on Specific Aspects of Regulatory Miura, H. Shimizu, Y. Suzuki, K. Morimoto, H. Ogura, K. Mure, K. Tests for Pharmaceuticals. Koshi and T. Sofuni (1999) Validation study of the in vitro Ishidate, M. Jr. (1998) Data Book of Chromosomal Aberration Test micronucleus test in a Chinese hamster lung cell line (CHL/IU), In Vitro, Life-Science Information Center, Tokyo, Japan. Mutagenesis, 14, 569-580. Kalweit, S., D. Utesch, W. von der Hude and S. Madle (1999) Miller, B.M., E. Pujadas and E. Gocke (1995) Evaluation of the Chemically induced micronucleus formation in V79 cells-compari- micronucleus test in vitro using Chinese hamster cells, Environ. son of three different test approaches, Mutat. Res., 439, 183-190. Mol. Mutagen, 26, 240-247. Kirsch-Volders, M. (1997) Towards a validation of the micronucleus Miller, B., S. Albertini, F. Locher, V. Thybaud and E. Lorge (1997) test, Mutat. Res., 392, 1-4. Comparative evaluation of the in vitro micronucleus test and the in Kirsch-Volders, M., T. Sofuni, M. Aardema, S. Albertini, D. vitro chromosome aberration test: industrial experience, Mutat. Eastmond, M. Fenech, M. Ishidate Jr., E. Lorge, H. Norppa, J. Res., 392, 45-59. Surralles, W. von der Hude and A. Wakata (2000) Report from the Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and In Vitro Micronucleus Assay Working Group, Environ. Mol. survival: Application to proliferation and cytotoxicity assays, Mutagen, 35, 167-172. J.lImmunol. Methods, 65, 55-63. Kirsch-Volders, M., T. Sofuni, M. Aardema, S. Albertini, D. Organization for Economic Cooperation and Development (OECD) Eastmond, M. Fenech, M. Ishidate Jr., S. Kirchner, E. Lorge, T. (1997) In vitro mammalian chromosome aberration test, OECD Morita, H. Norppa, J. Surralles, A. Vanhauwaert and A. Wakata guideline for the testing of chemicals, No. 473, OECD, Paris. (2003) Report from the in vitro micronucleus assay working Von der Hude, W., S. Kalweit, G. Engelhardt, S. McKiernan, P. group, Mutat. Res., 540, 153-163. Kasper, R. Slacik-Erben, H.G. Miltenburger, N. Honarvar, R. Marzin, D. (1997) The position of the in vitro micronucleus test with- Fahrig, B. Görlitz, S. Albertini, S. Kirchner, D. Utesch, F. Pötter- in the battery of screening for genotoxic potential determination Locher, H. Stopper and S. Madle (2000) In vitro micronucleus and the regulatory guidelines, Mutat. Res., 392, 175 -181. assay with Chinese hamster V79 cells-results of a collaborative Matsuoka, A., N. Yamazaki, T. Suzuki, M. Hayashi and T. Sofuni study with in situ exposure to 26 chemical substances, Mutat. (1993) Evaluation of the micronucleus test using a Chinese ham- Res., 438, 137-163. ster cell line as an alternative to the conventional in vitro chromo-

151