ToxSci Advance Access originally published online on December 27, 2006
Toxicological Sciences 2007 96(2):214-217; doi:10.1093/toxsci/kfl193
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Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part II: Performances of the In Vitro Micronucleus Test Compared to the Mouse Lymphoma Assay and the In Vitro Chromosome Aberration Assay
* Servier Group, Drug Safety Assessment, 45403 Orléans-Gidy, France
Servier Group, Institut de Recherches Internationales Servier IRIS, 92400 Courbevoie, France
1 To whom correspondence should be addressed at Biologie Servier, BP 43255, 45403 Fleury-les-Aubrais, France. Fax: +33 2 38 23 86 50. E-mail: elisabeth.lorge{at}fr.netgrs.com.
Received October 13, 2006; accepted December 13, 2006
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ABSTRACT |
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The in vitro micronucleus test is commonly used in the early stages of pharmaceutical development as a predictive tool for the regulatory mouse lymphoma assay or in vitro chromosome aberration test. The accumulated data from this assay leads to the suggestion that it could be used as an alternative to the chromosome aberration test or the mouse lymphoma assay in the regulatory genotoxicity battery. In this paper, we present the results of the in vitro micronucleus test on L5178Y mouse lymphoma cells with 25 compounds from Servier research and have compared these results to those obtained in the genotoxicity regulatory battery. All the negative compounds were also negative in the in vitro micronucleus assay. Among the 14 positive compounds, two of them, positive in the mouse lymphoma assay, were found negative in the in vitro micronucleus test. However, this apparent discordance was likely to be due to cytotoxicity- or high concentration–related false positive responses in the mouse lymphoma assay. In addition, we confirmed that the in vitro micronucleus assay is useful for detecting aneugens, especially, when cells in metaphasis and multinucleated cells are also scored and when cells are allowed to recover after the long treatment. On this series of compounds, the in vitro micronucleus assay showed high sensitivity and possibly a better specificity than the mouse lymphoma assay. Thus, the in vitro micronucleus assay was shown to be at least as adequate as the mouse lymphoma assay or the in vitro chromosome aberration test to be used in the standard genotoxicity battery.
Key Words: genotoxicity; in vitro tests; in vitro micronucleus assay.
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INTRODUCTION |
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The in vitro micronucleus test started to be used in genotoxicity in the 1970s. A long experience of this system was first obtained from early studies on human lymphocytes (Fenech et al., 1985, Norppa et al., 1993
, Van Hummelen and Kirsch-Volders, 1992), and now the in vitro micronucleus assay on human lymphocytes is widely used for biomonitoring studies. Since then, due to its simplicity, it has been widely used in genotoxicity testing, especially, as a screening tool for pharmaceutical research. Data from this test have been accumulated through literature (Garriott et al., 2002) and through several collaborative validation studies (Lorge et al., 2006; Matsushima et al., 1999; Von der Hude et al., 2000). These studies have shown the accuracy of the in vitro micronucleus test for detecting clastogens and aneugens and have confirmed that different cells are suitable for scoring micronuclei, including the L5178Y mouse lymphoma cell line, allowing comparisons with the mouse lymphoma assay. A standard protocol was designed at the International Workshops on Genotoxicity Testing (Kirsch-Volders et al., 2000, 2003), and an Organization for Economic Co-operation and Development guideline is now in preparation. It has been shown recently that the in vitro chromosome aberration test and the mouse lymphoma assay was lacking in specificity (Kirkland et al., 2005). The limitations of the in vitro chromosome aberration test have also been pointed out (Hilliard et al., 1998). At present, the need for the in vitro micronucleus assay in the standard International Conference on Harmonisation regulatory minimal battery (ICH, 1997) is addressed, and it was recently suggested that it could be used as an alternative to the chromosome aberration test or the mouse lymphoma assay (COM guidance, 2000; IVGT, 2006). In this paper, we present data collected from in vitro micronucleus assays performed on L5178Y mouse lymphoma cells to assess the advantages of the in vitro micronucleus test compared to the in vitro chromosome aberration test and/or the mouse lymphoma assay. |
MATERIALS AND METHODS |
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The tests were all performed, and data were analyzed according to the most recent recommendations for the in vitro micronucleus assay (Kirsch-Volders et al., 2000
, 2003) and to regulatory guidelines for the tests of the genotoxicity battery (ICH, 1997; OECD, 1997). See supplementary data related to this paper on the Web site. For reasons of confidentiality, compounds are coded in this paper. |
RESULTS OF THE IN VITRO MICRONUCLEUS ASSAY |
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Negative Results
Eleven compounds were found negative in the in vitro micronucleus assay on mouse lymphoma L5178Y cells (Table 1). They had different pharmacological activities and were intended for therapeutic uses as diverse as cardiotherapy, vascular diseases, neurology, and psychiatry. Most of the highest concentrations tested were cytotoxic and covered a range from 3.75 to 5000 µg/ml. This set of compounds was representative of a variety of pharmaceuticals.
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Positive Results
Fourteen compounds were found positive or equivocal in the in vitro micronucleus assay (Table 1). Positive concentrations ranged from 5 to 4690 µg/ml. Three compounds (C, K, and Z) were positive or equivocal in all treatment schedules. Those compounds induced high numbers of micronucleated cells. Three (A, J, and L) were positive only after the 3-h treatments, including one specifically positive without S9 and one specifically positive in the presence of S9. Four compounds (D, G, M, and Q) were positive specifically in the 28-h treatment and three other positive compounds (EB, EC, and ED) were tested only in the 28-h treatment. For these later compounds and compound Q, as well as compound Z positive in all treatment conditions, an increase of multinucleated cells and/or cells in metaphasis was also observed, and they all needed a recovery period after the 28-h treatment to be detected for the induction of micronuclei.
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DISCUSSION |
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Among the 11 negative compounds in the in vitro micronucleus assay, nine were also negative in all other tests used (i.e., at least in the Ames test and in the mouse lymphoma assay for nine compounds). For the two remaining compounds (B and EA), pharmacologically and chemically unrelated, the only positive response was found in the mouse lymphoma assay, and compound EA, also tested in the in vitro chromosome aberration test, was negative. In both cases, the highest concentrations tested were very high (
1000 µg/ml). For compound B, the statistically significant increase in the mutant frequency in the mouse lymphoma assay found in the 24-h treatment at 2000 and 2500 µg/ml remained moderate (387 and 441 mutants per 106 cells vs. 175 in the control, corresponding to an induction factor of 2.2 and 2.5, respectively) and was associated with a severe cytotoxicity (Relative Total Growth of 20 and 14%, respectively). However, the response was confirmed as positive when applying the Global Evaluation Factor (Moore et al., 2003). These concentrations could not be achieved in the in vitro micronucleus assay at 28-h treatment for cytotoxic reasons. This suggests either that the difference in calculation of the cytotoxicity could be the source of discordant results or that the apparent positive result in the mouse lymphoma assay could be a cytotoxicity-related effect observed at very high and nonachievable concentrations in humans. This may imply that the level of cytotoxicity in the mouse lymphoma assay may not be the most adequate. Thus, the relevance of the positive result in the mouse lymphoma assay may be questionable, and with additional negative results on an in vitro chromosome aberration test, this compound could be considered to have a low or no level of concern. Compound EA was repeatedly found positive in the mouse lymphoma assay from 23 to 1500 µg/ml only in the 24-h treatment time without any relationship to the concentration. In the in vitro micronucleus assay, it was found negative after the 3-h treatments and the 28-h treatment with or without a 21-h recovery. This may suggest that the result in the mouse lymphoma assay was a spurious effect due to some partially soluble impurity or some specific issue related to the 24-h incubation time. Eight of the fourteen positive compounds were positive in the in vitro micronucleus assay and in the mouse lymphoma assay at the same treatment times, including two compounds also found slightly positive or equivocal at the 3-h treatments, one in the in vitro micronucleus assay and the other in the mouse lymphoma assay. Positive concentrations were similar in both tests. For four positive compounds in the mouse lymphoma assay (Q, Z, EB, and EC, from two groups of related compounds), a 21-h recovery was found necessary after the long treatment to detect them as positive in the in vitro micronucleus test. However, the number of cells in metaphasis and/or multinucleated cells was increased after the long treatment without recovery. All together, these results strongly suggest an aneugenic mode of action for these compounds. The recovery time allowed cells to get over the mitotic inhibition indicated by the increase in the number in metaphasis and/or in the number of multinucleated cells.
The positive compounds in the in vitro micronucleus test were, when tested, also positive in the chromosome aberration test, except the possible aneugens.
Two compounds (A and D) were positive and equivocal, respectively, in the in vitro micronucleus test although they were negative in the mouse lymphoma assay and in the chromosome aberration test because they induced no structural aberrations. However, both induced polyploidy on human lymphocytes in the chromosome aberration test. A borderline increase in the number of micronucleated cells was also found in mouse bone marrow for compound D. These compounds were presumably aneugens, acting either on mitosis itself or on subsequent processes leading to daughter cells. However, they were not detected in the mouse lymphoma assay. These assays, performed in the 1990s, did not include a 24-h treatment, which was later recognized to help to detect aneugens. Thus, the equivocal response in the in vitro micronucleus test with compound D was not susceptible to being reproduced in the mouse lymphoma assay. The mitotic inhibition due to aneugenic effects may have precluded the detection of these two compounds as positive in the mouse lymphoma assay even with a 24-h treatment. The low concentrations selected on the basis of cytotoxicity were an additional indicator of a possible mitotic inhibition.
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CONCLUSIONS |
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The protocol used, including 3- and 24-h treatments, as recommended (Kirsch-Volders et al., 2003
), was shown to be suitable to accurately detect clastogens and aneugens on L5178Y cells. However, for two sets of compounds, it was found useful to allow cells to recover from the long treatment for 21 h to detect the formation of micronuclei and to record other signs of aneuploidy, like the number of multinucleated cells and/or the number of cells in metaphasis. Although no recovery has been included in the IWGT recommendations for the long treatment in the in vitro micronucleus assay, it may be accurate to revise this in a more flexible way to allow a recovery period for suspected aneugens, which, in our experience, would have been missed without a recovery period after the long treatment. In this study, 84% (21/25) of the results were concordant between the in vitro micronucleus test and the mouse lymphoma assay. The discordant results (4 compounds/25) were probably due to cytotoxicity- or high concentration–related false positive responses for two compounds in the mouse lymphoma assay and the inadequacy of the mouse lymphoma assay to detect aneugens after a 3-h treatment for two other compounds.
Thus, the in vitro micronucleus test gave results as relevant as those in the mouse lymphoma assay. Moreover, it allowed the detection of aneugens and was more accurate with respect to cytotoxicity-related borderline results in the mouse lymphoma assay. The concordance with the general judgment of each compound was raised to 92%.
When calculating the relative predictivity (Kirkland et al., 2005) on this series of compounds, it can be stated that a positive compound in the in vitro micronucleus assay has every chance of being positive in the genotoxicity battery, and a negative compound in the in vitro micronucleus assay is eight times more likely to be anongenotoxic. This illustrates the better specificity of the in vitro micronucleus test compared to the mouse lymphoma assay.
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SUPPLEMENTARY DATA |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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