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Mouse Lymphoma Thymidine Kinase Gene Mutation Assay: Follow-Up International Workshop on Genotoxicity Test Procedures, N e w Orleans, Louisiana, April 2 0 0 0 \ M a r t h a M . M o o r e , 1 * Masamitsu H o n m a , 2 Julie Clements, 3 Karen Harrington-Brock, Takumi A w o g i , 5 G e o r g e Bolcsfoldi, 6 r ^ M a r i a Cifone, 7 D e b o r a h C o l l a r d , 4 Michael Fellows, 8 K a l h r y n Flanders, 7 ^ Bhaskar G o l l a p u d i , 9 . P e t e r Jenkinson, 1 0 Paul K i r b y , 1 1 Stephan Kirchner, 1 2 Joann Kraycer, 1 ^ S t e p h e n M c E n a n e y , 3 W o l f g a n g Muster, 1 2 Brian M y h r , 7 * * \ M k h a e f O ' D o n o v W ^ J o Oliver,14 Marie-Claude Ouldelhkim,15 N c a m a l a Pant, 1 1 Robert Preston, 1 * Colin R i a c h , 1 7 * * Richard S a n , 1 8 Hiroyctsu S h i m a d a , a n d Leon F. Stankowski, Jr. 7 1 National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas 2 Nationa! Institute of Health Sciences, Division of Genetics & Mutagenesis, Tokyo, Japan 3 Covance Laboratories, Ltd., Harrogate, North Yorkshire, United Kingdom ^National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 5 Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan 6 Genetic Toxicology, Safety Assessment, AstraZeneca, R&D, Sodertalje, Sweden 7 Covance Laboratories, Inc., Vienna, Virginia 8 Safety Assessment, AstraZeneca, R&D Charnwood, Loughborough, United Kingdom 9 The Dow Chemical Company, Health and Environmental Research Laboratory, Midland, Michigan 10 Safepharm Laboratories Ltd., Derby, United Kingdom 17 Sitek Research Laboratories, Rockville, Maryland '2F. Hoffmann-La Roche Ltd., Basel, Switzerland 13 Calvert Preclinical Services Corporation, Olyphant, Pennsylvania u GlaxoWellcome R&D, Ware, Hertfordshire, United Kingdom '^Aventis, Paris, France 16 Johnson & Johnson Pharmaceutical Research and Development, Spring House, Pennsylvania lz lnveresk Research, Tranent, Scotland ,8 BioReliance Corporation, Rockville, Maryland 19 Developent Research Laboratories, Daiichi Pharmaceutical Co., Ltd., Edogawa-ku, Tokyo, Japan \ The Mouse Lymphoma Assay (MLA) Workgroup of -the International Workshop on Genotoxicity Test Procedures held a second harmonization meeting just prior-to the U.S. Environmental Mutagen Society Meeting in New Orleans, LA, in April 2000. The discussion focused on several important aspects of the MLA, including; 1) cytotoxicity measures and their determination, 2] use of a 24-hr treatment, 3) the ability of the assay to detect aneugens, and 4] concentration selection. Prior to the meeting the group developed Microsoft Excel Workbooks for data entry.'Ten laboratories entered their data into the workbooks (primarily as coded chemicals). The Excel Workbooks were used to facilitate data analysis by generating an extensive set of graphs that were evaluated by the meeting participants. Based on the Workgroup's previous agreement that a single cytotoxicity measure should be established for both the microwell and.soft

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agar versions of the assay, the Workgroup analyzed the submitted data and unanimously agreed that the relative total growth (RTG) should be used as the cytotoxicity measure for concentration selection and data evaluation. The Workgroup also agreed that the various cytotoxicity measures should be calculated using the same methods regardless of whether the soft ^ agar or microwell version of the assay was used. In 1 the absence of sufficient data to make a definitive • .„_ determination, the Workgroup continued to endorse < : the International Committee on Harmonization recom- \ mendation for the use of 24-hr treatment and made some specific 24-hr treatment protocol recommendations. The Workgroup recognized the ability of the MLA to detect at least some aneugens and also developed general guidance and requirements for appropriate concentration selection. Environ. Mol. Mutagen. 40:292-299, 2002. © 2002 Wiley-Liss, Inc.

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INTRODUCTION The mouse lymphoma assay (MLA) using the thymidine kinase (Tk) gene is the most widely used of the various in vitro mammalian cell gene mutation assays. There are currently two equally acceptable methodologies for performing the assay, one using soft agar medium for cloning and enumeration of mutants [Turner et al., 1984], and the other using liquid medium and 96-well microwell plates [Cole et al., 1983, 1986]. While these two methods are basically the same, there are some differences in how mutant frequencies are derived and also in the way that cytotoxicity has been defined. Because of the importance of the MLA in genotoxicity assessment, it is necessary that an internationally harmonized guideline be established for the conduct of and interpretation of data from the assay. The second International Workshop on Genotoxicity Test Procedures (IWGTP) was held in Washington, DC, in the spring of 1999. The Mouse Lymphoma Assay (MLA) Workgroup, comprised of experts from Japan, Europe, and the United States, reached consensus on a number of important issues and also developed a strategy for data analysis, follow-up discussion, and the development of further recommendations [Moore et al., 2000]. The panel identified three main areas requiring further evaluation and discussion. These included: 1) the conduct of a data-based analysis to result in a final recommendation for the cytotoxicity measure; 2) the issues related to the International Committee for Harmonization (ICH) recommended use of a 24-hr treatment time (including the ability of the assay to detect aneugens); and 3) the criteria for data acceptability, variability and statistical analysis. A follow-up meeting by the MLA Workgroup was held in April 2000, in New Orleans, LA. For this second workgroup meeting, in addition to the official IWGTP workgroup members from the Washington meeting, a number of additional MLA experts were invited to provide data for decision-making and to participate in the discussion, development of the consensus, and in the publication. For the 2000 Workshop we focused on the first two issues (see above). In addition, recognizing the importance of test agent concentration selection in the conduct of optimal mouse lymphoma

•Correspondence tor Martha M, Moore, NCTR/DGRT, HFT-120, 3900 NCTR Road, Jefferson, AR 72079. E-mail: [email protected] **Unfortunately, Brian Myhr and Colin Riach were unable to participate in the workshop discussions. They did, however, provide laboratory data for the workshop and provided comments prior to the meeting and reviewed and contributed to the manuscript. Received 24 June 2002; provisionally accepted 19 August 2002; and in final form 17 September 2002 DOI 10.1002/em.l0122


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assays the Workgroup discussed and reached consensus on the important aspects of concentration selection. In this article, we report the consensus reached during the 2000 MLA Workgroup meeting. WORKGROUP APPROACH To provide a scientific basis for evaluation, discussion, and decision-making, the Workgroup requested data from 10 laboratories conducting either the agar or the microwell versions of the MLA. To facilitate analysis of the data, and the graphical presentation of the data, a workbook for entering primary MLA data was developed using the software program Microsoft Excel. Data for approximately 550 experiments evaluating approximately 170 chemicals were submitted and approximately 1,200 graphs generated for analysis. For proprietary reasons, the identity of many of the chemicals is not shown. The specific issues, the analyses, the discussions, and the consensus statements are described below. CYTOTOXICITY The agar and microwell versions of the MLA have evolved to include several important differences in the methods used to assess cytotoxicity. These differences are shown schematically in Figure 1. Both methods use a 3- or 4-hr treatment time, after which the chemical is removed by centrifugation and the cells are resuspended in fresh medium. For the agar method, the first cell count takes place approximately 24 hr after the initiation of the chemical exposure and the cell density for each culture is readjusted, generally to 0.2 or 0.3 X 106 cells per ml of medium. Treated cultures with densities less than 0.2 or 0.3 X 10 6 cells per ml of medium are generally not adjusted in their density and usually have sustained too much cytotoxicity to carry through the full experiment for mutant enumeration. For each treatment culture, the relative suspension cell growth (compared to control) is calculated. On the second day following treatment the cultures are again counted, adjusted in density, and prepared to clone for mutant enumeration. The total 2-day suspension growth of each culture is calculated and each treated culture is compared to the control. This value is referred to as the relative suspension growth (RSG). Cultures are cloned in soft agar medium with and without a selective agent to enumerate mutants and to calculate the mutant frequency (number of mutants per 10 6 cloneabte cells). The relative plating efficiency in soft agar medium for each culture is determined (relative to the negative control) and multiplied by the RSG to obtain a relative total growth (RTG). The RTG is generally used in the agar method as the measure of survival [Clive and Spector, 1975] . In the microwell procedure, there are two methods used. In Method 1 (see Fig. 1), a cell count is taken immediately

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P/Number of cells plated per well. The mutant frequency is then calculated: MF = [PE(mutant)/PE(viable)] X 10 6 . The Agar Method: RTG can be calculated by multiplying the RSG by the relative PE. As described above, there is one very significant difference between the agar method and microwell Method I. Microvjell Method 1: This difference impacts the calculation of the RSG and RTG. In the agar method, the first cell count and adjustment to cell density takes place the day following treatment. Therefore, the RSG and the RTG, in the agar method, are calculated to include any differences that may occur in cell growth between the chemically treated and control cultures. These differences are determined from the initiation of the treatment through the cloning phase of the experiment. However, in the microwell Method 1 the cultures are adjusted in density following treatment and the RS, RSG, and RTG are calculated using the relative cell growth and platFig. 1. Schematic outline for the agar method and the two versions of the ing efficiency that occurs following the end of the treatment. microwell method. The three methods differ in how the cells are handled In other words, any differential growth that occurs between immediately following treatment. In the agar method, the cells are washed free of test, compound, and resuspended in fresh medium and allowed to the negative controls and the treatment cultures during the grow until the next day. In microwell Method 1, the cells are washed free treatment phase of the assay is not factored into the calcuof test compound and the density is adjusted when the cells are resus- lation for microwell Method 1. pended in fresh medium. A sample of each cell culture is then plated for In microwell Method 2 (Fig. 1), the cell density is not plating efficiency and calculation of the relative survival (RS). In microadjusted in the cultures immediately following treatment well Method 2 the treated cell cultures are washed free of test compound and a sample taken for RS without adjusting the cell density. For all three and the calculated RSG and RTG values do include all of methods the cell cultures are counted and density adjusted on days 1 and the growth from the start of the treatment. That is, for 2. For all three methods the relative suspension growth (RSG) is calculated microwell Method 2 the RSG and RTG values are the same as the 2-day cell culture growth relative to the control culture. Cloning for as those obtained in the agar method. the enumeration of mutant frequency is conducted following the day-2 cell It should be noted that for both agar and microwell density adjustment, using either the soft agar or microwell procedure. For all three methods the relative total growth (RTG) is calculated according to method 24-hr treatments, the cultures should be counted and the methods of Clive and Spector [1975] and includes both the RSG and adjusted at the end of the 24-hr treatment period. the relative plating efficiency at the time of cloning for mutant quantitation. The first issue addressed by the Workgroup was to decide If microwell Method 1 is used, the RS, RSG and RTG must be adjusted by if users of microwell Method 1 (as shown in Fig. 1) must the relative culture growth during the treatment phase. adjust their RS, RSG, and RTG values to include the differential growth that can occur during treatment. This adafter the treatment, wash, and resuspension and the cell justment should be made by comparing the cell density in density of each culture is adjusted. A sample is then taken each treated culture with that of the negative control immefrom each culture and plated at low density, in 96-well diately following treatment. By comparing the growth of plates, to determine a plating efficiency. It is this value, the each treated culture relative to the control, it is possible to relative survival (RS), that has generally been used as the calculate a relative growth during treatment factor that can cytotoxicity measure for the microwell method. Just as in then be used to adjust the RS, RSG, and RTG obtained in the agar method, the cultures are counted and the density the rest of the experiment. As an example, if following the adjusted both 1 and 2 days following the initiation of treatment period the negative control has a cell density of treatment. Following the 2-day expression period, an RSG 0.6 X ] 0 6 cells/ml and the treated culture has a density of is calculated (based on the cell growth from the end of the 0.3 X 10 6 cells/ml, then the relative growth during treattreatment to the second day posttreatment). To determine ment for that treated culture is 0.5 (or 50%). If the RS for mutant frequencies, cultures are plated in 96-well plates, that culture is 0.4, then the adjusted RS is the RS multiplied with and without trifluorothymidine (TFT) selection. In the by the relative growth during treatment or 0.4 X 0.5 = 0.20 microwell method the plating efficiency and the mutant (or 20%). The RSG is adjusted in the same manner. The frequency are calculated using the Poisson distribution. The adjusted RTG is obtained by multiplying the adjusted RSG plating efficiency (PE) in both the mutant selection plates by the relative plating efficiency at the time of mutant and the viability plates is calculated as follows: From the selection. zero term of the Poisson distribution, the probable number As a first step for this discussion, the Workgroup used the of clones/well (P) is equal to the -In (EW/TW), where Microsoft Excel Workbook to generate the adjusted RS, EW = empty wells and TW = total wells. The PE = RSG, and RTG values for the microwell data and graphi-


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Fig. 3. Comparison of the relative survival (RS) and the adjusted RS for 12 experiments conducted in different laboratories using the microwell method and 24-hr treatment. Nonproprietary chemicals are identified on the graph.

Fig, 5. Comparison of the relative suspension growth (RSG) and the adjusted RSG for 12 experiments conducted in different laboratories using the microwell method and 24-hr treatment. Nonproprietary chemicals are identified on the graph.

cally compared the nonadjusted and adjusted values. Graphs were generated using the same numerical values on each axis so that if there were no differences between the nonadjusted and the adjusted values the relationship would be linear and fall on a 45° angle. It should be noted that the majority of the data supplied was for coded chemicals. Nonproprietary chemicals are identified on the individual graphs. As can be seen in Figures 2-7, for some chemicals the adjusted values are similar to the unadjusted values but in other cases there are dramatic differences between the

two. The graphs shown in Figures 2-7 are representative of the patterns seen in the larger dataset. Based on this analysis, the Workgroup unanimously agreed with the need to account for the fact that the treated cultures often do not attain the same final cell density as the control culture during the treatment phase of the assay. Therefore, laboratories must base their RS, RSG, and RTG on the relative growth/plating efficiency that occurs in all phases of the assay, including the treatment phase. For laboratories that count cells and


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adjust cell density following treatment (microwell Method 1), this requires that the relative growth during treatment be included in the RS, RSG, and RTG value. That is, these laboratories need to adjust their RS, RSG, and RTG values as described above. During the 1999 meeting the Workgroup unanimously agreed that there should be one standard cytotoxicity measure for describing the dose response and the highest concentration required for defining a negative response. At that time, the group could not unanimously agree on the measure


Fig. 8. Comparison of the RSG (squares) and RTG (diamonds) as measures to define the cytotoxicity component of the mutant frequency (MF) dose response. Experiments from different laboratories using the agar version of the assay and 3- or 4-hr treatment. Nonproprietary chemicals are identified on the graph.

to use. While valid arguments can be made for both the RS and the RTG as cytotoxicity measures, the decision was complicated by the fact that the agar method is not usually performed in a manner that obtains an RS value. As an additional option, the proposal was raised and the group agreed to investigate the use of the RSG as the cytotoxicity measure. Using the data obtained from the various laboratories a large number of graphs were generated. Figures 8-10 show representative patterns of the mutant frequency plotted relative to the different measures of cytotoxicity. For the microwell data the RS, RSG, and RTG values are all adjusted as previously discussed. In some cases the three measures of cytotoxicity are approximately the same and in other cases there are substantial differences. An analysis of Figures 9 and 10 reveals that when there are differences between the values, either the RS or the RTG can be the lowest of the values. It should be noted that the RSG can either be higher or lower than the RS, but it will always be higher than the RTG. Based on the agreed need to select a single measure and an analysis of the data, the Workgroup unanimously agreed that the reported standard cytotoxicity measure should be the RTG. As in the above recommendation, this RTG should be calculated to include the relative growth during treatment. The Workgroup also recognized that other measures of toxicity (e.g., RS and RSG) may be used as supporting data and can often be very useful in identifying possible modes of action for the test chemical. These measures should also be adjusted as outlined above.

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The Workgroup agreed that optimal cell growth during the 24-hr treatment period is critical. We recommend that the serum concentration be sufficiently high to ensure proper growth. The majority of the laboratories find that 5-10% horse serum provides optimal growth. Expression Time

The Workgroup recommended a 2-day expression period following the 24-hr treatment. As noted during the previous meeting, this recommendation is based on relatively limited data and the issue should receive further investigation. Requirement for 2 4 - H o u r Treatment

Fig. 10. Comparison of the RS (diamonds), RSG (squares), and RTG (triangles) (all adjusted as descnbed in the text) as measures to define the cytotoxicity component of the mutant frequency dose response. Experiments from different laboratories using the microwell version of the assay and 24-hr treatment. Nonproprietary chemicals are identified on the graph.

The data available for Workgroup evaluation did not provide a clear basis upon which to judge the need for 24-hr treatment. The Workgroup agreed to continue their endorsement of the ICH statement concerning the need for 24-hr treatment [ICH, 1997]. This ICH recommendation states "a continuous treatment without metabolic activation for approximately 24 hours is needed in case of a negative result for the short treatment without metabolic activation." The Workgroup was concerned that some individuals may interpret this to require a 24-hr treatment without S9 in situations where the short treatment time response is negative without S9 but is positive with S9. Therefore, the Workgroup clarified this recommendation to state that if a positive result was observed in either of the short-term treatments, then a 24-hr treatment should not be required. However, the Workgroup recognizes that there may be situations when a 24-hr treatment may be needed in order to define the profile of the test material. Cytotoxicity Limits

2 4 - H R TREATMENT Cell N u m b e r

The Workgroup recommended that the number of cells used for treatment must provide a minimum of 1 X 107 cells


In the absence of any information to conclude otherwise, the Workgroup recommends that the same cytotoxicity limits apply to short-term and 24-hr treatments. This issue may need to be reviewed as the database for 24-hr treatments increases.

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Detection of Aneuploidy It is clear that the MLA detects at least some aneugens. Positive mutagenicity data was presented to the Workgroup for a number of aneugens, including thiabendazole, carbendazim, griseofulvin (Fig. 10), noscapine (data not shown), and chloral hydrate (Fig. 8). The efficiency of the MLA for detecting aneugens is less clear, however, and requires further investigation. It should be noted that, at least for chloral hydrate, a 24-hr treatment is not required to detect its mutagenicity. CONCENTRATION SELECTION The selection and spacing of the test agent concentrations is a critical factor in the proper conduct of the MLA. It is desirable to have more than one data point that can be used to confirm a positive or negative response. The Workgroup agreed that the assay may be conducted using either single or multiple cultures per concentration point. The strategy for the number of concentrations used, and the selection and spacing of concentrations, can vary based on the toxicity range of the test material being evaluated and the degree to which the chemical does or does not increase mutant frequency. For toxic test materials, the highest concentration level should induce a nominal 80% reduction in RTG. Concentration levels that induce more than a 90% reduction in RTG are usually excluded from the evaluation. However, as noted below, there are circumstances where the data points obtained at less than 10% RTG can be useful in the final evaluation. While it is generally advisable to obtain data points covering the entire 100-10% RTG range, the absolute requirement for attaining this complete dose response varies with the test material being evaluated When a test material induces large increases in mutant frequency, it is generally sufficient to provide data points anywhere within the 1 0 0 10% RTG range. For test materials that are not mutagenic or that induce weak mutagenic responses it is advisable to place emphasis on selecting concentrations that are expected to produce higher toxicity. This increases the probability of obtaining data points that can be used to make a definitive evaluation, that is, data points in the approximately 10-20% RTG range. Therefore, it is normal practice for laboratories to attempt to achieve a maximum concentration with RTG values between 10-20%. However, as already indicated, if a chemical clearly satisfies the criteria for a positive response, there is no reason to require a concentration resulting in 10-20% RTG. The Workgroup unanimously agreed that there are some circumstances under which a chemical may be determined to be nonmutagenic when there is no culture showing an RTG value between 10-20% RTG. These situations are


outlined as follows: 1) There is no evidence of mutagenicity (e.g., no dose response or mutant frequencies above those seen in the historical background ranges) in a series of data points within 100% to 20% RTG and there is at least one negative data point between 20% and 25% RTG. 2) There is no evidence of mutagenicity (e.g., no dose response or mutant frequencies above those seen in the historical background ranges) in a series of data points between 100% to 25% and there is also a negative data point between 10% and 1% RTG. The Workgroup agreed that when the mutant frequency is increasing above the background frequency (yet has not reached a level to be determined positive), it is necessary to attain an RTG within the 10-20% range. This may require a repeat experiment in which the concentration range is modified to increase the probability of attaining datapoints within the 10-20% RTG range. The Workgroup also agreed that significant increases in mutant frequencies seen only at RTG < 1 0 % , but with no evidence of mutagenicity at RTG > 1 0 % , do not constitute a positive result. DATA ACCEPTABILITY CRITERIA, VARIABILITY, STATISTICAL ANALYSIS The Workgroup agreed to continue with the process started at the Washington IWGTP meeting. The major issues to be addressed in the next IWGTP MLA Workshops include establishing data acceptability criteria and discussing the various statistical approaches to data analysis and reaching consensus as to how MLA data should be analyzed. Since the 2000 meeting in New Orleans, the Workgroup collected a large multiple laboratory dataset for negative and positive controls and has used the experiments represented in the Excel Workbook to evaluate a variety of statistical analyses. The Workgroup met in 2001 in conjunction with the U.S. Environmental Mutagen Society Meeting in San Diego, CA, and also met in June 2002 in Plymouth, England, at the Third International Workshop for Genotoxicity Testing. The summary from the Plymouth meeting is currently in preparation. DISCLAIMER This article has been reviewed by the National Center for Toxicological Research of the Food and Drug Administration and the National Health and Environmental Effects Research Laboratory of the U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

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MLA Protocol Harmonization REFERENCES Clive D, Spector JFS. 1975. Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells. Mutat Res 31:17-29 Cole J, Ariett CF, Green MHL, Lowe J, Muriel W. 1983. A comparison of the agar cloning and microtritration techniques for assaying cell survival and mutation frequency in L5178Y mouse lymphoma cells. Mutat Res 111:371-386. Cole J, Muriel WJ, Bridges B A. 1986. The mutagenicity of sodium fluoride to L5178Y (wild-type and TK+'~ 3.7.2C) mouse lymphoma cells. Mutagenesis 1:157-167. ICH. 1997. Topic S2B genotoxicity: a standard battery for genotoxicity testing of pharmaceuticals. International Conference on the Harmonisation of Technical Requirements for Registration of Pharma-


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ceuticals for Human Use. Step 4 Guideline, Brussels, July 1997. (See website http;//wwwifpma,irg.ichl.html) Moore, MM, Honma M, Clements J, Awogi T, Bolcsfoldi G, Cols: J, Gollapudi B, Harrington-Brock K, Mitchell A, Muster W, Myhr M, O'Donovan M, Ouldelhkim M-C, San R, Shimada H, Stankowski LF Jr. 2000. The mouse lymphoma thymidine kinase locus (tk) gene mutation assay: International Workshop on Genotoxicity Test Procedures (IWGTP) Workgroup Report. Environ Mol Mutagen 35:185-190. Turner NT, Batson AG, Clive D. 1984. Procedures for theL5178Y/TK +/ to TK"'" mouse lymphoma assay. In: Kilbey et al., editors. Handbook of mutagenicity test procedures, 2nd ed. Amsterdam: Elsevier. p 239-268.

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