- Email: [email protected]
Suitable top concentration for tests with mammalian cells: Mouse lymphoma assay workgroup
Mutation Research 723 (2011) 84–86
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
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Suitable top concentration for tests with mammalian cells: Mouse lymphoma assay workgroup Martha M. Moore a,∗ , Masamitsu Honma b , Julie Clements c , Takumi Awogi d , George R. Douglas e , Freddy van Goethem f , Bhaskar Gollapudi g , Aoi Kimura h , Wolfgang Muster i , Mike O’Donovan j , Rita Schoeny k , Shinobu Wakuri l a
Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U. S. Food and Drug Administration, HFT-120, 3900 NCTR Rd., Jefferson, AR, USA National Institute of Health Sciences, Division of Genetics & Mutagenesis, Tokyo, Japan c Covance Laboratories, Ltd., Harrogate, North Yorkshire, United Kingdom d Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan e Mechanistic Studies Division, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, ON, Canada f Janssen, Research and Development, Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium g The Dow Chemical Company, Midland, MI, USA h Shin Nippon Biomedical Laboratories Ltd., Drug Safety Research Laboratories, Kagoshima, Japan i F. Hoffmann-La Roche Ltd., Basel, Switzerland j AstraZeneca R&D, Alderley Park, Macclesfield, Cheshire, United Kingdom k Office of Water, U.S. Environmental Protection Agency, Washington, DC, USA l Hatano Research Institute, Food and Drug Safety Center, Kanagawa, Japan b
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Article history: Received 4 April 2011 Accepted 5 April 2011 Available online 14 April 2011 Keywords: Mouse lymphoma assay Top concentration Thymidine kinase mutation
a b s t r a c t The Mouse Lymphoma Expert Workgroup of the International Workshop for Genotoxicity Tests (IWGT) met in Basel, Switzerland in August of 2009. The Workgroup (WG) was tasked with discussing the appropriate top concentration for non-pharmaceuticals that would be required for the conduct of the mouse lymphoma assay (MLA) when sufficient cytotoxicity [to between 10 and 20% relative total growth (RTG)] has not been attained. The WG approached this task by (1) enumerating the various regulatory decisions/use for MLA data, (2) discussing the appropriate assays to which MLA data and assay performance should be compared and (3) discussing all the proposals put forth concerning the top concentration for non-pharmaceuticals. In addition, one of the members presented a summary of a re-evaluation of the National Toxicology Program MLA data using the IWGT harmonized guidance that was underway as a separate (non IWGT) activity, being conducted by two members of the Expert WG. The WG was asked to vote on each of the various proposals for top concentration for when cytotoxicity is not concentration limiting. While there was general agreement that the top concentration for non-pharmaceuticals should be re-evaluated and likely lowered from the current recommended levels, there was no agreement on a specific new recommendation. Published by Elsevier B.V.
Contents 1. 2. 3. 4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory decisions utilizing MLA data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion of the appropriate in vivo endpoint to which MLA data should be compared . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proposals for top concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Review of older MLA data using the IWGT WG recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
∗ Corresponding author. Tel.: +1 870 543 7050; fax: +1 870 543 7393. E-mail address: [email protected] (M.M. Moore). 1383-5718/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.mrgentox.2011.04.001
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Disclaimer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The Mouse Lymphoma Expert WG of the International Workshop for Genotoxicity Tests (IWGT) met in Basel, Switzerland in August of 2009. The WG has met previously in Washington, D.C., USA in 1999 [1], New Orleans, Louisiana, USA in 2000 [2], Plymouth, England in 2002 [3], Aberdeen, Scotland in 2003 [4] and San Francisco, California, in 2005 [5]. While the WG membership for these various meetings has changed over time, there is a core group of experts that has been involved in all of the meetings, and every meeting has included a balance of individuals from Europe, Japan and the United States. The WG has tackled a number of important mouse lymphoma assay (MLA) issues and has reached consensus on relative total growth (RTG) as the appropriate measure of cytotoxicity for the assay [2], 10–20% RTG as the appropriate maximum cytotoxicity [2], the appropriate strategy for test concentration selection [2], criteria for individual assay acceptance [4], criteria for determining positive and negative responses [4] and the need for a 24 h treatment when the test agent is not positive in the short (3 or 4 h) treatment [5]. In the Basel Meeting, the WG was tasked with discussing the appropriate top concentration for the evaluation of nonpharmaceuticals. This discussion was prompted because of a proposal from the International Committee for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) to lower the top concentration for pharmaceutical safety evaluation to 1 mM or 500 g/ml (whichever is lower) for the in vitro mammalian assays. The WG approached this task by (1) enumerating the various regulatory decisions/use for mouse lymphoma assay (MLA) data, (2) discussing the appropriate assays to which MLA data and assay performance should be compared and (3) discussing all the proposals put forth concerning the top concentration for non-pharmaceuticals. In addition, one of the members presented a summary of a re-evaluation of the National Toxicology Program MLA data using the IWGT harmonized guidance that was underway as a separate (non IWGT) activity, being conducted by two members of the Expert WG [6]. The WG members were asked to vote on each of the various proposals for top concentration when cytotoxicity, solubility, osmolarity or pH is not concentration limiting. Because of the short meeting time and the absence of specific data supporting the various proposals, it was not possible for the WG to reach a consensus. The current paper includes an overview of the discussion and the outcomes of the WG votes for each proposal. 2. Regulatory decisions utilizing MLA data In order to provide a background for discussing the appropriate top concentration for the MLA, the WG outlined the various types of regulatory decisions that involve genetic toxicology data in general and the MLA in particular. In the pharmaceutical arena, preclinical safety evaluation utilizes a recommended battery of genetic toxicology tests, including the MLA as one alternative for the in vitro mammalian tests. While the focus of this evaluation is for detecting potential carcinogens there is also an interest in detecting potential mutagens. In the broader chemical arena, genetic toxicology tests also are used to predict whether chemicals will be carcinogens or mutagens. Some regulatory agencies consider mutation to be an adverse health outcome and have policies that deal with both carcinogens and mutagens. In some cases, even when the rodent cancer or heritable mutation data are available, in vitro mutation assays also are used as a part of the weight of the evidence evaluation as to whether a particular chemical is likely to be a human
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carcinogen or mutagen, and to assist with the probability descriptors. Genetic toxicology information plays a key role in the mode of action evaluation for chemicals that are carcinogens and is also a factor in overall hazard and risk assessments. Pharmaceutical labels and chemical safety data sheets also include a summary of the evidence from genetic toxicology assays. 3. Discussion of the appropriate in vivo endpoint to which MLA data should be compared With the various regulatory uses of MLA data in mind, the WG discussed the appropriate in vivo endpoint(s) against which assay performance should be assessed. While the rodent cancer bioassay is often used as this “gold standard”, the WG emphasized that cancer is a complex multi-step disease and mutation is only one potential factor in cancer etiology. Therefore, it would not be expected that the MLA, or any other genetic toxicology assay, would provide a perfect cancer prediction. Some members of the WG emphasized that a more appropriate endpoint would be in vivo gene mutation (i.e., the transgenic rodent gene mutation assay, for which there is an extensive database [7]). 4. Proposals for top concentration WG members agreed that the current top concentration (10 mM) for testing agents that are not limited by cytotoxicity, solubility, osmolarity or pH needs to be re-considered and further evaluated. WG members identified several proposals for the appropriate top concentration that would be required for the conduct of the MLA when sufficient cytotoxicity (between 10 & 20% RTG) has not been attained. While the focus of this meeting was for all chemicals that might be tested in the MLA for regulatory purposes including both pharmaceuticals and non-pharmaceuticals, the proposal to limit the top concentration to 1 mM or 500 g/ml (whichever is lower) originated from the ICH which deals with guidance for regulating pharmaceuticals. It was observed that pharmaceuticals often have high molecular weights while the average molecular weight of the chemicals that go through testing in the chemical/agrochemical world is approximately 250. To start the discussion, participants, both WG (12 members) and audience (12 individuals), were asked to indicate their support for the ICH proposal for pharmaceuticals to lower top concentration from 10 mM to 1 mM. Overall 67% of the people attending the MLA WG discussion supported lowering the top concentration to 1 mM or 500 ug/ml (whichever is lower) for pharmaceuticals. Following this initial discussion several different proposals were raised concerning the top concentration for non-pharmaceuticals and the WG experts were asked to vote on the proposals. For each proposal the WG members were asked to indicate whether they (1) were ready to support the proposal based on available information or (2) felt that the proposal had merit and should be further developed, investigated and evaluated. The proposals and voting outcomes for the WG members are as follows: Proposal 1 (: Lower top concentration for non-pharmaceuticals to 1 mM or 500 g/ml (whichever is lower)). Although some felt that this is a viable proposition warranting further evaluation, 50% of WG members were ready to endorse this now. Proposal 2 (: Use a top concentration of 1000 g/ml or 10 mM (whichever is lower)). Although only one WG member was ready
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to endorse this now, 50% of WG members considered it is a viable proposition warranting further evaluation. Proposal 3 (: Lower top concentration to 2 mM). Although only one WG member was ready to endorse this now, more than 50% of WG members considered it is a viable proposition warranting further evaluation. Proposal 4 (: For complex mixtures, the top concentration proposed to be 5000 g/ml (or as high as considered appropriate in each case)). Almost 50% of WG members were ready to endorse this now.
compare in vitro results with in vivo mutagenicity data. It is anticipated that the information from such studies will provide a clearer understanding of the performance characteristics of the MLA and other in vitro mammalian tests, and provide a firmer basis for a consensus on the issues discussed in this WG. Funding All authors are funded by their respective organizations. Conflict of interest
5. Review of older MLA data using the IWGT WG recommendations Additional observations included the fact that the MLA WG has established acceptance criteria and data interpretation recommendations, but these criteria have not been generally applied to older published data. While not an official part of the MLA WG activity, it was noted that two members of the MLA WG were currently working on an evaluation of the NTP MLA database and a preliminary summary of that evaluation was presented. Following the IWGT meeting, this analysis has been completed [6] and the full analysis is in preparation for publication. Briefly, there are approximately 1900 experiments for 342 chemicals in the database. Approximately 17% of these experiments meet all of the IWGT MLA acceptance criteria as described by [4]. Lack of colony sizing data and overall low mutant frequencies of the positive control, prevented any chemicals being defined as clearly negative. 92 chemicals were determined to be positive based on their ability to induce mutant frequencies exceeding the global evaluation factor (GEF: 90 × 10−6 ) for the agar version of the assay. Three of these were only positive above 10 mM (the currently recommended top concentration) and therefore would not be considered positive by current standards. It should be noted that approximately 60% of the chemicals previously determined to be positive by the NTP did not meet the GEF and, therefore, were not called positive in this review. 6. Conclusions The WG members completed their discussion by outlining what would be required to reach consensus on any recommended changes to the current top concentration for the MLA. Issues needing further discussion include reaching agreement on the data that is required and the “gold standard” that should be used for the analysis and decision. There needs to be agreement as to what level of concordance is required, if the decision is to be made based on a correlation with a “gold standard” such as cancer or in vivo mutation. It will be necessary to formulate a strategy to obtain the necessary data, and all WG members agreed that the decision should be based on a complete analysis of all the available acceptable data. There are efforts currently under way to reanalyze older data, to retest chemicals that had been tested using older protocols, and
None. Disclaimer The views expressed are those of the authors and do not necessarily reflect the policy of their respective organizations. References [1] M.M. Moore, M. Honma, J. Clements, T. Awogi, G. Bolcsfoldi, J. Cole, B. Gollapudi, K. Harrington-Brock, A. Mitchell, W. Muster, B. Myhr, M. O’Donovan, M.-C. Ouldelhkim, R. San, H. Shimada, L.F. Stankowski Jr., The mouse lymphoma thymidine kinase locus (tk) gene mutation assay, in: International Workshop on Genotoxicity Test Procedures (IWGTP) Workgroup Report, Environ. Mol. Mutagen 35 (2000) 185–190. [2] M.M. Moore, M. Honma, J. Clements, K. Harrington-Brock, T. Awogi, G. Bolcsfoldi, M. Cifone, D. Collard, M. Fellows, K. Flanders, B. Gollapudi, P. Jenkinson, P. Kirby, S. Kirchner, J. Kraycer, S. McEnaney, W. Muster, B. Myhr, M. O’Donovan, J. Oliver, M.-C. Ouldelhkim, K. Pant, R. Preston, C. Riach, R. San, H. Shimada, L.F. Stankowski Jr., Mouse lymphoma thymidine kinase locus gene mutation assay, in: Followup International Workshop on Genotoxicity Test Procedures, New Orleans, LA, April 2000, Environ. Mol. Mutagen 40 (2002) 292–299. [3] M.M. Moore, M. Honma, J. Clements, G. Bolcsfoldi, M. Cifone, R. Delongchamp, M. Fellows, B. Gollapudi, P. Jenkinson, P. Kirby, S. Kirchner, W. Muster, B. Myhr, M. O’Donovan, M.-C. Ouldelhkim, K. Pant, R. Preston, C. Riach, R. San, L.F. Stankowski Jr., A. Thakur, S. Wakuri, I. Yoshimura, Mouse lymphoma thymidine kinase locus gene mutation assay, in: International Workshop on Genotoxicity Test Procedures Workgroup Report, Plymouth, England, Mutat. Res. 540 (2003) 127–140. [4] M.M. Moore, M. Honma, J. Clements, G. Bolcsfoldi, B. Burlinson, M. Cifone, J. Clarke, R. Delongchamp, R. Durward, M. Fellows, B. Gollapudi, S. Hou, P. Jenkinson, M. Lloyd, J. Majeska, B. Myhr, M. O’Donovan, T. Omori, C. Riach, R. San, L.F. Stankowski Jr., A. Thakur, F. Van Goethem, S. Wakuri, I. Yoshimura, Mouse lymphoma thymidine kinase gene,mutation assay, in: Follow-up Meeting of the International Workshop on Genotoxicity Tests 2003 Assay Acceptance Criteria, Positive Controls, and Data Evaluation, Aberdeen, Scotland, Environ. Mol. Mutagen 47 (2006) 1–5. [5] M.M. Moore, M. Honma, J. Clements, G. Bolcsfoldi, B. Burlinson, M. Cifone, J. Clarke, P. Clay, R. Doppalapudi, M. Fellows, B. Gollapudi, S. Hou, P. Jenkinson, W. Muster, K. Pant, D.A. Kidd, E. Lorge, M. Lloyd, B. Myhr, M. O’Donovan, C. Riach, L.F. Stankowski Jr., A.K. Thakur, F. Van Goethem, Mouse lymphoma thymidine kinase mutation assay, in: Meeting of the International Workshop on Genotoxicity Testing, San Francisco, 2005, Recommendations for 24-hr treatment, Mutat. Res. 627 (2007) 36–40. [6] M.R. Schisler, B.B. Gollapudi, M.M. Moore, Evaluation of the mouse lymphoma mutation assay (MLA) data of the U.S. National Toxicology Program (NTP) using International Workshop on Genotoxicity Testing (IWGT) criteria, Environ. Mol. Mutagen 51 (2010) 732. [7] OECD, Detailed review paper on transgenic rodent mutation assays, in: Series on Testing and Assessment Number 103 OECD, Paris, France, July 23, 2009.
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Minireview
Suitable top concentration for tests with mammalian cells: Mouse lymphoma assay workgroup Martha M. Moore a,∗ , Masamitsu Honma b , Julie Clements c , Takumi Awogi d , George R. Douglas e , Freddy van Goethem f , Bhaskar Gollapudi g , Aoi Kimura h , Wolfgang Muster i , Mike O’Donovan j , Rita Schoeny k , Shinobu Wakuri l a
Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U. S. Food and Drug Administration, HFT-120, 3900 NCTR Rd., Jefferson, AR, USA National Institute of Health Sciences, Division of Genetics & Mutagenesis, Tokyo, Japan c Covance Laboratories, Ltd., Harrogate, North Yorkshire, United Kingdom d Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan e Mechanistic Studies Division, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, ON, Canada f Janssen, Research and Development, Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium g The Dow Chemical Company, Midland, MI, USA h Shin Nippon Biomedical Laboratories Ltd., Drug Safety Research Laboratories, Kagoshima, Japan i F. Hoffmann-La Roche Ltd., Basel, Switzerland j AstraZeneca R&D, Alderley Park, Macclesfield, Cheshire, United Kingdom k Office of Water, U.S. Environmental Protection Agency, Washington, DC, USA l Hatano Research Institute, Food and Drug Safety Center, Kanagawa, Japan b
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Article history: Received 4 April 2011 Accepted 5 April 2011 Available online 14 April 2011 Keywords: Mouse lymphoma assay Top concentration Thymidine kinase mutation
a b s t r a c t The Mouse Lymphoma Expert Workgroup of the International Workshop for Genotoxicity Tests (IWGT) met in Basel, Switzerland in August of 2009. The Workgroup (WG) was tasked with discussing the appropriate top concentration for non-pharmaceuticals that would be required for the conduct of the mouse lymphoma assay (MLA) when sufficient cytotoxicity [to between 10 and 20% relative total growth (RTG)] has not been attained. The WG approached this task by (1) enumerating the various regulatory decisions/use for MLA data, (2) discussing the appropriate assays to which MLA data and assay performance should be compared and (3) discussing all the proposals put forth concerning the top concentration for non-pharmaceuticals. In addition, one of the members presented a summary of a re-evaluation of the National Toxicology Program MLA data using the IWGT harmonized guidance that was underway as a separate (non IWGT) activity, being conducted by two members of the Expert WG. The WG was asked to vote on each of the various proposals for top concentration for when cytotoxicity is not concentration limiting. While there was general agreement that the top concentration for non-pharmaceuticals should be re-evaluated and likely lowered from the current recommended levels, there was no agreement on a specific new recommendation. Published by Elsevier B.V.
Contents 1. 2. 3. 4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory decisions utilizing MLA data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion of the appropriate in vivo endpoint to which MLA data should be compared . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proposals for top concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Review of older MLA data using the IWGT WG recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
∗ Corresponding author. Tel.: +1 870 543 7050; fax: +1 870 543 7393. E-mail address: [email protected] (M.M. Moore). 1383-5718/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.mrgentox.2011.04.001
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Disclaimer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The Mouse Lymphoma Expert WG of the International Workshop for Genotoxicity Tests (IWGT) met in Basel, Switzerland in August of 2009. The WG has met previously in Washington, D.C., USA in 1999 [1], New Orleans, Louisiana, USA in 2000 [2], Plymouth, England in 2002 [3], Aberdeen, Scotland in 2003 [4] and San Francisco, California, in 2005 [5]. While the WG membership for these various meetings has changed over time, there is a core group of experts that has been involved in all of the meetings, and every meeting has included a balance of individuals from Europe, Japan and the United States. The WG has tackled a number of important mouse lymphoma assay (MLA) issues and has reached consensus on relative total growth (RTG) as the appropriate measure of cytotoxicity for the assay [2], 10–20% RTG as the appropriate maximum cytotoxicity [2], the appropriate strategy for test concentration selection [2], criteria for individual assay acceptance [4], criteria for determining positive and negative responses [4] and the need for a 24 h treatment when the test agent is not positive in the short (3 or 4 h) treatment [5]. In the Basel Meeting, the WG was tasked with discussing the appropriate top concentration for the evaluation of nonpharmaceuticals. This discussion was prompted because of a proposal from the International Committee for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) to lower the top concentration for pharmaceutical safety evaluation to 1 mM or 500 g/ml (whichever is lower) for the in vitro mammalian assays. The WG approached this task by (1) enumerating the various regulatory decisions/use for mouse lymphoma assay (MLA) data, (2) discussing the appropriate assays to which MLA data and assay performance should be compared and (3) discussing all the proposals put forth concerning the top concentration for non-pharmaceuticals. In addition, one of the members presented a summary of a re-evaluation of the National Toxicology Program MLA data using the IWGT harmonized guidance that was underway as a separate (non IWGT) activity, being conducted by two members of the Expert WG [6]. The WG members were asked to vote on each of the various proposals for top concentration when cytotoxicity, solubility, osmolarity or pH is not concentration limiting. Because of the short meeting time and the absence of specific data supporting the various proposals, it was not possible for the WG to reach a consensus. The current paper includes an overview of the discussion and the outcomes of the WG votes for each proposal. 2. Regulatory decisions utilizing MLA data In order to provide a background for discussing the appropriate top concentration for the MLA, the WG outlined the various types of regulatory decisions that involve genetic toxicology data in general and the MLA in particular. In the pharmaceutical arena, preclinical safety evaluation utilizes a recommended battery of genetic toxicology tests, including the MLA as one alternative for the in vitro mammalian tests. While the focus of this evaluation is for detecting potential carcinogens there is also an interest in detecting potential mutagens. In the broader chemical arena, genetic toxicology tests also are used to predict whether chemicals will be carcinogens or mutagens. Some regulatory agencies consider mutation to be an adverse health outcome and have policies that deal with both carcinogens and mutagens. In some cases, even when the rodent cancer or heritable mutation data are available, in vitro mutation assays also are used as a part of the weight of the evidence evaluation as to whether a particular chemical is likely to be a human
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carcinogen or mutagen, and to assist with the probability descriptors. Genetic toxicology information plays a key role in the mode of action evaluation for chemicals that are carcinogens and is also a factor in overall hazard and risk assessments. Pharmaceutical labels and chemical safety data sheets also include a summary of the evidence from genetic toxicology assays. 3. Discussion of the appropriate in vivo endpoint to which MLA data should be compared With the various regulatory uses of MLA data in mind, the WG discussed the appropriate in vivo endpoint(s) against which assay performance should be assessed. While the rodent cancer bioassay is often used as this “gold standard”, the WG emphasized that cancer is a complex multi-step disease and mutation is only one potential factor in cancer etiology. Therefore, it would not be expected that the MLA, or any other genetic toxicology assay, would provide a perfect cancer prediction. Some members of the WG emphasized that a more appropriate endpoint would be in vivo gene mutation (i.e., the transgenic rodent gene mutation assay, for which there is an extensive database [7]). 4. Proposals for top concentration WG members agreed that the current top concentration (10 mM) for testing agents that are not limited by cytotoxicity, solubility, osmolarity or pH needs to be re-considered and further evaluated. WG members identified several proposals for the appropriate top concentration that would be required for the conduct of the MLA when sufficient cytotoxicity (between 10 & 20% RTG) has not been attained. While the focus of this meeting was for all chemicals that might be tested in the MLA for regulatory purposes including both pharmaceuticals and non-pharmaceuticals, the proposal to limit the top concentration to 1 mM or 500 g/ml (whichever is lower) originated from the ICH which deals with guidance for regulating pharmaceuticals. It was observed that pharmaceuticals often have high molecular weights while the average molecular weight of the chemicals that go through testing in the chemical/agrochemical world is approximately 250. To start the discussion, participants, both WG (12 members) and audience (12 individuals), were asked to indicate their support for the ICH proposal for pharmaceuticals to lower top concentration from 10 mM to 1 mM. Overall 67% of the people attending the MLA WG discussion supported lowering the top concentration to 1 mM or 500 ug/ml (whichever is lower) for pharmaceuticals. Following this initial discussion several different proposals were raised concerning the top concentration for non-pharmaceuticals and the WG experts were asked to vote on the proposals. For each proposal the WG members were asked to indicate whether they (1) were ready to support the proposal based on available information or (2) felt that the proposal had merit and should be further developed, investigated and evaluated. The proposals and voting outcomes for the WG members are as follows: Proposal 1 (: Lower top concentration for non-pharmaceuticals to 1 mM or 500 g/ml (whichever is lower)). Although some felt that this is a viable proposition warranting further evaluation, 50% of WG members were ready to endorse this now. Proposal 2 (: Use a top concentration of 1000 g/ml or 10 mM (whichever is lower)). Although only one WG member was ready
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to endorse this now, 50% of WG members considered it is a viable proposition warranting further evaluation. Proposal 3 (: Lower top concentration to 2 mM). Although only one WG member was ready to endorse this now, more than 50% of WG members considered it is a viable proposition warranting further evaluation. Proposal 4 (: For complex mixtures, the top concentration proposed to be 5000 g/ml (or as high as considered appropriate in each case)). Almost 50% of WG members were ready to endorse this now.
compare in vitro results with in vivo mutagenicity data. It is anticipated that the information from such studies will provide a clearer understanding of the performance characteristics of the MLA and other in vitro mammalian tests, and provide a firmer basis for a consensus on the issues discussed in this WG. Funding All authors are funded by their respective organizations. Conflict of interest
5. Review of older MLA data using the IWGT WG recommendations Additional observations included the fact that the MLA WG has established acceptance criteria and data interpretation recommendations, but these criteria have not been generally applied to older published data. While not an official part of the MLA WG activity, it was noted that two members of the MLA WG were currently working on an evaluation of the NTP MLA database and a preliminary summary of that evaluation was presented. Following the IWGT meeting, this analysis has been completed [6] and the full analysis is in preparation for publication. Briefly, there are approximately 1900 experiments for 342 chemicals in the database. Approximately 17% of these experiments meet all of the IWGT MLA acceptance criteria as described by [4]. Lack of colony sizing data and overall low mutant frequencies of the positive control, prevented any chemicals being defined as clearly negative. 92 chemicals were determined to be positive based on their ability to induce mutant frequencies exceeding the global evaluation factor (GEF: 90 × 10−6 ) for the agar version of the assay. Three of these were only positive above 10 mM (the currently recommended top concentration) and therefore would not be considered positive by current standards. It should be noted that approximately 60% of the chemicals previously determined to be positive by the NTP did not meet the GEF and, therefore, were not called positive in this review. 6. Conclusions The WG members completed their discussion by outlining what would be required to reach consensus on any recommended changes to the current top concentration for the MLA. Issues needing further discussion include reaching agreement on the data that is required and the “gold standard” that should be used for the analysis and decision. There needs to be agreement as to what level of concordance is required, if the decision is to be made based on a correlation with a “gold standard” such as cancer or in vivo mutation. It will be necessary to formulate a strategy to obtain the necessary data, and all WG members agreed that the decision should be based on a complete analysis of all the available acceptable data. There are efforts currently under way to reanalyze older data, to retest chemicals that had been tested using older protocols, and
None. Disclaimer The views expressed are those of the authors and do not necessarily reflect the policy of their respective organizations. References [1] M.M. Moore, M. Honma, J. Clements, T. Awogi, G. Bolcsfoldi, J. Cole, B. Gollapudi, K. Harrington-Brock, A. Mitchell, W. Muster, B. Myhr, M. O’Donovan, M.-C. Ouldelhkim, R. San, H. Shimada, L.F. Stankowski Jr., The mouse lymphoma thymidine kinase locus (tk) gene mutation assay, in: International Workshop on Genotoxicity Test Procedures (IWGTP) Workgroup Report, Environ. Mol. Mutagen 35 (2000) 185–190. [2] M.M. Moore, M. Honma, J. Clements, K. Harrington-Brock, T. Awogi, G. Bolcsfoldi, M. Cifone, D. Collard, M. Fellows, K. Flanders, B. Gollapudi, P. Jenkinson, P. Kirby, S. Kirchner, J. Kraycer, S. McEnaney, W. Muster, B. Myhr, M. O’Donovan, J. Oliver, M.-C. Ouldelhkim, K. Pant, R. Preston, C. Riach, R. San, H. Shimada, L.F. Stankowski Jr., Mouse lymphoma thymidine kinase locus gene mutation assay, in: Followup International Workshop on Genotoxicity Test Procedures, New Orleans, LA, April 2000, Environ. Mol. Mutagen 40 (2002) 292–299. [3] M.M. Moore, M. Honma, J. Clements, G. Bolcsfoldi, M. Cifone, R. Delongchamp, M. Fellows, B. Gollapudi, P. Jenkinson, P. Kirby, S. Kirchner, W. Muster, B. Myhr, M. O’Donovan, M.-C. Ouldelhkim, K. Pant, R. Preston, C. Riach, R. San, L.F. Stankowski Jr., A. Thakur, S. Wakuri, I. Yoshimura, Mouse lymphoma thymidine kinase locus gene mutation assay, in: International Workshop on Genotoxicity Test Procedures Workgroup Report, Plymouth, England, Mutat. Res. 540 (2003) 127–140. [4] M.M. Moore, M. Honma, J. Clements, G. Bolcsfoldi, B. Burlinson, M. Cifone, J. Clarke, R. Delongchamp, R. Durward, M. Fellows, B. Gollapudi, S. Hou, P. Jenkinson, M. Lloyd, J. Majeska, B. Myhr, M. O’Donovan, T. Omori, C. Riach, R. San, L.F. Stankowski Jr., A. Thakur, F. Van Goethem, S. Wakuri, I. Yoshimura, Mouse lymphoma thymidine kinase gene,mutation assay, in: Follow-up Meeting of the International Workshop on Genotoxicity Tests 2003 Assay Acceptance Criteria, Positive Controls, and Data Evaluation, Aberdeen, Scotland, Environ. Mol. Mutagen 47 (2006) 1–5. [5] M.M. Moore, M. Honma, J. Clements, G. Bolcsfoldi, B. Burlinson, M. Cifone, J. Clarke, P. Clay, R. Doppalapudi, M. Fellows, B. Gollapudi, S. Hou, P. Jenkinson, W. Muster, K. Pant, D.A. Kidd, E. Lorge, M. Lloyd, B. Myhr, M. O’Donovan, C. Riach, L.F. Stankowski Jr., A.K. Thakur, F. Van Goethem, Mouse lymphoma thymidine kinase mutation assay, in: Meeting of the International Workshop on Genotoxicity Testing, San Francisco, 2005, Recommendations for 24-hr treatment, Mutat. Res. 627 (2007) 36–40. [6] M.R. Schisler, B.B. Gollapudi, M.M. Moore, Evaluation of the mouse lymphoma mutation assay (MLA) data of the U.S. National Toxicology Program (NTP) using International Workshop on Genotoxicity Testing (IWGT) criteria, Environ. Mol. Mutagen 51 (2010) 732. [7] OECD, Detailed review paper on transgenic rodent mutation assays, in: Series on Testing and Assessment Number 103 OECD, Paris, France, July 23, 2009.