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1. The COM and COC undertake routine horizon scanning exercises as part of their annual remit (see appended internet links at the end of this statement). The COM identified the use of in-vivo target organ mutagenicity studies as a subject for further consideration. The COM and COC agreed to hold a joint meeting on the use of data derived from in-vivo target organ mutagenicity studies in carcinogen risk assessment. An open meeting of the committees was held on the 9 June 2005. Attendees included a number of external experts who gave presentations and comments to the committees discussions. This brief statement has been drafted to record the main outcomes of the meeting. A full write up is being prepared for publication in a peer review journal. Introduction to current review 2. The interpretation and consequent risk assessment of rodent carcinogenicity data is currently aided by the evaluation of a battery of mutagenicity data.1 However there are examples of compounds for which equivocal data, or lack of concordance between mutagenicity and carcinogenicity data make it difficult to complete the risk assessment. This is of particular importance when the organ in which tumours are found is not one of those assessed during in vivo genotoxicity tests. Recent developments have facilitated the identification of target organ mutagenicity thus offering the potential to more closely define whether tumours seen are attributable to specific mutagenic events.2 Muta®mouse and Big Blue transgenic rodent assay systems 3. An increasing number of rodent carcinogens are being investigated using the Big Blue or Muta™Mouse transgenic systems. (For example the COM and COC have recently evaluated transgenic mutation assays as part of the evaluation of malachite green and leucomalachite green.2 An important contribution of these assays is that any tissue can be evaluated for the presence of mutations following the administration of a chemical by any exposure route. By demonstrating carcinogen target organ mutagenesis, it can then be inferred that conditions are favourable for DNA reactivity therein (eg the occurrence of site-specific metabolism). Site of contact mutagenesis can also be readily studied.4 Additionally, sequencing of both the lac genes and cII is now commonplace and this provides information on the more precise nature of the induced mutations. It is considered that these analyses will contribute to the understanding of target organ tumourigenesis and subsequent risk assessment.5 There are several examples in the published literature of how data from Big Blue or Muta™Mouse have aided carcinogen risk assessment.6-8 The Comet assay 4. The comet assay is now well established as a supplementary assay to the standard battery of genotoxicity tests and can be used to assist in evaluating chemical which have given equivocal results in other in-vivo mutagenicity tests or to investigate the potential mechanisms of tumourigenic responses.9,10 Guidelines and recommendations for performing the assay have recently been developed.11 The principle concerns that may arise in respect of the use of the comet assay are the relevance of the measured endpoint to the carcinogenic process, and the robustness and sensitivity of the method. DNA binding approaches 5. The COM considered the measurement of DNA binding by a number of methods
(eg postlabelling, radioimmunochemical and HPLC/mass spectrometry) in
1996.12 The COM agreed that these methods could provide useful data on
exposure to and uptake of DNA reactive chemicals and metabolites. More
recently the COC has considered the application of Accelerator Mass Spectrometry
(AMS) in the detection of DNA binding in biological samples.13 AMS is
the most sensitive technique for measuring the formation of adducts with
DNA. AMS technology allows the accurate measurement of very low levels
of radiolabelled chemicals (particularly 14C) in biological samples at
around 10-21 to 10-18 mole. The COC considered that one potential application
of AMS was in hazard identification. AMS has provided evidence for a lack
of DNA binding of 2-phenylphenol and its metabolites in rat bladder which
has been important information in concluding a non-genotoxic mechanism
for the carcinogenic effect of 2-phenylphenol and its sodium salt in rat
bladder.14 6. The symposium was attended by Committee members, relevant officials from government agencies and delegates from industry and academia, took the form of introductory presentations followed by round-table discussion groups. A programme was published.15 Overview of presentations 7. Professor John Heddle (York University, Toronto , Canada) gave the first presentation and provided a comprehensive overview of the usefulness of the transgenic mouse and rat mutation assays in carcinogenicity risk assessment. His principal observations were that the transgenes lac I and lac Z, are neutral genes which persist and accumulate mutations linearly over time, ensuring consistency in response which has allowed for the development of optimum protocols, notably the establishment of expression periods. The Committee noted the relevance of persistent mutations in transgenes to the initiation-promotion model of carcinogenesis. 8. Professor David Phillips (Institute of Cancer Research, Sutton) gave a brief synopsis on the use of DNA adduct detection methodologies in providing evidence of genotoxic mechanisms of action. These techniques are constantly being improved. DNA adduct formation represents mutagenic and carcinogenic potential, but it is recognized that the presence of DNA adducts in a particular tissue does not necessarily correlate with tumour induction. 9. Dr Brian Burlinson (HLS) summarized recent developments in the use of the COMET assay, which was gaining in popularity as a second tissue in vivo mutagenicity assay, to supplement the in-vivo bone marrow assay for chromosomal aberrations or micronuclei. One advantage was that a number of tissues including any tumour target organ could be included in any study. It was noted that an draft OECD guideline was currently under consideration. 10. Dr Phil Carthew (Unilever) presented the pathologists view of the usefulness of target organ mutagenicity data. He pointed out that lack of concordance between mutation frequency, DNA adducts and tumour burden indicate a need to understand more definitively the importance of other steps in the carcinogenic process. However, it is anticipated that data from transgenic mutation assays will be able to provide answers to critical questions, principally with view to hypothesis testing and mode of action evaluation. Group discussions 11. Delegates then split into two groups for the ensuing discussions. Group 1 considered methodological developments, robustness, sensitivity and target organ specificity of the assays under scrutiny, whilst Group 2 addressed specifically the use of target organ mutagenicity data in carcinogenicity risk assessment through questions such as ' how can these data be used to understand the aetiology/ pathogenesis of rodent tumours?' and ' are these data likely to be more useful for the evaluation of some tumours types and/or target organs? Conclusions from Group discussions 12. Group 1 (methods) derived conclusions on the use of the assays considered during the symposium with regard to the screening of chemicals for potential in-vivo mutagenesis. It was concluded that the transgenic mutation assays were sufficiently robust for general use and that moves should be made to optimise assay conditions and develop protocol guidelines. There were some concerns regards overall sensitivity and this may be particularly relevant when using unusual tissues or chemicals about which little is known (eg nature of DNA damage, ADME profile). Nevertheless, it was felt that the limitations of transgenic mutation assays were fairly well understood and this added to a general feeling of confidence in their use. However, it was recognised that the expense of conducting the transgenic assays meant that it was likely that the COMET assay would be used more frequently, even though this was more suited to detecting clastogens and potentially less sensitive at identifying point mutagens. The COMET assay was considered to be satisfactorily validated in most tissues. DNA adducts were not considered useful in a risk assessment scenario, and more likely to be of value for weight of evidence approaches to understanding carcinogenic mechanisms. 13. Group 2 (risk assessment) derived conclusions on the use of target organ mutation assays in carcinogen risk assessment, ie as part of mode-of-action assessments. It was concluded that the in-vivo target organ mutagenesis/ genotoxicity assays provided important information for the mode-of-action evaluations for rodent cancer target organs and thus contribute to carcinogen risk assessment. It was considered important to ensure adequate conduct of studies particularly with regard to information on target cell exposure and potential modes of action. In general, positive results which are not due to high dose cytotoxicity, inflammation or reactive oxygen DNA damage support a non-threshold genotoxic mode of action and that negative results need to be interpreted with regard to the sensitivity of the study used. Clearly, concordant results for several in-vivo approaches increase the confidence of conclusions reached. Overall conclusion 14. The COM and COC agreed that data from adequately conducted in-vivo carcinogen target organ mutagenicity and genotoxicity studies (which included information from investigations using transgenic animals, the comet assay, and approaches to measuring DNA binding (eg postlabelling and radiolabel methods)) can provide valuable information of use in the mode-of action of carcinogenic responses seen in rodents. Such studies can provide supporting information for use by regulatory authorities in carcinogen risk assessment on a case-by-case basis.
October 2005
References 1. COM (2000). Guidance on a strategy for testing of chemicals for mutagenicity.
December 2000. http://www.advisorybodies.doh.gov.uk/com/guidance.pdf 3. COM, COC (2004). Joint COM & COC Statement on Mutagenicity and
Carcinogenicity of Malachite Green (MG) and Leucomalachite Green (LMG)
COM/04/S4 & COC/04/S7 - December 2004 http://www.advisorybodies.doh.gov.uk/com/mglmg.htm 4. von Pressentin MdM, Kosinka W, Guttenplan JB (1999). Mutagenensis
induced by oral carcinogens in lacZ (Muta®Mouse) tongue and other
oral tissues. Carcinogenesis, 20 (11), 2167-2170. 5. COM (2005). Discussion paper MUT/05/4. Development of the cII transgenic
mutation assay. Comparison with lacI and lacZ systems. http://www.advisorybodies.doh.gov.uk/pdfs/mut054.pdf 6. Suzuki T, Miyata Y, Saeki K, Kawazoe Y, Hayashi M and Sofuni T (1998).
In-vivo mutagenesis by the hepatocarcinogens quinoline in the lacZ transgenic
mouse: evidence for its in-vivo genotoxicity. Mutat Res, 412 (2), 161-166. 7. Suzuki T, Wang X, Miyata Y, saeki K, Kohara A, Kawazoe Y, Hayashi
M and Sofuni T (2000). Hepatocarcinogen quinoline induces G:C to C:G transversions
in the cII gene in liver of lamda/LacZ transgenic mice (Muta™Mouse).
Mutat Res 456, 73-81. 8. Turner SD, Tinwell H, Piergorsch W, Schmezer P and Ashby J (2001).
The male rat carcinogens limonene and sodium saccharin are not mutagenic
to male Big Blue rats. Mutagenesis, 16 (4), 329-332. 9. Hartmann A, Schumacher M, Plappert-Helbig U, Lowe P, Suter W, and
Mueller L (2004). Use of alkaline in-vivo comet assay for mechanistic
genotoxicity investigations. Mutagenensis, 19 (1), 51-59. 10. Seikhashi K, Yamamoto A, Matusmura Y et al (2002). Comparative investigation
of multiple organs of mice and rats in the comet assay. Mutat Res, 517,
53-74. 11. Hartmann A, Agurell E, Beevers C et al (2003). Recommendations for
conducting the in-vivo alkaline Comet assay. 4th International comet assay
workshop. Mutagenesis, 18 (1), 45-51. 12. Mulholland C, Battershill J and Fielder RJ (1997). Symposium on the
significance of low level exposures to DNA adduct inducing chemicals,
Department of Health, UK, May 1996. Mutagenesis, 12 (4), 305-312. 13. COC (2000). Accelerator mass spectrometry - an aid to carcinogen
risk assessment COC statement COC/00/S2 - May 2000, http://www.advisorybodies.doh.gov.uk/coc/ams.htm 14. COM (2004). Statement on 2-Phenylphenol and its Sodium Salt 15. COM/COC (2005). The use of Target organ mutagenicity data in carcinogenicity
risk assessment. Programme for meeting 9 June 2005. http://www.advisorybodies.doh.gov.uk/pdfs/Prog090605.pdf
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