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MINUTESPresent:
CONTENT
1. The Chair welcomed Dr Lesley Hetherington to the secretariat, attending her first meeting, Dr Elsie Damien and Mr Jeremy Tinkler from MHRA who were attending for the discussion on item 4 (Metal-on-Metal, MoM, hip replacements). The chairman welcomed Mr Barry Maycock from FSA who was drafting a paper for the meeting on benzimidazole common mechanism grouping of chemicals. The Chairman noted that this was Dr Karin Fletcher's last meeting before moving to a new post. He thanked her for a number of excellent papers and work on the joint COC/COM symposium. 2. Apologies for absence were received from Dr D Gatehouse, Dr N Gooderham, Dr D Andrew (PSD, Mrs Daniels attending) and Dr H Stemplewski (MHRA). 3. Members were reminded of the need to declare any interests before discussion of items. ITEM 2: MINUTES OF MEETING ON 13 OCTOBER 2005 (MUT/MIN/05/3) 4. Members agreed the minutes subject to some minor editorial changes. The secretariat were asked to check the mutagenicity data regarding tests with furan in bacteria (para 20) and the solubility of zinc oxide in dilute hydrochloric acid (para 31) and to amend the minutes if necessary. ITEM 3: MATTERS ARISING (NOT COVERED BY LATER AGENDA ITEMS):
5. The finalised statement was published on the COM internet site and forwarded to the ACP who agreed the conclusions reached by COM. A manuscript by the DH Toxicology Unit and the secretariat had been submitted to Mutagenesis and had been recommended for publication subject to minor alteration. The chair told members that the former chair of ACP had written to him thanking COM for their advice on biomonitoring and also on proquinazid. ITEM 4: BIOLOGICAL EFFECTS OF WEAR DEBRIS GENERATED FROM METAL ON METAL BEARING SURFACES: EVIDENCE FOR GENOTOXICITY (MUT/06/2) 6. No interests were declared. 7. The Chairman asked Dr Elsie Damien (MHRA) to provide an overview of metal-on-metal (MoM) hip replacement. Dr Damien noted that hip replacement is a highly successful operation which had been undertaken for over 40 years. Key benefits to individuals were increased motion and load bearing with relief from pain. The National Joint Registry (NJR) had documented 50,000 operations (45,000 primary and 5,000 revision) in 2004. There were incomplete data on the reasons for revision operations. In 2005, the NJR had recorded 63,000 hip replacement operations (33,000 in the NHS and 17,000 in the independent sector). The first generation MoM prostheses had unpredictable performance because of inconsistencies in materials and manufacturing. From the 1970's metal-on- polyethylene hip replacements had been used but considerable problems due to wear of the plastic component, the generation of wear debris, resultant periprosthetic osteolysis, asceptic loosening, failure of treatment and consequent need for revision surgery had been reported. In the 1990's the 2nd generation MoM implants had been introduced and over 300,000 hip replacements had been undertaken in the last 10 years. MoM hip resurfacing had been introduced in 1991 as a conservative treatment for use in younger patients where bone and soft tissue were preserved. The National Institute for Clinical Excellence (NICE) had issued guidance to surgeons that less was known regarding the medium to long term reliability and safety of hip resurfacing. 8. Dr Damien noted that in 2004 there were 574 combinations of cup brand and stem brand used in hip replacement surgery. There were 101 brands of femoral stem and 88 different brands of acetabular cup available in 2004. The established problems with MoM hip replacement included wear debris in surrounding tissue, metal sensitivity , enlargement of lymph nodes due to presence of wear debris. Emerging problems included genetic damage (which had been referred to COM), impairment of the immune system, cell death, particulate and ionic wear debris generation. With regard to emerging problems, there was, at present, no proven link with adverse health effects in humans. 9. The Chairman asked for any generic comments before considering the three papers cited in the Committee on Safety of Devices paper on metal hip replacements. Members noted the wide diversity of MoM hip replacement devices in use and agreed that only generic conclusions could be drawn with regard to metals used in such devices. Members raised the subject of patient choice and how this would be exercised given any advice from COM. MHRA considered that most patient choice concerned choice of surgeon and not the type of metal hip replacement device to be used. Members considered that COM advice to MHRA would need to be considered in the context of the benefits associated with hip replacement. MHRA responded that a risk benefit assessment would be undertaken. It was noted that for medical devices adverse effects were often identified post marketing where information on bio-compatibility accumulated. 10. The Chairman asked for specific comments on the three papers identified by the Committee on Safety of Devices.
11. Chromosome translocations and aneuploidy in peripheral blood lymphocytes were compared between a group of revision arthroplasty patients (n=31, mean age=71±13.4y, average implantation time 11.5 years, range= 3-21y) and controls undergoing total hip athroplasty (n=-30, mean age =63.9±12.7y). All patients had osteoarthritis except two at primary arthroplasty. All took non steroidal anti inflammatory medicines (NSAIDs). 11 patients had cobalt-chromium (Co-Cr) prostheses, 13 had titanium- aluminium-vanadium- (TiAlV), six had stainless steel (SS), and one a hybrid titanium- Co/Cr prosthesis. [In a subsequent paper (see para Ladon et al below) it was reported that all these patients had metal-on-polyethylene prostheses.] Adjusted analyses reported a statistically significant five-fold increase in aneuploidy in patients with Ti (without any increase in translocations). In contrast adjusted analyses reported a 2.5 fold increase in aneuploidy and a 3.5 fold increase in translocations in patients with Co-Cr prostheses. No increase in either end point was reported for stainless steel. 12. Members considered that the number of patients included in the study was relatively small and that it would not be possible to draw any definite conclusions regarding differences between types of MoM hip replacement devices from the available results. It was agreed that the analysis using high resolution inductively-coupled mass spectrometry (ICPMS) for concentrations of metals in blood had been adequately undertaken. Members considered that the evaluation of aneuploidy and chromosomal aberrations had been generally adequately reported, although members would be interested to see full details of how the studies were undertaken and reported, so that it would be possible to consider how the aneuploid index was derived and how the results of the non-disjunction assays were reported. Thus it was noted that 300 cells were used in metaphase analysis, but it was not apparent whether this also applied to the detection of non-disjunction or aneuploidy.
13. 95 patients with metal on metal total hip arthroplasty (Metasul®; head and articulation (Co-Cr high carbon), acetabular cup (large cup shaped cavity on the lateral surface of the oscoxae in which the head of the femur articulates); polyethylene; stem Protasul S30 (stainless steel)) were recruited. Patients with existing prostheses, previous radiotherapy, or chemotherapy were excluded. Blood samples (10 ml) were obtained prior to operation (95) and at 6 months (80), 1 year (89), and 2 years (54) post operation. Another 5 ml sample was taken at each time point for trace metal analysis. Cultures were set up within 24 hour of collection. Post operative blood levels of Co-Cr were elevated at 2 years. The highest level of Cr was at 2 years and the highest level of Co was at 1 year. A much smaller but statistically significant increase in Molybdenum was reported at the time points used. There was a statistically significant increase in translocations and aneuploidy at all time points after operation. This was evident if the data from both scorers were combined and if the data from the single scorer of both translocations and aneuploidy (both chromosome gain and loss) were analysed separately. The increase in aneuploidy was much greater than that of chromosome translocation and both were progressive over time. 14. Members agreed that more patients had been studied in this study compared to Doherty et al 2004. The measurement of metal concentrations in blood had been adequately undertaken. Members noted that very few details of the determination of aneugenicity had been reported and agreed that further information should be requested from the authors. The evaluation of chromosomal aberrations had been adequately undertaken and reported. 15. Members noted that the evidence from these two studies supported the involvement of released chromium and cobalt in the observed chromosomal effects associated with MoM hip replacement, although it was not possible on these data to conclude whether this was due to release of soluble ions or particulate metals. The Chairman asked members to consider the available ex-vivo study.
16. This study examined the proposal that there would be metal-specific DNA damage following incubation of synovial fluid from patients undergoing revision arthroplasty. It was considered appropriate to use the Comet assay to measure DNA strand breaks, cross links and alkali labile sites in primary fibroblasts from synovial fluid. 24 Patients were included in the study at revision surgery. There were synovial fluid samples from six patients with Co-Cr MoM hips, six with Co-Cr metal on polyethylene knee replacements, six patients with Stainless Steel hip replacements and six control patients with no hip or knee replacements. 17. Members agreed that the Comet assays had been adequaltely undertaken. All six samples from Co-Cr MoM hip revisions induced a statistically significant increase in DNA damage. Four/six samples from Co-Cr-on-polyethylene knee joints induced statistically significant DNA damage. None of the samples from SS-on-polyethylene prostheses induced statistically significant DNA damage. All samples from osteoarthritic control joints caused a low level but statistically significant increase in DNA damage. 18. The level of Cr in synovial fluid from MoM hips at revision was between 0.95-6.88 µM and Co varied from 0.92-2.64 µM. In the group with Co/Cr-on-polyethylene implants concentrations of chromium varied between 0.07-2.06 µM and those of Co between 0.01-0.62 µM. In the SS implant group, Cr levels were reported to vary between 0.07-2.76 µM whilst Co were below the detection limit in four cases and 0.05 µM in the two other patients. Low but measurable concentrations of Cr were documented in the osteoarthritic group whereas the level of Co was below the limit of detection in all individuals in the osteoarthritic group. It was noted that the authors argued the data were consistent with an interaction between Co and Cr and this would explain why no DNA damage is seen in studies using SS implants. A further reference (De Boeck M et al, Mutagenesis, 18, 177-186, 2003) had been cited by Davies et al to support the proposal that there were metal-metal interactions involved in the aetiology of the observed DNA damage. Members agreed the data suggested a plausible hypothesis but no definite conclusions could be drawn. The secretariat noted that a further study on in-vitro effects of metals extracted from worn hip and knee replacements on micronucleus formation and aneuploidy had been identified but had not yet been retrieved for committee discussion (Daley B et al, J Bone Joint Surg, Br, 86, 598-606, 2004). 19. The Chairman asked members to consider the supporting data submitted. Members noted a preliminary study where there was evidence for a higher incidence in chromosome aberrations in bone marrow samples adjacent to the prosthesis (ie the femur) compared to iliac crest marrow from the same patients (Case CP et al, Clin Orthop Relat Res, 329 (suppl), S269-279, 1996) but agreed that it was unclear from the paper which MoM hip replacements had been studied. A further preliminary report whch had been published in abstract form only (Jenkin L et al, J of Bone and Joint Surgery, 81B (suppl III), 319, 1999) documented a higher incidence of 14:18 translocations in peripheral blood lymphocytes in patients undergoing revision hip arthroplasty. 20. Members commented that the available information suggested that metal-on-metal hip replacement results in elevated blood levels of Co and Cr ions (Milosev et al J Orthop Res, 2005, 23, 526-535, 2005). Post-mortem histological evaluations had shown that widespread metal debris in individuals with SS and Co-Cr implants which could be detected even when there was no apparent wear of the replacement hip. Metal debris was detected in both local and distant lymph nodes, bone marrow, liver and spleen. (Case CP et al J of Bone and Joint Surgery, 76-B, 701-712, 1994.) In a further post-mortem histological evaluation study metallic wear particles were more prevalent in patients who had a failed hip arthroplasty compared with patients with a primary hip or knee replacement. (Urban RM et al, J of Bone and Joint Surgery, 82-A, 457-477, 2000). 21. Members briefly discussed potential mechanisms by which metal ions could induce the observed effects which included effects on DNA repair and fidelity and induction of oxidative DNA damage. 22. The Chairman asked members to consider the questions posed in paragraph 31 of the covering paper. The Committee agreed that there was good evidence for an association between metal-on-metal and metal-on-polyethylene hip replacements and increased genotoxicity in patients. In this respect members noted the consistent negative findings with regard to Stainless Steel implants. The Committee agreed that the evidence for increased blood levels of Chromium and Cobalt which had potential for carcinogenicity in humans gave rise to concern but it was not possible to make any definite conclusions regarding which metal ions or interactions between meta ions or particulate metals might be responsible for the observed responses. In respect of the last question, members considered there was evidence to suggest a possible interaction between chromium and cobalt ions and possible mutagenicity/DNA damage. ITEM 5: A COMPARISON OF THE RELATIVE SENSITIVITIES OF THE IN-VIVO RAT LIVER UDS ASSAY AND THE IN-VIVO COMET ASSAY (MUT/06/3) 23. The COM had requested a discussion paper on the comparison of the in-vivo rat liver UDS assay and the in-vivo COMET assay during the horizon scanning discussion in October 2005. MUT/06/3 had been based on published literature and presented data for 16 compounds, all of which had been shown to be carcinogenic in rodents. The initial focus had been on comparing response in the liver but the data presented had included the response in the COMET assay in other tissues. The evaluation had been complicated as for each compound there were significant differences in the dose levels, routes, species and strain of rodent used between the UDS and COMET studies. In addition most rat liver UDS studies conformed to the OECD guideline whilst there was no equivalent standard for the COMET assays. 24. The Chairman asked for general comments on the approach used and the available studies before reviewing data for individual compounds. 25. Members recalled that the original stimulus for the current work had originated from the discussion at the joint COM/COC target organ mutagenicity meeting held in June 2005 where attendees had expressed reservations over the utility of the UDS assay. A broad interpretation of the current review paper, accepting the results as presented based only on response in rat liver was that there was a good degree of concordance in positive results with six chemicals (aflatoxin, benzidine, 2,4 diaminotoluene, 1,2 dimethylhydrazine, diethylnitrosamine, methylmethane sulphonate), negative results in three chemicals (acrylamide, benzidine and o-anisidine), with discordant results in chorodibromomethane. 26. Members considered that a significant reservation in reaching these conclusions related to the quality of the available COMET assays and in particular the use of isolated nuclei in the COMET assay as reported by Sasaki Y et al (Mutation Research, 391, 215-231, 1997) and Sekihashi K et al (Mutation Research, 517, 53-74, 2002). Members noted that procedures were still being developed for different organs in the COMET assay and hence it was difficult to draw any conclusions on the utility of the assay at the present time. Members also commented that in general intra peritoneal dosing the rat liver UDS and COMET assays could complicate the interpretation of data. HSE commented that it might be possible to obtain information from industry which would be informative with regard to the assessment of the utility of the rat liver UDS and COMET assays. Members also commented that the approach used in the discussion paper was relevant to empirical comparisons between in-vivo mutagenicity assays but that any discussion on overall testing strategy also needed to include consideration of using in-vivo assays in the context of the data provided by the in-vitro assessment of mutagenicity. HSE commented that the COM strategy stage 2 in-vivo assays started with an in-vivo bone marrow assay whilst the EU regulatory approach didn't state which in-vivo assay was to be used first. Members agreed that this would be considered when the COM strategy was reviewed which was expected to be initiated next year. There was discussion on how the in-vivo COMET assay could be used in the context of a general screen for mutagenicity. The secretariat suggested that one possible way forward was to screen the eight most common cancer target organs identified by Gold and colleagues in their Carcinogenicity Potency Database. 27. The Chairman asked members to provide comments on the individual chemicals reviewed. With regard to acrylamide, although the data suggested a negative result for both rat liver UDS and for mouse liver COMET (using ip administration), it was considered, based on the relatively poor results with concurrent positive control (MMS), that the COMET assay had underperformed in this instance. Members commented that positive COMET data were available for glycinamide, the metabolite of acrylamide. It was agreed that the COM statement should make reference to these latter data. Overall it was felt that there was no further need to examine acrylamide in the context of the current review of UDS and COMET assay data. With regard to benzyl acetate it was noted that COMET data identified the carcinogen target organ, namely the bladder, and this suggested added value could be derived from appropriately conducted COMET assays. With regard to benzo(a)pyrene, the Food Standards Agency noted that the discussion paper had not linked the available data on mutagenicity from rat and mice with the differing cancer target organs in these species. This would also be an important aspect in any future consideration of selection of in-vivo mutagencity assays for investigating mode of action of carcinogenic responses seen in rodents. 28. With regard to chlorodibromomethane (a water disinfection by-product), members recalled that the COM and COC had considered this compound, which induced malignant liver tumours in rats, in detail in 1994/5, and had concluded that it was not a genotoxic carcinogen on the basis of adequate negative bone marrow MN assays and rat liver UDS assays. The available COMET data indicated a clear positive result in both rats and mice in the liver. However members expressed reservations regarding the conduct of these assays which used isolated nuclei and considered that a repeat test for rat liver COMET would be appropriate supported, if possible, by a repeat rat liver UDS assay conducted concurrently. With regard to cyclophosphamide, members thought that the rat liver UDS showed some very limited evidence for a positive result, although no definite conclusions could be reached on the basis of the available data. With regard to MNNG, members noted the apparent weak positive result in the rat liver UDS, and clear positive in the mouse liver COMET assay but noted that a clear positive was obtained in bone marrow assays with this compound. Members commented on the added value of the COMET assay to detect cancer target organs whilst reviewing the data on o-anisidine. 29. The Chairman asked members to consider the overall questions posed in MUT/06/3. Members agreed broadly that the available data was consistent with the view that rat liver UDS assay and the rat liver COMET assay had provided similar responses in the available tests with a limited number of known rodent carcinogens. It was agreed that further repeat rat liver COMET assay was necessary for chlorodibromomethane. Members agreed that no further evaluation of acrylamide was required at the present time. 30. The committee was informed that a short statement would be drafted for members consideration. ITEM 6: BACKGROUND VARIATION IN MICRONUCLEI IN PERIPEHRAL BLOOD LYMPHOCYTES: DISCUSSION PAPER ON RISK FACTORS (MUT/06/1) 31. The COM had recently identified the need for further evaluation of the factors affecting the formation of micronuclei in peripheral blood lymphocytes (PBLs) before the results of biomonitoring studies of environmental exposure to chemicals could be evaluated. 32. The discussion paper overviews the published literature specifically in relation to studies which provide information on the background variance of MN formation in PBLs. The review includes studies investigating the development of the cytokinesis block MN assay (CBMN assay) including measuring MN formation in mononucleated and binucleated cells and the identification of numerical chromosomal changes in the CBMN assay, and the effects of age, smoking, sex and micronutrients on CBMN. A number of other studies reported data on the influence of methylenetetrahydrofolate reductase (MTHFR) genotype on the formation of MN in PBLs and the effects of cofactors for MTHFR activity on MN formation. 33. An important set of retrieved papers came from the Human Micronucleus project (HUMN) which was initiated in 1997. The idea for HUMN was conceived by Dr Stefan Bonassi (Italian National Cancer Institute) and Dr M Fenech (CSIRO Division of Human Nutrition, Adelaide, SA Australia). Following an announcement there was direct approach to scientists and a workshop held during the ICEM in Toulouse 1997. Forty-five scientists expressed an interest in participating. The main objectives were:
34. A total of 32 publications which contributed information on background
variance of MN in PBLs were identified through PuBMed literature searches
and cross referencing retrieved papers. Members were also told that some
additional comments from Dr Fenech who has reviewed the COM covering paper
had been tabled. 36. Members were impressed with this work and asked where there any differences in scoring slides at the different time periods used in the study. It was noted that the slide quality differed between 24h and 48 h slides with a higher cell density at 48 h. Members commented that it would be useful to evaluate the MN formation in mononucleate cells which escaped stimulation by PHA and to use centromere specific staining procedures to evaluate aneuploidy. It was hoped that an abstract of the work would be published at the forthcoming joint BTS/UKEMS meeting. 37. The Chairman asked members to overview the background variables for MN formation in peripheral blood lymphocytes. Members agreed that the most important variable was age which was most likely due to changes in DNA repair efficiency and chromosome loss with age. It was not evident from the available information why the age related pattern of MN formation in peripheral blood lymphocytes happened. Members agreed that female gender was associated with higher MN formation, although this was not evident in all females. Members were surprised that only heavy smoking (ca 30 cigarettes/day) was associated with increased MN formation in peripheral blood lymphocytes. With regard to micronutrients, members considered that there was good evidence from cross sectional and intervention studies to suggest that plasma or serum folate and/or vitamin B12 were associated with MN formation. There was less evidence with regard to plasma/serum vitamin C, but an association could not be excluded. No conclusions could be reached on other micronutrients although it was possible that micronutrients which influenced the extent of oxidative DNA damage would also affect MN formation in peripheral blood lymphocytes. 38. Members agreed that methylene tertrahydrofolate reductase (MTHFR) genotypes appeared to have an effect on homocysteine formation (which is required for the formation of methionine and subsequent methylation of DNA). Thus C677T TT genotype was associated with an approximate 2 fold increase in MTHFR activity compared to CC or CT genotypes. 39. Members noted the important effect of the number of MN scorers and their experience in the determination of MN frequency. There was also limited evidence that the amount of folate in cultures also affected MN formation. 40. The Committee discussed the modelling of factors affecting MN formation in peripheral blood lymphocytes undertaken by the HUMN project group. It was noted that there was considerable correlation between the parameters included in the four general groupings of host (eg age, gender, continent), exposure to genotoxic agents, methodological parameters and criteria for identification of binucleated cells and scoring MN. This resulted in the sum of the variance being above 100%. Members noted that on a simplistic basis if the variance due to exposure was considered to be 45%, this would leave just over half of the variance, ie 55% to be due to other factors of which methodological parameters predominated. The overall approximate four fold variance in the dose-response gradient noted in the scoring test experiment undertaken by laboratories participating in the HUMN project could explain the findings COM had observed in floriculturalists. 41. The Chairman referred members to the comments submitted by Dr Fenech. Members were grateful for Dr Fenech's input and agreed the his ranking of variables which influence MN formation in peripheral blood lymphocytes, although no information on the effect of alcohol had been presented to the Committees. The secretariat had obtained a number of papers which would need to be considered at the next meeting in May. It was noted that alcohol might act by reducing vitamin B12 levels or there might be an effect due to acetaldehyde accumulation in individuals with aldehdye dehydrogenase-2 polymorphisms. No definite conclusions could be drawn at this stage. Members considered Dr Fenech's comment that biomonitoring studies should be adequately powered to detect at least 5MN/1000 binucleated cells. Members agreed it would be useful to determine the average variance across all available control determinations, to determine a power curve and from this the minimum fold increase for a positive response. Members agreed the comments made by Dr Fenech on the design of studies and also the need for the determination of MN in mononucleated periperhal blood lymphocytes (which is a biomarker for potential aneuploidy). Members noted the submitted paper from Dr Fenech and colleagues on the association between MN formation and risk of cancer and looked forward to reviewing the published document. 42. The Chairman asked members to consider the questions posed in para 31 of the covering paper. Members agreed that good progress had been made towards identifying the factors which influenced MN formation in peripheral blood lymphocytes although more review work was required on the potential effects of alcohol. It was agreed following a suggestion from HPA, that the review work should be extended to consider the background variables affect chromosomal aberrations in peripheral blood lymphocytes. Members considered that it would be useful to compare the available data for chromosomal aberrations with that already reviewed for MN formation. ITEM 7: HESI PROPOSED WORK ON THE RELEVANCE AND FOLLOW-UP OF POSITIVE RESULTS IN IN-VITRO GENETIC TOXICOLOGY TESTING (MUT/06/6) 43. The Chairman alerted members to this project mainly for their information. Members did not agree with many of the statements (eg on the use of weight of evidence approach to mutagenicity assessment). It was noted that the US Food and Drug Administration had issued guidance on weight of evidence assessment. Members agreed to note the work of the HESI group. ITEM 8: ANY OTHER BUSINESS 44. One member raised concerns regarding the status of the draft OECD guideline for the in-vitro micronucleus test. The committee had previously forwarded comments on the unit for statistical analysis in the assay. Members agreed to pass additional comments to the National coordinator for OECD activities. Members were also made aware that the secretariat would forward an OCED discussion document on the cell transformation assay for members comments. The COM had previously been strongly opposed to the use of cell transformation assays in genotoxicity testing. 45. Members were alerted to the COT/COC discussion paper on the Royal College of Environmental Pollution report on crop spraying and health of residents and bystanders. The secretariat informed members that any comments from COM members would be incorporated into the COT/COC review. 46. Members and Departmental assessors were made aware of the UKEMS MSc modular course in Genetic Toxicology which was organised by the University of Wales at Swansea. There was particular interest in encouraging participation from regulatory departments. ITEM 9: PAPERS FOR INFORMATION/WRITTEN COMMENTS 47. Neri M et al, Mutation Research, 612, 1-13, 14-19, 2006 (MUT/06/5) 48. Annual report 2005 (MUT/06/4) ITEM 10: DATE OF NEXT MEETING 49. 18 MAY 2006.
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