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| Chairman: Professor P Farmer |
Members: |
Secretariat:
Mr J Battershill (HPA secretariat) Mr S Robjohns (HPA minutes) Dr D Benford (FSA secretariat) Mrs J Cleverly (Administrative) |
|
Assessors: |
In attendance : |
Observers:
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| Item |
Paragraph |
|
|
1. |
Announcements/Apologies for absence |
1 - 3 |
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2. |
Minutes of the meeting on 1 February 2007 (MUT/MIN/07/1) |
4 |
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3. |
Matters Arising (not covered by later agenda items): |
5 - 7 |
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4 |
4.1 Presentation: The TK6 GADD45a-GFP Genotoxicity assay 4.2 Additional summary information on GADD45a-GFP assay |
8 - 22 |
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5. |
Development of an
OECD guideline for the in-vitro micronucleus Assay (MUT/07/11) |
23 - 38 |
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6. |
Use of LED for potency
ranking of in-vivo mutagens (MUT/07/08) |
39 - 46 |
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7. |
Ethaboxam (Partial review). Consideration of initial data |
47 |
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8. |
Mutagenicity of terephthalic acid - Additional cytogenetics data |
48 - 59 |
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9. |
Draft working paper on formaldehyde (written comments) (MUT/07/12) |
60 |
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10.
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Any other business |
61 |
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11. |
Date of next meeting: 4 October 2007 |
62 |
ITEM 1: ANNOUNCEMENTS/APOLOGIES FOR ABSENCE
1. The Chair congratulated Mrs Janice Cleverly from HPA who had gained promotion within the HPA and thanked her for her good work with the committee. The Chair welcomed Dr R Walmsley (University of Manchester), Dr K Burnett (DH Toxicology unit), Dr P Edwards (HPA), Dr S Dutton (HSE), Mr Barry Maycock (FSA), Dr D Mason (FSA), Dr N Rajapakse (FSA), Mr S Samuels (PSD), Dr A Wolfreys (UCB-Celltech), Dr D Kirkland (Covance) and Dr Dal-Soo Kim (data holder for Ethaboxam).
2. Apologies for absence were received from Dr J Clements (COM member), Dr C Allen (COM member), Mrs R Glazebrook (COM member), Dr B Viegas (National Assembly for Wales), and Dr D Andrew (PSD).
3. Members were reminded of the need to declare any interests before discussion of items.
ITEM 2: MINUTES OF MEETING ON 1 February 2007 (MUT/MIN/07/1)
4. Members agreed the minutes subject to some minor editorial changes.
ITEM 3: MATTERS ARISING (NOT COVERED BY LATER AGENDA ITEMS)
3.1 COM project on Mixtures
5. Members were updated on the progress of an ongoing project on mixtures. The COM had asked for a further paper on interactions between DNA reactive mutagens. Dr Lovell was thanked for his contribution to this project . The chair noted that relatively few papers had been retrieved which presented data on DNA adduct measuments following combined exposures to mutagens and asked members to forward any relevant information they were aware of to the secretariat. The Committee was informed that some papers on modulation of mutagenic responses had been retrieved but these would not be included in the paper being drafted for the October COM meeting.
3.2 Benzimidazoles
6. The COM was informed that a statement on benzimidazoles and a common mechanism group had been published.
3.3 Formaldehyde
7. The committee was told that a draft working paper on formaldehyde had been tabled (MUT/07/12) for written comments from members.
ITEM 4: PRESENTATION: THE TK6 GADD45a GFP GENOTOXICITY ASSAY (MUT/07/14).
Additional information (Hastwell et al Mutation Research 2006 MUT/07/10)
8. Dr Richard Walmsley from the University of Manchester gave a presentation on the TK6 GADD45a GFP genotoxicity assay to the committee. The Chair noted a number of members declared non personal interests but the information under discussion was not compound specific and considered all members could participate in the discussion and conclusion.
9. Dr Walmsley thanked the COM for the opportunity to make a presentation and outlined the areas he had been asked to cover which included validation of the GADD45a biomarker, methods and appearance of results, the interpretation of results, the rationale for using the GADD45a assay as a genotoxicity hazard screen, the use of S-9 metabolic activation, and likely use in the current approaches to mutagenicity screening. Dr Walmsley made a distinction between genetic endpoints arising from genome damage (cell death, mutations aberrations and cancer) and response endpoints sensing or repairing genome damage (such as induction of GADD45a). Members were shown a reproduction of a figure from Kirkland et al (Mutation Research 584 (1-2), 1-256.) outlining the high sensitivity (ie identification of carcinogens) resulting from current strategies for mutagenicity testing but low specificity (ie identification of non carcinogens). Dr Walmsley noted that a number of regulatory authorities acknowledge the low specificity of current strategies and in particular the performance of the in vitro chromosomal aberration test in mammalian cells. The end result was loss of potentially useful compounds and unnecessary in vivo testing of a number of compounds.
10. The GADD 45a GFP assay had been developed as an early screen for mutagenicity and other forms of genome damage that had the potential to reduce the number of false positive results in in-vitro genotoxicity testing and thus also the potential to reduce the number of follow-up in-vivo animal tests.GADD45a was described as a well characterised human gene with a central role in genomic integrity. A new reporter had been constructed that included the p53 response element from intron 3 and thus used p53 dependent, genotoxic specific induction of human GADD45a expression in TK6 cells. Increased gene expression in response to DNA damage in this assay could be detected by induction of the Green Fluorescent Protein Gene (GFP) and increased cellular fluorescence.
11. The assay was conducted using microplate with four compounds (at nine two-fold dilutions) with internal positive controls. Plate set up takes approximately 20 minutes. Cells (75 µl of a suspension containing 1x106 TK cells in RPMI-1640 medium) were incubated and fluorescence read at 24 hours and 48 hours after initiation. The upper test concentration 1000 µg/ml or 10 mM. Results of chemical testing in this assay are displayed via a simple graphical output. A modified assay had been developed where cells were incubated in the presence of exogenous metabolising fraction for 24 hour prior to harvesting and resuspension in phosphate buffered saline prior to measurement of fluorescence. The GFP fluorescence (excitation 435 nm, emission 535 nm) divided by absorbance (600 nm or 620 nm) was termed the "Brightness" and was a measure of GFP induction. Absorbance also served as a measure of reduction in proliferative potential or relative suspension growth. Results of chemical testing in this assay are displayed via a simple graphical output. A genotoxicity threshold was selected at 1.5 relative GFP induction. Dr Walmsley noted this threshold was selected at a level that was 3 times the standard deviation in untreated samples and was said to demonstrate biological relevance by showing a good separation of genotoxic and non-genotoxic chemicals. He noted that Brightness values taken at the 24/48 hour time point represent the integrated measure of all GFP production during exposure.
12. Regarding cytotoxicity, the software was designed to flag up 20% inhibition (80% RSG) as a cytotoxic effect. This was described as a small effect that did not mean 20% cells were dead. The cells would have had time to complete at least one cell cycle. The user would be alerted to look at the data when 20% inhibition had occurred. A 30% RSG inhibition indicated that many of the cells had not divided and that about 90% would be dead. Dr Walmsley noted that measurements of oxygen consumption and propidium iodide exclusion could be used to aid in the assessment of cytotoxicity. He reported that the assay could allow testing to a higher degree of cytotoxicity than other mammalian in-vitro assays.
13. Dr Walmsley referred to the published validation study (appended to MUT/07/14). He noted that all validation chemicals had been tested four times, up to 10 mM or to the limit of solubility or cytotoxicity, whichever was lower. For validation purposes, the GADD45a assay had been used to test 34 genotoxic agents with a known mechanism of action. This included 10 direct acting genotoxins, 10 aneugens, 7 nucleotide synthesis inhibitors, 4 topoisomerase inhibitors and 3 that induced reactive oxygen species. Thirty one of the 34 genotoxic agents induced the GADD45a reporter. The 3 compounds that gave negative results were didanosine (equivocal regulatory mutagenicity data), thiabendazole (which Dr Walmsley reported might not have aneugenic activity), and methyl viologen (reactive oxygen species generator). Overall Dr Walmsley considered the assay had high sensitivity for identification of potential in vivo genotoxicants.
14. Additionally, 41 non-genotoxic agents were tested, including 30 non-genotoxic agents with no positive in-vitro genotoxicity data and 11 cytotoxic positive compounds that had produced positive in-vitro chromosome results associated with cytotoxicity, but were negative in vivo (ie 'false positives'). The GADD45a assay was reported to give negative results for all the non-genotoxic agents included in the validation study. 15 agents were not cytotoxic and did not induce reporter induction at 10 mM. 18 compounds tested to cytotoxic levels also did not induce the reporter. The 11 compounds that were negative in vivo, but had given a positive in-vitro micronucleus tests, were all negative in the GADD45a assay.
15. A table comparing the results obtained with the GADD 45a GFP assay with available data on Ames, mouse lymphoma assay, the in vitro chromosome aberration and micronucleus tests and the in vivo chromosomal aberration assay and micronucleus tests was presented. Differences in results between currently used genotoxicity assays and the GADD 45a GFP assay included lack of response in the Ames tests for eukaryotic genotoxins including aneugens and nucleotide DNA synthesis inhibitors, and false positive chemicals in the in vitro mammalian assays. Dr Walmsley reported that based on the validation study the GADD 45a GFP assay had shown evidence of high sensitivity (95%) without loss of specificity (100%) for predicting genotoxic carcinogenesis. He noted a further study using 62 marketed pharmaceuticals had been undertaken and a manuscript was in preparation. A further study of eight non-toxic unique positives (in in vitro chromosomal aberration or mouse lymphoma assays) had reported negative results in 6/8 chemicals examined.
16. There were practical limits to the top concentration for screening ie 100 µm, thus 'weak' genotoxins could be missed at that screening concentration eg EMS (156 µm) and ENU (250 µm). Another limitation of the original assay was that it was not compatible with the use of S9 as an enzyme activating system (ie it is fluorescent and light absorbing). Thus, a modified protocol had been developed where S9 was removed after exposure. Initial studies with cyclophosphamide had indicated the modified protocol was suitable for testing compounds requiring exogenous metabolic activation. Further studies had been conducted with other compounds (graphical data presented to COM) which involved the use of exogenous activation and the modified protocol. These results would be published at a later date. Dr Walmsley concluded by noting a number of pharmaceutical companies were now running the GADD 45a GFP assay as part of pregulatory testing screening where the attributes of high throughput and early hazard alert using minimal amounts of test material were of value in drug discovery. He thanked the COM for the opportunity to make a presentation and acknowledged the contributions of co workers.
17. The COM considered this to be an interesting presentation and that initial results had suggested both good specificity and sensitivity. Members were interested to see the results when the dataset had been expanded, including further exogenous activation results. Members asked for more information on the validation of the exogenous metabolising fraction to be used in the GADD 45a GFP assay. This would involve studies on source of exogenous metabolising fraction (eg rat or hamster liver) and the level of incorporation in the assay and effect of exogenous metabolising fraction on the growth of TK cells in the assay and influence on the flow cytometry method.
18. Members were surprised that aneugens had given positive results in this assay, particularly at levels producing high cytotoxicity as reported in the Hastwell paper (MUT/07/14). It was suggested that the positive response for aneugens could be an indirect cell stress response. Members asked about false positives in the GADD 45a GFP assay. Dr Walmsley presented some data for a number of chemicals which were undergoing further examination and the results had not yet been published. Members queried whether a GADD 45a response was seen in starvation? Dr Walmsley reported that high levels of cytotoxicity did not induce a false positive response in this assay. The committee noted that there was a need to be careful of how the results obtained from tests with this assay was presented in terms of specificity and sensitivity, which would be dependent on the criteria and particular rules used to calculate these values.
19. The COM also noted that care would be needed in not representing the value of 1.5 used to separate genotoxins and non-genotoxins as an actual biological threshold. Regarding validation, members noted that care would also have to be taken on choosing the criteria for non-genotoxic compounds and that these would have to conform to a recognised consensus view.
20. In response to a question from Dr Walmsley, the secretariat noted the pre-screening assays had not been included in the COM guidance and this was an aspect to be considered during the proposed revision of the guidance (due to start next year). In relation to the overall COM strategy and guidance for testing for mutagenicity it was possible that the GADD 45a GFP assay could be used as an early in-vitro screen which could reduce the need for an in-vivo test eg if there was evidence that a positive result in mammalian cells could be considered artifactual.
21. The Chair thanked Dr Walmsley for his presentation and welcomed future publications on the assay.
Post meeting note
22. After the meeting Dr Walmsley had forwarded further slides (which had been presented to the COM) to the secretariat containing some pre-publication data to address members' question of how specific the assay was for genotoxic compounds. The additional data suggested that the sensitivity was around 81% and the specificity around 94% for rodent carcinogenicity and also suggested that cytotoxicity did not induce false positives for genotoxicity. The further information noted the need for further work with regard to fluorescent compounds.
ITEM 5: DEVELOPMENT OF AN OECD GUIDELINE FOR THE IN-VITRO MICRONUCLEUS ASSAY (MUT/07/11)
23. No interests were declared. The Chair welcomed representatives from IGG.
24. COM members had previously contributed to the development of an OECD guideline for the in-vitro micronucleus test. The secretariat was aware that an OECD working group was to be set up with UK representation to take this subject forward. It was intended that the COM could provide generic advice on a number of areas to the working group ie areas that had mainly been identified by the US EPA. The COM was asked to provide comments which would aid in developing a UK position for discussions to be held in Atlanta USA in October 2007. Of main concern was advice on the need for confirmatory testing, the uses of the cytokinesis block in test in mammalian cell lines, and the appropriate level of toxicity to determine the maximum concentrations for testing. Other potential areas identified included the use of exogenous metabolic activation, repeat testing when the first assay is negative, exposure times, redrafting of the guidance on the differentiation between tests using lymphocytes and cell lines and clearer methodological descriptions.
25. A draft discussion paper was provided that took suggested approaches from the published evidence, predominantly from the 3rd International Workshop on Genotoxicity Testing publication (IVMNT) by Kirsch-Volders M et al, 2003 (Mutation Research 540, 153 - 163) and a number of publications of data evaluations, notably by the French branch of the Environmental Mutagen Society (Lorge E et al, 2006. Mutation Research, 607, 13 - 136), and a commentary from E Parry & J Parry (Mutation Research 2006, 5 - 8).
26. The Chair suggested the Committee discuss the topics in MUT/07/11 in the order presented in the draft discussion paper. He asked that observers from the IGG be present at the meeting table to answer members questions.
27. Regarding the use of cell lines and confirmatory testing, the 3rd IWGT recommended a confirmatory test for negative and equivocal first tests (3 - 6 hour exposure) using continuous exposure (2 - 2.5 cell cycles and possibly up to 3 cell cycles for certain groups of chemicals such as nucleoside analogues) and a repeat of the first study with modified exogenous metabolic activation.
28. In the case of using human lymphocytes, the 3rd IWGT meeting recommended
the first test should involve a 20 hour exposure to the test compound
in the absence of exogenous metabolic activation. A test with exogenous
metabolic activation could follow this test (if protocols without metabolic
activation are negative or equivocal) or be carried out at the same time.
If the test with exogenous metabolic activation is negative then the repeat
test should involve similar testing conditions, but with a longer PHA
stimulation of lymphocytes (cf 48 hour for repeat test compared with 24
hour for initial test in the presence of exogenous metabolic activation).
29. Members considered the main discussion topic concerning the current draft OECD guideline regarding the use of peripheral blood lymphocytes related to confirmation of initial negative or equivocal tests (using 48h cultures post PHA stimulation) with a 24 hour culture post PHA stimulation substantially increased the size of the study. Members considered this element of confirmation using two PHA stimulation periods probably related to the development of the cytokinesis block micronucleus assay in biomonitoring for exposure to genotoxicants. Members heard from IGG representatives that the main rationale for inclusion of the 24 PHA stimulation came from colleagues in Europe. It was noted a new publication from Dr M Fenech (Nature Protocols, 2007, 2 (5), 1084-1104) might be helpful with regard to negotiations concerning this aspect of the draft OCED protocol. Members agreed that removal of the 24 hour PHA stimulation would be the best course of action or possibly an amendment to the draft OCED protocol to limit the need to undertake this part of the confirmatory testing.
30. Regarding the use of the cytokinesis block in tests with mammalian cell lines, the consensus view from the 3rd IWGT report was for the inclusion of the cytokinesis block (CB) for both cell lines and human lymphocytes. Additional recommendations form the 3rd IWGT noted the value of the CB for identifying the mode of action for genotoxic chemicals.
31. Lorge E et al 2006, subsequently published a SFTG (French Branch of the European Environmental Mutagen Society) international collaborative study on the in-vitro micronucleus tests, which specifically investigated the role of cytochalasin B in the IVMNT. It was noted that the cytokinesis block by application of cytochalasin B to lymphocytes ensures that cells that have undergone one cell division are counted for micronuclei and that unsynchronised mammalian cell lines continuously undergo cell division.
32. The study involved 38 participating laboratories from Europe, Japan, and the USA with tests in four cell types (lymphocytes, CHO, CHL and L5178Y) in the presence and absence of cytochalasin B. A range of chemicals were selected including clastogens, base and nucleoside analogues, aneugens, and/or polyploidy inducers and non-genotoxic compounds. The authors noted that MN frequencies in the presence of cytochalasin B cytokinesis block counted MN in binucleate cells, whereas counts for MN in the absence of cytochalasin B counted in mononucleate cells. Thus, the expected background frequency of MN in binucleate cells in the presence of cytochalasin B would be double that in the same cell line in the absence of cytochalasin B. With L5178Y cells a doubling of MN frequency was noted in control cultures in the presence of CB. It was also noted that DMSO affected the frequency of MN formation in these cells. The authors reported that scoring micronucleated cells in both mononucleated and binucleated cells was the preferred approach for identifying aneugens. However, overall, Lorge et al 2006 reported that the use of cytochalasin B had no effect on the detection of micronuclei in cell lines exposed to genotoxins.
33. Garriott M et al, 2002 and Phelps J et al, 2002 had also published data that were consistent with the conclusions reached by Lorge E et al, 2006. Further, a commentary by Parry J & Parry E 2006 had noted that the application of the assay using CB offered the method for distinguishing between clastogens and aneugens and thus could provide additional information on the mode of action for micronuclei induction.
34. Members considered the main discussion topic concerning the use of cytokinesis block with cell lines concerned the use of this procedure to determine the level of cytostasis. Members heard from IGG representatives that this aspect could significantly expand the determination of cytotoxicity part of the protocol particularly if other indicators of cytotoxicity approaches such as confluency, apoptosis, necrosis, and metaphase counting were also used. Members agreed with IGG representatives that the most appropriate approach would be redraft the OECD guideline to include a summary of the strengths and weaknesses of each approach and to allow investigators to select the most appropriate approach on a case-by-case basis. Members also stressed the need to score both binucleate and mononucleate cells, particularly if an aneugenic effect was suspected. For positive results, the COM noted that further investigation, such as with kinetochore probing could be conducted in mononucleate or binucleate cells to determine the mode of action, and identify aneugens.
35. Regarding the appropriate level of cytotoxicity/cytostasis reported in the draft OCED guideline to be between 50%-70%, members noted the maximum concentration for testing, the 3rd IWGT report recommended 50 - 60% toxicity based on a reduction in cytokinesis-block proliferation index (CMPI). The recommendation for the upper limit of 60% cytotoxicity was based on data for aneugens included as a personal communication. There were no published data available in the discussion paper (MUT/07/11) that suggested that 70% cytotoxicity would be appropriate.
36. Members noted two published papers from Sheila Galloway (Environmental and Mol Mutagenesis, 2000, 35, 191-201 and Mutation Research, 1998, 400, 169-186.) which had been published to assist in determination of maximum levels of cytotoxicity in mammalian cell clastogenicity tests. These publications reported 50% to be considered as an acceptable upper level of cytotoxicity for this assay. Members agreed with comments from the IGG representatives in response to a question from the Chair, that the most appropriate way forward would be to draft the OECD guideline to provide information on the strengths and weaknesses of using different levels of cytotoxicity in the micronucleus tests and to note that exposure to aneugens tended to be associated with steep dose-response curves, which could make it difficult to achieve a precise upper limit of cytotoxicity. Closely spaced concentrations should be used if there is a steep concentration-effect relationship. It was noted that the detailed comments provided by the Japanese authorities might be helpful when considering the most appropriate redrafted text. There would also be a need to explain the biological relevance for any positive results obtained at cytotoxic concentrations. Additionally, one IGG representative noted in response to a question from the Chair that ICH might suggest a reduction in the upper test concentration to 1mM from 10mM for the IVMNT and that that this might conflict with the OECD test guideline.
37. The COM also agreed that there was a need to redraft the OECD guideline section on blood donors for the human lymphocyte protocol to allow both the pooling of blood and the use of single donor samples.
38. The Chair thanked members for their comments and representatives
from the IGG for their contributions. He agreed that the COM would provide
further assistance in completing the process of ratification of the OECD
guideline if requested.
ITEM 6: FURTHER CONSIDERATION OF APPROACHES TO RANKING IN-VIVO MUTAGENS:
THE LOWEST EFFEECTIVE DOSE (LED FOR IN VIVO GENOTOXICITY); A POSSIBLE
APPROACH TO MUTAGEN POTENCY RANKING (MUT/07/08)
39. No interests were declared.
40. The COM and COC had previously discussed approaches to wider dissemination of their advice regarding Comparative Risk Assessment. The COC had agreed in principle to use the Margin of Exposure (MOE) as an additional tool to aid in the communication of risks in relation to genotoxic carcinogens at its November 2006 meeting. However, there had been no overall agreement by COC on the banding approach that could potentially be used to aid in the risk communication in relation to genotoxic carcinogens.
41. The COM had discussed a pragmatic approach to ranking in-vivo mutagens, which did not have carcinogenicity data, based on a suggested approach using the Lowest Effective Dose (LED) developed by Sanner & Dybing 2005 and had agreed that a further paper could be produced. It was acknowledged that there were problems with the LED approach suggested by Sanner & Dybing and that there were arguments against the proposal ie it would involve the use of data from a wide number of end points with varying sensitivity, that the approach depended upon available published data, and there would also be a publication bias. These were considered to be valid criticisms, but the desired outcome was broad categories, which might be helpful for pragmatic for risk ranking. The COM agreed that some further consideration could be given to chemicals categorised as IARC group 1 and 2A carcinogens. Members also asked for a consideration of chemicals within structural classes.
42. The secretariat had undertaken further work in light of members' comments with regard to developing a generic approach to ranking in-vivo mutagens. One outcome had been that it was not possible to derive LED potency boundaries for any in-vivo tests other than in the rodent micronucleus test (MUT/07/08).
43. The secretariat had prepared a suggested approach to ranking in-vivo mutagens using the Lowest Effective Dose (LED) for the rodent bone marrow and peripheral blood micronucleus tests. Due to time limitations it was evident that it would not be practical to use the IARC and EU classification databases (too small and not in an easy format for abstraction of LEDs). The secretariat had considered that the publication of a large evaluation of the rodent micronucleus studies by the US EPA, although in the 1990s, was the most suitable database to use (Mavournin KH et al, Mutation Research, 1990, 239, 29-80). The Japanese Environmental Mutagen Society evaluation of rodent micronucleus tests for IARC groups 1, 2A and 2B represented another useful source of relevant data (Morita T et al, Mutation Research, 1997, 389, 3-122).
44. The secretariat suggested the following steps, which the COM were asked to consider.
A. Consider all available data. If carcinogenicity data are available, then rank according to T25 etc. If there are no carcinogenicity data, determine ip LED value for the most appropriate mouse micronucleus test (usually lowest LED value, unless the study is considered inadequate). Pragmatic definitions of potency bands for ip mouse MN assays, £ 2 mg/kg bw (high), 2 ³ 200 mg/kg bw (medium) and > 200 mg/kg (low).
B. If oral study available (but no ip study), adapt estimate for LED according to percentage absorption. For compounds with no oral absorption data, consider using an algorithm for estimating oral absorption (or a default value).
C. Consider if LED value is close to a potency boundary and determine if consideration of molecular weight would result in placement in a higher potency category. One approach would be to derive ratio for molecular weight of the compound under consideration with a known low molecular weight mutagen eg MMS ca 100 and use this as a way for determining potency banding.
D. Consider percent induction of micronucleated PCEs at the LED value. If above 1%, consider placing in a higher potency group.
45. Another suggestion was that other dose levels could be used for the derision of potency bands/and or reduce the number of potency bands from three to two.
46. There was no overall COM agreement for the approach suggested by the secretariat. Members were concerned at the number of factors used to derive a potency banding and for what purpose the banding was being undertaken. Some members considered that potency ranking based on doses expressed as g/kg was inappropriate and considered that potency based on molar doses was more appropriate. Members noted that an approach based on molar doses would not be suitable for mixtures. In answer to questions from members, the secretariat explained that the primary purpose was prioritisation for risk management, although it had always been agreed that the primary policy was ALARP. The secretariat would further consider this approach and the potential incorporation of molar doses in the proposed scheme and bring it back for additional discussion at a later meeting.
ITEM 7: ETHABOXAM (PARTIAL REVIEW). CONSIDERATION OF INITIAL DATA FROM STUDIES REQUESTED BY COM [IN CONFIDENCE ITEM] -
47. The minutes of this item will be published when a statement is published. NOW AVAILABLE SEE BELOW paragraph 62.
ITEM 8: MUTAGENICITY OF TEREPHTHALIC ACID - ADDITIONAL CYTOGENETIC DATA (MUT/07/12)
48. No interests were declared. The Chair welcomed the observer from BP. He noted the item could be taken in open session, although the annexes to the paper were confidential.
49. Terephthalic acid (TPA) is used as a starting material in the manufacture of polyethylene terephalate (PET). PET may be used to coat the internal surface and welded joints of food cans. PET can as also be used in the manufacture of beverage bottles.
50. In 2001, the COM concluded that the limited in-vitro data and absence of toxicokinetic data in the in-vivo micronucleus assay were insufficient to determine the mutagenic potential of TPA. Therefore, the committee recommended that an adequately conducted in-vitro cytogenetics test in mammalian cells was needed before any definite conclusions could be reached ie whether the bladder tumours seen in the rat carcinogenicity bioassay arose from a non-genotoxic mechanism.
51. In 2006, the COM reviewed data which had been submitted to address the committee concerns expressed in 2001. In vitro cytogenetic data were provided. An initial study using terephthalic acid was found to be positive following 20 hour incubation in the presence of exogenous metabolic activation with S9. Since pH was limiting in this study, a second study was conducted using sodium terephthalate. The use of the sodium salt of TPA did not affect the pH of the assay buffer, permitting the use of a concentration up to the maximum for this assay. Although the study author considered the assay to be negative, small but significantly increases in the percentage of aberrant cells were observed following 3 hours of incubation in the presence of S9. Members were concerned that a relatively small reduction of 1 pH unit could not fully account for the clastogenicity observed in the first study. While the criteria for a positive response had not been fulfilled in the second study, the low incidence of aberrations in the control meant it was not possible to determine that terephthalic acid produced no effect. It was noted that 100 metaphases had been scored in the controls and at each dose group level. Members agreed that this should be increased to 200 to aid the interpretation of the data.
52. A mouse metabolism study had also been submitted to address concerns regarding bone marrow exposure in the mouse micronucleus study, which had been reviewed at the 2001 meeting. The committee considered that this study was not helpful in demonstrating bone marrow exposure; however, there was sufficient evidence of toxicity in the original micronucleus study to demonstrate systemic exposure. The findings of the micronucleus study were supported by a further in vivo study. Members agreed that this unscheduled DNA synthesis (UDS) study had been adequately conducted and was negative.
53. The committee considered that overall the evidence was consistent with a non-genotoxic mechanism for the bladder tumours seen in the rat carcinogenicity study; however, this conclusion would be reinforced if further investigation of the in vitro cytogenetics was performed.
54. As requested by the COM, counts of an additional 100 metaphases (where possible) had been conducted. The additional counts were reported in Annex A of MUT/07/9, and an overview report that combined the original counts with the additional counts was provided at Annex B.
55. The combination of the original and additional counts resulted in slight variations in the frequency of aberrant cells throughout the study. Although statistically significant increases in the aberrant cells persisted, they remained within the historical range for the negative control data.
56. The COM was asked to comment on the additional information provided.
57. Members considered that the additional metaphase counts had reaffirmed the previous result of a suggestion for a weak clastogenic effect in vitro and that the mechanism for this effect was unclear. The low incidence of aberrations in the control made interpretation of the response difficult, but overall members did not consider the criteria for a positive effect had been fulfilled.
58. The committee agreed that the two in-vivo studies were adequate and negative. Overall, the COM agreed that terephthalic acid was not an in-vivo mutagen and that the additional evidence was consistent with their previous conclusion of a non-genotoxic mechanism for the bladder tumours seen in the rat carcinogenicity study.
59. A revised working paper would be circulated to COM members for comment before being sent to the COT.
ITEM 9: DRAFT WORKING PAPER ON FORMALDEHYDE (MUT/07/09)
60. A draft working paper on formaldehyde was circulated to members for written comments.
ITEM 10: ANY OTHER BUSINESS
61. Members were informed that HSE had requested a COM review of a proposed submission from industry on acrylamide which had, in part, been produced in response to the COM evaluation of a number of studies on acrylamide (http://www.advisorybodies.doh.gov.uk/com/acryla.htm). FSA requested a full evaluation of the mutagenicity of acrylamide.
ITEM 11: DATE OF NEXT MEETING
62. 4 October 2007
*****************************
IEM 7: PARTIAL REVIEW OF ETHABOXAM: CONSIDERATION OF INITIAL DATA FROM STUDIES REQUESTED BY COM. MUT/07/13 and additional data in presentation from data holders representative.
1. The Chair asked the data holder (LG Life Sciences), their representative (HLS) and the observer from JSCI regulatory group (attending as an observer) to leave the room whilst the COM discussed the areas of questioning to raise. The Chair noted the previous declaration of interest from Dr Clare (regarding involvement in a number of previous studies on ethaboxam). He considered that Dr Clare had no involvement in the current studies submitted by the data holder or in the laboratory where they were conducted and agreed Dr Clare could take part in the discussions and conclusions of the COM.
COM consideration of areas for discussion
2. The COM considered there were a number of questions relating to the interpretation of the in vitro MN assay with regard to the derivation of NOELs in binucleate and mononucleate cells and the appropriate statistical level of significance to apply. With regard to the repeat intraperitoneal in vivo bone marrow MN assay in mice, members considered that the justification of the top dose needed further explanation as there was evident excessive toxicity and mortalities reported at the top dose of 300 mg/kg bw in the main study. Members also agreed that HLS should be questioned on the selection of tissue levels in the testes as a surrogate for the bone marrow and whether in vitro data on non disjunction were available. (Subsequently submitted during the COM meeting of 15 May 2007)
Presentation by representative of data holder
3. HLS reported the data from the repeat in-vitro MN assay in peripheral blood lymphocytes (PBLs) using both 24 hour and 48 hour PHA stimulation. The lowest NOEL for binucleate and mononucleate cells of 4 µg/ml were documented after 48 hour stimulation. FISH analysis using probes for chromosomes 1,8,11,17 and 18 in binucleate cells reported NOELS after 24 hour and 48 hour PHA stimulation of 7 µg/ml and 4 µg/ml respectively. Abnormal cells (categorised according to Marshall Mutation Research, 372, 233-245, 1996). At 7 µg/ml (24 hour PHA stimulation ) and 4 µg/ml (48 hour PHA stimulation), there was evidence of chromosome loss and gain reported but no evidence of non disjunction. Overall the lowest NOELs for binucleate cells were interpreted to be 6 µg/ml (at 24 hour PHA stimulation) and 3 µg/ml (for 48 hour PHA stimulation).
4. With regard to the repeat in-vivo bone marrow MN assay in mice (groups of 7 male mice dosed intraperitoneally twice at 50 mg/kg bw, 150 mg/kg bw or 300 mg/kg bw (24 hours apart) with bone marrow sampling 24 hours and 48 hours after the last dose), a small statistically significant (P<0.01) increase in the group mean incidence of micronucleated polychromatic erythrocytes (MNPCEs) was reported at the 24 hour and 48 hour sampling times after the second intraperitoneal dose of 300 mg/kg bw ethaboxam (in 1% methylcellulose/0.1% Tween 80). The trend analysis was statistically significant at the 48 hour sampling time point. A statistically significant reduction in percentage of polychromatic erythrocytes (PCEs) had been documented at both 150 mg/kg bw and 300 mg/kg bw ethaboxam (P <0.001 or <0.0001) with a statistically significant trend (P<0.001). HLS reported that the initial dose setting study had reported animal deaths at 400 mg/kg bw and that the maximum tolerated dose (MTD) had been achieved at 300 mg/kg bw. In the main study three animals dosed with 300 mg/kg bw had died and a further animal had been killed in extremis at the top dose of 300 mg/kg bw. The increase in MNPCEs at the top dose of 300 mg/kg bw reported at both the 24 hour and 48 hour sampling time points was not considered biologically relevant as it was only seen at the high dose where there was significant systemic toxicity and mortality reported, the individual and group mean data were within the historical control ranges and the low vehicle control MNPCE rate increased the statistical significance.
5. The toxicokinetic analysis for ethaboxam revealed evidence of exposure in the plasma, liver, spleen and testes. Tissue concentrations had not been measured in the bone marrow due to the small amount of tissue. HLS considered that tissue concentrations of ethaboxam were at least equivalent to testes and this was supported by evidence of bone marrow toxicity (reduced PCE frequency). Overall HLS suggested ethaboxam did not induce biologically significant increases in PCEMN and therefore no subsequent FISH analysis for whole chromosomes had been undertaken.
6. HLS concluded that the request for additional data from COM had been adequately fulfilled. NOELs for binucleate PBLs were interpreted to be 6 µg/ml (24 hour stimulation) 3 µg/ml (48 hour stimulation). There was no evidence for induction of non disjunction. Ethaboxam was not mutagenic in a repeat intraperitoneal bone marrow MN assay in mice with additional toxicokinetic data in directly and indirectly exposed tissues. Overall HLS concluded there was no evidence for genotoxicity in vivo.
COM questions for HLS
7. The Chair asked members to raise the questions outlined in paragraph 2 above regarding the presentation and data submitted to COM.
8. Members considered that further evaluation of the dose-response data for the in vitro MN assay in PBLs using 5% statistical significance suggested that NOELS would be 4 µg/ml (24 hour stimulation) and 2 µg/ml (48 hour stimulation). HLS noted the dose spacing in the study was very small and the increases seen at low concentrations of ethaboxam were small compared to concurrent controls.
9. Members asked for comments on the process for and selection of the top dose level for the repeat in vivo bone marrow MN assay in mice. HLS considered the process had been adequate and the top dose level (300 mg/kg bw) satisfactory. Members considered the level of toxicity at the top dose level was excessive. Members asked for an explanation of the cause of the small increase in MNPCEs seen in the study. HLS noted the response in animals was within the historical control range, and the response had been seen at highly toxic doses at the limit of the MTD and could be interpreted as a toxic-stress related response. Members asked if further investigation using procedures to identify whole chromosomes would be useful. HLS considered there were too few MNPCES on slides at the top dose level for any meaningful analysis for the presence of whole chromosomes.
10. The COM asked for more explanation regarding the assertion that tissue concentrations in the testes could be regarded as equivalent to bone marrow. HLS considered that testes were representative of peripherally exposed tissue and hence similar to bone marrow. HLS noted that higher tissue levels in liver and spleen might in part represent surface retention of ethaboxam following intraperitoneal dosing. Members asked if tissues had been specially prepared to avoid such contamination by intraperitoneally dosed ethaboxam. HLS reported that standard operating procedures had been used. COM members considered that surface retention of ethaboxam in spleen and liver was unlikely at 24 hours post dose. Overall the time course seen for all tissues and plasma suggested a reduction in tissue concentrations at 6-12 hours post dose, a peak at between 12-24 hours post dose, with a relatively slow elimination (compared to plasma) for liver, spleen and testes. Overall COM members were not convinced that testes could be selected as a representative tissue of bone marrow.
11. The data holder, HLS and JSCI withdrew from the meeting.
COM consideration and conclusions
12. The Chair suggested the COM consider the suggested areas for discussion outlined in the covering paper.
The interpretation of new in vitro MN data in PBLs
13. The COM agreed that NOELS were slightly lower from the submitted test data than reported; approximately 4 µg/ml (24 hour PHA stimulation) and 2 µg/ml (48 hour PHA stimulation).
The need for additional in vitro data on aneuploidy and chromosome disjunction
14. Members noted no evidence for non disjunction had been reported. [Post meeting note; further evaluation of these data suggest one possible interpretation that chromosome gain may have resulted from polyploidy cells which subsequently lost chromosomes or from some other aberration of segregation. Chromosome loss would also occur at the same time. Nuclear slippage would allow mononucleate cells to progress through mitosis resulting in polyploidy cells. Overall, the mechanism of ethaboxam induced effects in-vitro was unclear and needed to be resolved.]
The interpretation of the new in vivo MN data in mice given ethaboxam via intraperitoneal dosing
15. The small statistically significant increase in MNPCEs seen in the repeat study at high doses (2 x 300 mg/kg bw in 1% methylcellulose/0.1%Tween 80) at resulting in severe toxicity and increased mortality at both the 24 hour and 48 hour post dose bone marrow sampling times is a similar result to the first intraperitoneal study where small statistically significant increase in MN in the bone marrow was reported in mice (following intraperitoneal dosing of 2 x 486 mg/kg bw (in aqueous 1% methylcellulose) with sampling at 48 hours post dose). Severe toxicity and increased mortality were also reported in the first intraperitoneal bone marrow mouse MN assay with ethaboxam. The COM noted the evidence for aneugenicity of ethaboxam in vitro and considered that there was no adequate explanation for the small increases in bone marrow micronuclei seen in the intraperitoneal in vivo tests with ethaboxam. The COM could not exclude a direct aneugenic effect of ethaboxam in these studies. The COM did not agree that the increase in MNPCEs seen in these studies could definitely be related to a toxic-stress response. Thus overall the top dose level in the repeat intraperitoneal bone marrow MN assay of 300 mg/kg bw should be regarded as a LOAEL. The COM agreed that further evaluation of the slides from the repeat bone marrow MN assay at the top dose level with additional procedures to identify whole chromosomes should be undertaken if sufficient micronuclei could be identified.
The need for additional in vivo MN data in other tissues (eg spleen)
16. The COM agreed that further information could be provided for other tissues but this would require additional in vivo studies in mice using intraperitoneal dose levels below 2 x 300 mg/kg bw. There would need to be additional toxicokinetic evaluation undertaken. The COM considered that such a request would need to come from the Advisory Committee on Pesticides (ACP).
The use of tissue concentration data for risk assessment
17. The COM did not agree from the data submitted, that ethaboxam concentrations in testes could be used as a surrogate for bone marrow. The COM noted the absence of bone marrow tissue levels from the repeat bone marrow MN assay in mice. The COM suggested that in this instance an initial risk assessment could be undertaken using the peak plasma levels of ethaboxam at the LOAEL for MN induction in bone marrow reported in the repeat intraperitoneal bone marrow MN assay, although further evaluation of the suitability of this approach for risk assessment of site of contact aneugenicity would need to be considered by the ACP.
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MEETING BETWEEN ETHABOXAM DATA HOLDERS AND COM MEMBERS
Held on 17 August 2007 at Department of Health, Room LG16, Wellington House, London
Present
Professor P Farmer (COM chair)
Dr G Clare (COM)
Mr J Battershill (COM Secretariat)
Mr S Samuels (PSD)
Mr K Mistry (DH administrative arrangements)
Representatives for the data holder and advisers
Item 1: Introductory comments
1. The Chair welcomed attendees to the meeting which had been arranged by agreement between the COM secretariat and the representatives of the data holder. The Chair noted the primary objective of the meeting was to consider the proposals outlined in the updated COM statement on ethaboxam (June 2007) for further investigation of the potential for aneugenicity of ethaboxam in vitro and in vivo. (This statement had been finalised but publication was subject to agreement from ACP/PSD. The secretariat noted the data holder had provided a rebuttal to the statement but had not suggested any alterations to the wording of the statement).
2. The data holder's representative asked to make a statement from the data holder. Thus the data holder reported that the small increases in micronucleus formation seen in the in vivo i.p study in mice at the highest dose administered (2x 300 mg/kg bw) was due to toxic stress and not to ethaboxam. In answer to a question from the data holder's representative, the secretariat gave a summary of the information which had been forwarded to the ACP.
3. COM members and secretariat noted that the COM has considered a precautionary hazard assessment was most appropriate in this instance given the results of the in vitro studies and evidence for small increases in micronuclei in two in vivo studies in mice.
4. The COM chair noted that the COM role was to provide scientific advice to ACP. Regulatory advice to ministers was provided by ACP. The ACP would make recommendations on additional studies needed to support the risk assessment of ethaboxam.
Item 2: Consideration of further in vitro studies
5. COM members asked for information on the mode of action of ethaboxam as a fungicide (i.e. which proteins were inhibited in fungi and what species specificity existed). This might help to evaluate potential targets for aneugenicity in mammalian cells. The data holder's representatives commented that fungicides which targeted oomycetes were quite specific in their action but little was known regarding mode of action. It was noted that studies undertaken at University Departments had identified the cytoskeleton and mitochondria as potential targets in effects in sensitive fungi. The precise molecular targets were unknown. COM members felt that it was possible that microtubules were targeted in mammalian cells because of the halt in the cell cycle between metaphase and anaphase, but agreed this was speculative. It was noted that the fate of micronucleated cells was of interest since ethaboxam seemed to be a novel type of aneugen in vitro. Thus the continued ability of the cells to divide (e.g. did the mononucleate cells become polyploid?) and also the ability of ethaboxam to induce apoptosis was briefly discussed. This would further characterise the effects of ethaboxam in mammalian cells and may be of potential use in the risk assessment process.
6. The data holder's representatives noted this suggestion and was receptive to the ideas with regard to mechanistic work but felt that consideration of the in vivo data on ethaboxam from micronucleus studies was of a higher priority.
7. The chair noted that ethaboxam seemed to be quite unique in its spectrum of effects in the in vitro aneugenicity studies and this was a valid reason for further investigating mechanism.
Item 3: Further consideration of in vivo studies with ethaboxam
8. COM members considered that further evaluation of the pattern of micronuclei type e.g. aneugenic (centromere positive) or clastogenic from the high dose animals and controls would be informative. Thus if there was evidence of a shift in the pattern from control towards aneugenic in the high dose animals this would support the conclusion that the micronucleus formation at this dose level was due to the aneugenic action of the test compound. However if the pattern was predominantly similar to controls or clastogenic this would support a general toxicity based mechanism for the induction of micronuclei.
9. The data holder's representatives considered that undertaking such a study would be difficult, given the small number of micronuclei on the slides from the high dose animals and the technical difficulties in getting acceptable FISH staining of the slides. It was agreed this was a possible further investigation which could be undertaken.
10. Attendees heard from PSD that the evidence of sperm reduction and testicular atrophy in the data package on ethaboxam might be re-evaluated if an aneugenic mechanism was confirmed. It was important to exclude such a mechanism for the formation of micronuclei seen in the i.p. mouse studies. Given the data showing evidence for testicular exposure following i.p. dosing some member states might consider a category 2 mutagen classification was appropriate. This would be overcome if the effects seen in the i.p. mouse micronucleus studies were demonstrated to be due to a general toxic mechanism. The secretariat added that investigation of plausible general toxic mechanisms perhaps due to i.p. dosing of a poorly absorbed material might be relevant.
11. The data holder's representatives thanked COM members for their comments and considered re evaluation of the slides from the existing mouse micronucleus test at 300 mg/kg bw and controls was an option but there would be difficulties in obtaining a definite answer from such an approach. It was noted a further option would be to undertake a retest using i.p. dosing of mice for micronucleus formation. This proposal was further discussed under AOB during this meeting.
Item 4: Further mouse micronucleus studies using other tissues
12. COM members noted that further testing using spleen, liver or testes would be feasible, these tissues being selected based on the toxicokinetic data. The data holder's representative noted this option but considered there would be technical difficulties in undertaking such studies.
Item 5: Further toxicokinetic data
13. The secretariat noted that pooling bone marrow tissue had been previously undertaken in submissions and queried if there was any further consideration of this as an option. The data holder's representative reported that such an approach might be considered if any further in vivo testing was undertaken to define a NOEL for micronucleus formation. COM members noted that the use of plasma levels, on the basis of the kinetic studies seen by COM, would provide the conservative risk assessment, in the absence of levels of ethaboxam in the bone marrow.
Item 6: AOB and concluding remarks
14. PSD noted that there were two subjects to be addressed, namely obtaining a NOEL for risk assessment and evaluation of the potential in vivo aneugenicity of ethaboxam for hazard classification.
15. Attendees discussed the suggestion for an additional ip mouse micronucleus study with kinetic investigations. One option was to retest ethaboxam using dose levels of 225 mg/kg bw (x 2) and 300 mg/kg bw (x 2) given intraperitoneally to mice. If such a study provided a negative outcome for micronucleus formation, then this would add to a weight of evidence argument that the previous studies had shown a general toxic mechanism for micronuclei induction. The Chair commented that this might not be definitive but could be used to forward a weight of evidence argument. PSD commented that all options the data holder could take (eg evaluation of micronuclei from slides of existing studies in mice or undertaking further studies in mice) would add to a weight of evidence argument but this would need to be evaluated by the ACP. The decision on which approach to use was largely for the data holder to decide. Since ethaboxam was not approved for use in the UK, there was no urgency for ACP to provide regulatory advice to Departments.
16. The secretariat reported that minutes of the meeting would be circulated for agreement and comment to COM members who had attended this meeting and would then be forwarded to the ACP for use in its deliberations. The finalised minutes would go to COM members for information and would be published on the COM internet site when the COM statement was published.
HPA COM secretariat
August 2007
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ACTIONS
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Item
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Action
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Responsibility
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4. TK6 GADD45a-GFP Genotoxicity assay
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Draft working paper |
Secretariat
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5. Development of an OECD guideline value for the
in-vitro micronucleus test
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Forward COM comments to IGG group |
Secretariat
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6. The LED for in vivo genotoxicity, a possible
approach to mutagen potency ranking
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Consider further discussion paper |
Secretariat
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7. Ethaboxam (Partial review) consideration of initial
data requested by COM
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Draft working paper and send to company |
Secretariat
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8. Mutagenicity of terephthalic acid - additional
cytogenetics data
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Finalise working paper and send to COT |
Secretariat/FSA
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| Top |
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| copyright: © | updated: 22 October 2007 |
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