The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup

Mutation Research - Genetic Toxicology and Environmental Mutagenesis

Volumn 783, Issue , 2015, Pages 23-35

  Gollapudi, B Bhaskar ^a   Lynch, Anthony M ^b   Heflich, Robert H ^c   Dertinger, Stephen D ^d   Dobrovolsky, Vasily N ^c   Froetschl, Roland ^e   Horibata, Katsuyoshi ^f   Kenyon, Michelle O ^g   Kimoto, Takafumi ^h   Lovell, David P ⁱ   Stankowski, Leon F ^j   White, Paul A ^k   Witt, Kristine L ^l   Tanir, Jennifer Y ^m  

^a Eponent   (United States)

^b GLAXOSMITHKLINE   (United Kingdom)

^c NATIONAL CENTER FOR TOXICOLOGICAL RESEARCH   (United States)

^d LITRON LABORATORIES   (United States)

^e FEDERAL INSTITUTE FOR DRUGS AND MEDICAL DEVICES   (Germany)

^f NATIONAL INSTITUTE OF HEALTH SCIENCES   (Japan)

^g PFIZER GLOBAL RESEARCH AND DEVELOPMENT   (United States)

^h TEIJIN PHARMA LIMITED   (Japan)

ⁱ ST GEORGE'S UNIVERSITY OF LONDON   (United Kingdom)

^j BioReliance   (United States)

^k HEALTH CANADA   (Canada)

^l NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES   (United States)

^m ILSI HEALTH AND ENVIRONMENTAL SCIENCES INSTITUTE   (United States)

more..

Author keywords

CD59; Flow cytometry; Glycosylphosphatidylinositol; Mutation; Red blood cells; Reticulocytes

Indexed keywords

CD59 ANTIGEN; ANTIBODY; MEMBRANE PROTEIN; PHOSPHATIDYLINOSITOL GLYCAN-CLASS A PROTEIN;

ANALYTIC METHOD; ANIMAL CELL; ANIMAL EXPERIMENT; ARTICLE; BLOOD SAMPLING; CONSENSUS; CONTROLLED STUDY; ERYTHROCYTE; FEMALE; GENOTOXICITY; GENOTOXICITY ASSAY; HAZARD ASSESSMENT; IN VIVO PIG A ASSAY; MALE; NONHUMAN; PRIORITY JOURNAL; RAT; RISK ASSESSMENT; ANIMAL; CHEMISTRY; EDUCATION; FLOW CYTOMETRY; GENETICS; HEMOGLOBINURIA; HEMOLYTIC ANEMIA; HUMAN; METABOLISM; MUTATION; PATHOLOGY; PROCEDURES; STANDARDS;

MAMMALIA; RATTUS; SUIDAE;

ANEMIA, HEMOLYTIC; ANIMALS; ANTIBODIES; EDUCATION; ERYTHROCYTES; FLOW CYTOMETRY; HEMOGLOBINURIA; HUMANS; MEMBRANE PROTEINS; MUTATION; RATS;

EID: 84928810972     PISSN: 13835718     EISSN: 18793592     Source Type: Journal    
DOI: 10.1016/j.mrgentox.2014.09.007     Document Type: Article

References (66)

1. Bryce S.M., Bemis J.C., Dertinger S.D. In vivo mutation assay based on the endogenous Pig-a locus. Environ. Mol. Mutagen. 2008, 49:256-264.
2. Phonethepswath S., Bryce S.M., Bemis J.C., Dertinger S.D. Erythrocyte-based Pig-a gene mutation assay: demonstration of cross-species potential. Mutat. Res. 2008, 657:122-126.
3. Miura D., Dobrovolsky V.N., Kasahara Y., Katsuura Y., Heflich R.H. Development of an in vivo gene mutation assay using the endogenous Pig-A gene: I. Flow cytometric detection of CD59-negative peripheral red blood cells and CD48-negative spleen T-cells from the rat. Environ. Mol. Mutagen. 2008, 49:614-621.
4. Miura D., Dobrovolsky V.N., Mittelstaedt R.A., Kasahara Y., Katsuura Y., Heflich R.H. Development of an in vivo gene mutation assay using the endogenous Pig-A gene: II. Selection of Pig-A mutant rat spleen T-cells with proaerolysin and sequencing Pig-A cDNA from the mutants. Environ. Mol. Mutagen. 2008, 49:622-630.
5. Schuler M., Gollapudi B.B., Thybaud V., Kim J.H. Need and potential value of the Pig-a in vivo mutation assay - a HESI perspective. Environ. Mol. Mutagen. 2011, 52:685-689.
6. Rossi W.F., Ware R.E. The molecular basis of paroxysmal nocturnal hemoglobinuria. Blood 1995, 86:3277-3286.
7. Kinoshita T., Inoue N., Takeda J. Defective glycosyl phosphatidylinositol anchor synthesis and paroxysmal nocturnal hemoglobinuria. Adv. Immunol. 1995, 60:57-103.
8. Dobrovolsky V.N., Miura D., Heflich R.H., Dertinger S.D. The in vivo Pig-a gene mutation assay, a potential tool for regulatory safety assessment. Environ. Mol. Mutagen. 2010, 51:825-835.
9. Miura D., Dobrovolsky V.N., Kimoto T., Kasahara Y., Heflich R.H. Accumulation and persistence of Pig-A mutant peripheral red blood cells following treatment of rats with single and split doses of N-ethyl-N-nitrosourea. Mutat. Res. 2009, 677:86-92.
10. Kimoto T., Suzuki K., Kobayashi X.m., Dobrovolsky V.N., Heflich R.H., Miura D., Kasahara Y. Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl-N-nitrosourea: direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression. Mutat. Res. 2011, 723:36-42.
11. Walker V.E., Jones I.M., Crippen T.L., Meng Q., Walker D.M., Bauer M.J., Reilly A.A., Tates A.D., Nakamura J., Upton P.B., Skopek T.R. Relationships between exposure, cell loss and proliferation, and manifestation of Hprt mutant T cells following treatment of preweanling, weanling, and adult male mice with N-ethyl-N-nitrosourea. Mutat. Res. 1999, 431:371-388.
12. Lambert I.B., Singer T.M., Boucher S.E., Douglas G.R. Detailed review of transgenic rodent mutation assays. Mutat. Res. 2005, 590:1-280.
13. Bhalli J.A., Pearce M.G., Dobrovolsky V.N., Heflich R.H. Manifestation and persistence of Pig-a mutant red blood cells in C57BL/6 mice following single and split doses of N-ethyl-N-nitrosourea. Environ. Mol. Mutagen. 2011, 52:766-773.
14. Dobrovolsky V.N., Shaddock J.G., Mittelstaedt R.A., Manjanatha M.G., Miura D., Uchikawa M., Mattison D.R., Morris S.M. Evaluation of Macaca mulatta as a model for genotoxicity studies. Mutat. Res. 2009, 673:21-28.
15. Dobrovolsky V.N., Elespuru R.K., Bigger C.A.H., Robison T.W., Heflich R.H. Monitoring humans for somatic mutation in the endogenous PIG-A gene using red blood cells. Environ. Mol. Mutagen. 2011, 52:784-794.
16. Peruzzi B., Araten D.J., Notaro R., Luzzatto L. The use of PIG-A as a sentinel gene for the study of the somatic mutation rate and of mutagenic agents in vivo. Mutat. Res. 2010, 705:3-10.
17. Araten D.J., Nafa K., Pakdeesuwan K., Luzzatto L. Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria genotype and phenotype are present in normal individuals. Proc. Natl. Acad. Sci. U. S. A. 1999, 96:5209-5214.
18. DeMarini D.M., Brockman H.E., de Serres F.J., Evans H.H., Stankowski L.F., Hsie A.W. Specific-locus mutations induced in eukaryotes (especially mammalian cells) by radiation and chemicals: a perspective. Mutat. Res. 1989, 220:11-29.
19. Dertinger S.D., Heflich R.H. In vivo assessment of Pig-a gene mutation - recent developments and assay validation. Environ. Mol. Mutagen. 2011, 52:681-684.
20. Kimoto T., Horibata K., Chikura S., Hashimoto K., Itoh S., Sanada H., Muto S., Uno Y., Yamada M., Honma M. Interlaboratory trial of the rat Pig-a mutation assay using an erythroid marker HIS49 antibody. Mutat. Res. 2013, 755:126-134.
21. Dertinger S.D., Bryce S.M., Phonethepswath S., Avlasevich S.L. When pigs fly: immunomagnetic separation facilitates rapid determination of Pig-a mutant frequency by flow cytometric analysis. Mutat. Res. 2011, 721:163-170.
22. Dertinger S.D., Phonethepswath S., Weller P., Nicolette J., Murray J., Sonders P., Vohr H.-W., Shi J., Krsmanovic J., Gleason C., Custer L., Henwood A., Sweder K., Stankowski L.F., Roberts D.J., Giddings A., Kenny J., Lynch A.M., Defrain C., Nesslany F., van der Leede B.-J.M., Van Doninck T., Schuermans A., Tanaka K., Hiwata Y., Tajima O., Wilde E., Elhajouji A., Gunther W.C., Thiffeault C.J., Shutsky T.J., Fiedler R.D., Kimoto T., Bhalli J.A., Heflich R.H., MacGregor J.T. International Pig-a gene mutation assay trial: evaluation of transferability across 14 laboratories. Environ. Mol. Mutagen. 2011, 52:690-698.
23. Cammerer Z., Bhalli J.A., Cao X., Coffing S.L., Dickinson D., Dobo K.L., Dobrovolsky V.N., Engel M., Fiedler R.D., Gunther W.C., Heflich R.H., Pearce M.G., Shaddock J.G., Shutsky T., Thiffeault C.J., Schuler M. Report on Stage III Pig-a mutation assays using N-ethyl-N-nitrosourea - comparison with other in vivo genotoxicity endpoints. Environ. Mol. Mutagen. 2011, 52:721-730.
24. Shi J., Krsmanovic L., Bruce S., Kelly T., Paranjpe M., Szabo K., Arevalo M., Atta-Safoh S., Debelie F., LaForce M.K., Sly J., Springer S. Assessment of genotoxicity induced by 7,12-dimethylbenz(a)anthracene or diethylnitrosamine in the Pig-a, micronucleus and Comet assays integrated into 28-day repeat dose studies. Environ. Mol. Mutagen. 2011, 52:711-720.
25. Lynch A.M., Giddings A., Custer L., Gleason C., Henwood A., Aylott M., Kenny J. International Pig-a gene mutation assay trial (Stage III): results with N-methyl-N-nitrosourea. Environ. Mol. Mutagen. 2011, 52:699-710.
26. Bhalli J.A., Shaddock J.G., Pearce M.G., Dobrovolsky V.N., Cao X., Heflich R.H., Vohr H.-W. Report on Stage III Pig-a mutation assays using benzo[a]pyrene. Environ. Mol. Mutagen. 2011, 52:731-737.
27. Stankowski L.F., Roberts D.J., Chen H., Lawlor T., McKeon M., Murli H., Thakur A., Xu Y. Integration of Pig-a, micronucleus, chromosome aberration, and Comet assay endpoints in a 28-day rodent toxicity study with 4-nitroquinoline-1-oxide. Environ. Mol. Mutagen. 2011, 52:738-747.
28. Dertinger S.D., Phonethepswath S., Weller P., Avlasevich S., Torous D.K., Mereness J.A., Bryce S.M., Bemis J.C., Bell S., Portugal S., Aylott M., MacGregor J.T. Interlaboratory Pig-a gene mutation assay trial: studies of 1,3-propane sultone with immunomagnetic enrichment of mutant erythrocytes. Environ. Mol. Mutagen. 2011, 52:748-755.
29. Torous D.K., Phonethepswath S., Avlasevich S.L., Mereness J., Bryce S.M., Bemis J.C., Weller P., Bell S., Gleason C., Custer L.L., MacGregor J.T., Dertinger S.D. In vivo flow cytometric Pig-a and micronucleus assays: highly sensitive discrimination of the carcinogen/noncarcinogen pair benzo(a)pyrene and pyrene using acute and repeated-dose designs. Environ. Mol. Mutagen. 2012, 53:420-428.
30. Tweats D.J., Blakey D., Heflich R.H., Jacobs A., Jacobsen S.D., Morita T., Nohmi T., O'Donovan M.R., Sasaki Y.F., Sofuni T., Tice R. Report of the IWGT working group on strategy/interpretation of regulatory in vivo tests II. Identification of in vivo-only positive compounds in the bone marrow micronucleus test. Mutat. Res. 2007, 627:92-105.
31. Avlasevich S.L., Phonethepswath S., Labash C., Carlson K., Torous D.K., Cottom J., Bemis J.C., MacGregor J.T., Dertinger S.D. Diethylnitrosamine genotoxicity evaluated in Sprague Dawley rats using Pig-a mutation and reticulocyte micronucleus assays. Environ. Mol. Mutagen 2014, 55:400-406.
32. Bhalli J.A., Ding W., Shaddock J.G., Pearce M.G., Dobrovolsky V.N., Heflich R.H. Evaluating the weak in vivo micronucleus response of a genotoxic carcinogen, aristolochic acids. Mutat. Res. 2013, 753:82-92.
33. Dertinger S.D., Phonethepswath S., Avlasevich S.L., Torous D.K., Mereness J., Bryce S.M., Bemis J.C., Bell S., Weller P., MacGregor J.T. Efficient monitoring of in vivo Pig-a gene mutation and chromosomal damage: summary of 7 published studies and results from 11 new reference compounds. Toxicol. Sci. 2012, 130:328-348.
34. Fiedler R.D., Weiner S.K., Schuler M. Evaluation of a modified CD71 MicroFlow® method for the flow cytometric analysis of micronuclei in rat bone marrow erythrocytes. Mutat. Res. 2010, 703:122-129.
35. Organization for Economic Cooperation and Development (OECD) OECD Environment, Health and Safety Publications Series on testing and assessment. Number 34: guidance document on the validation and international acceptance of new or updated test methods for hazard assessment, ENV/JM/MONO(2005) 14 2005, Available from: http://search.oecd.org/officialdocuments/displaydocumentpdf/?cote=env/jm/mono(2005)14&doclanguage=en (accessed 16.02.14).
36. Miura D., Shaddock J.G., Mittelstaedt R.A., Dobrovolsky V.N., Kimoto T., Kasahara Y., Heflich R.H. Analysis of mutations in the Pig-a gene of spleen T-cells from N-ethyl-N-nitrosourea-treated Fisher 344 rats. Environ. Mol. Mutagen. 2011, 52:419-423.
37. Dertinger S.D., Phonethepswath S., Franklin D., Weller P., Torous D.K., Bryce S.M., Avlasevich S., Bemis J.C., Hyrien O., Palis J., MacGregor J.T. Integration of mutation and chromosomal damage endpoints into 28-day repeat dose toxicology studies. Toxicol. Sci. 2010, 115:401-411.
38. Lemieux C.L., Douglas G.R., Gingerich J., Phonethepswath S., Dertinger S.D., Phillips D.H., Arlt V.M., White P.A. Simultaneous measurement of benzo[a]pyrene-induced Pig-a and lacZ mutations, micronuclei and DNA adducts in Muta™Mouse. Environ. Mol. Mutagen. 2011, 52:756-765.
39. Nafa K., Bessler M., Castro-Malaspina H., Jhanwar S., Luzzatto L. The spectrum of somatic mutations in the PIG-A gene in paroxysmal nocturnal hemoglobinuria includes large deletions and small duplications. Blood Cells Mol. Dis. 1998, 24:370-384.
40. Mortazavi Y., Merk B., McIntosh J., Marsh J.C.W., Schrezenmeier H., Rutherford T.R., BIOMED II Pathophysiology and Treatment of Aplastic Anaemia Study Group The spectrum of PIG-A gene mutations in aplastic anemia/paroxysmal nocturnal hemoglobinuria (AA/PNH): a high incidence of multiple mutations and evidence of a mutational hot spot. Blood 2003, 101:2833-2841.
41. Ware R.E., Rosse W.F., Hall S.E. Immunophenotypic analysis of reticulocytes in paroxysmal nocturnal hemoglobinuria. Blood 1995, 86:1586-1589.
42. Robinson D.R., Goodall K., Albertini R.J., O'Neill J.P., Finette B., Sala-Trepat M., Moustacchi E., Tates A.D., Beare D.M., Green M.H.L., Cole J. Analysis of in vivo hprt mutant frequency in circulating T-lymphocytes in the normal human population: a comparison of four datasets. Mutat. Res. 1994, 313:227-247.
43. Dobrovolsky V.N., Shaddock J.G., Heflich R.H. 7,12-Dimethylbenz[a]anthracene-induced mutation in the Tk gene of Tk+/- mice: automated scoring of lymphocyte clones using a fluorescent viability indicator. Environ. Mol. Mutagen. 2000, 36:283-291.
44. Dobrovolsky V.N., Shaddock J.G., Heflich R.H. Mutagenicity of γ-radiation, mitomycin C, and etoposide in the Hprt and Tk genes of Tk+/- mice. Environ. Mol. Mutagen. 2002, 39:342-347.
45. Puig J.G., Mateos F.A., Torres R.J., Buño A.S. Purine metabolism in female heterozygotes for hypoxanthine-guanine phosphoribosyltransferase deficiency. Eur. J. Clin. Invest. 1998, 28:950-957.
46. Torres R.J., Puig J.G. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency: Lesch-Nyhan syndrome. Orphanet. J. Rare Dis. 2007, 2:48.
47. Aral B., de Saint Basile G., Al-Garawi S., Kamoun P., Ceballos-Picot I. Novel nonsense mutation in the hypoxanthine guanine phosphoribosyltransferase gene and nonrandom X-inactivation causing Lesch-Nyhan syndrome in a female patient. Hum. Mutat. 1996, 7:52-58.
48. Organization for Economic Cooperation and Development (OECD) OECD Guidelines for the testing of chemicals: repeated dose 28-day oral toxicity study in rodents, Test Guideline 407 2008, Available from: http://www.oecd-ilibrary.org/docserver/download/9740701e.pdf?expires=1392572011&id=id&accname=guest&checksum=075A45981B66F469CA4BE79F4801E9DC (accessed 16.02.14).
49. Organization for Economic Cooperation and Development (OECD) OECD Guidelines for the testing of chemicals: repeated dose 90-day oral toxicity study in rodents, Test Guideline 408 1998, Available from: http://www.oecd-ilibrary.org/docserver/download/9740801e.pdf?expires=1392572294&id=id&accname=guest&checksum=935B786566602D91984235CC5532A36 (accessed 16.02.14).
50. Organization for Economic Cooperation and Development (OECD) OECD Guidelines for the testing of chemicals: transgenic rodent somatic and germ cell gene mutations assays, Test Guideline 488 2013, Available from: http://www.oecd-ilibrary.org/docserver/download/9713251e.pdf?expires=1392571255&id=id&accname=guest&checksum=C7400BE10E1B8C223781FCB41BBB876B (accessed 16.02.14).
51. Dobrovolsky V.N., Heflich R.H., Ferguson S.A. The frequency of Pig-a mutant red blood cells in rats exposed in utero to N-ethyl-N-nitrosourea. Environ. Mol. Mutagen. 2012, 53:440-450.
52. Phonethepswath S., Franklin D., Torous D.K., Bryce S.M., Bemis J.C., Raja S., Avlasevich S., Weller P., Hyrien O., Palis J., MacGregor J.T., Dertinger S.D. Pig-a mutation: kinetics in rat erythrocytes following exposure to five prototypical mutagens. Toxicol. Sci. 2010, 114:59-70.
53. Dertinger S.D., Phonethepswath S., Avlasevich S.L., Torous D.K., Mereness J., Cottom J., Bemis J.C., MacGregor J.T. Pig-a gene mutation and micronucleated reticulocyte induction in rats exposed to tumorigenic doses of the leukemogenic agents chlorambucil, thiotepa, melphalan, and 1,3-propane sultone. Environ. Mol. Mutagen. 2014, 55:299-308.
54. Dertinger S.D., Avlasevich S.L., Torous D.K., Bemis J.C., Phonethepswath S., Labash C., Carlson K., Mereness J., Cottom J., Palis J., MacGregor J.T. Persistence of cisplatin-induced mutagenicity in hematopoietic stem cells: implications for secondary cancer risk following chemotherapy. Toxicol. Sci. 2014, 140:307-314.
55. Dobrovolsky V.N., Boctor S.Y., Twaddle N.C., Doerge D.R., Bishop M.E., Manjanatha M.G., Kimoto T., Miura D., Heflich R.H., Ferguson S.A. Flow cytometric detection of Pig-A mutant red blood cells using an erythroid-specific antibody: application of the method for evaluating the in vivo genotoxicity of methylphenidate in adolescent rats. Environ. Mol. Mutagen. 2010, 51:138-145.
56. Kimoto T., Chikura S., Suzuki-Okada K., Kobayashi X.m., Itano Y., Miura D., Kasahara Y. Effective use of the Pig-a gene mutation assay for mutagenicity screening: measuring CD59-deficient red blood cells in rats treated with genotoxic chemicals. J. Toxicol. Sci. 2012, 37:943-955.
57. Kimoto T., Chikura S., Suzuki K., Kobayashi X.m., Itano Y., Horibata K., Honma M., Dobrovolsky V.N., Heflich R.H., Miura D., Kasahara Y. Further development of the rat Pig-a mutation assay: measuring rat Pig-a mutant bone marrow erythroids and a high throughput assay for mutant peripheral blood reticulocytes. Environ. Mol. Mutagen. 2011, 52:774-783.
58. Bhalli J.A., Shaddock J.G., Pearce M.G., Dobrovolsky V.N. Sensitivity of the Pig-a assay for detecting gene mutation in rats exposed acutely to strong clastogens. Mutagenesis 2013, 28:447-455.
59. Hayashi M., Dearfield K., Kasper P., Lovell D., Martus H.-J., Thybaud V. Compilation and use of genetic toxicity historical control data. Mutat. Res. 2011, 723:87-90.
60. Guérard M., Koenig J., Festag M., Dertinger S.D., Singer T., Schmitt G., Zeller A. Assessment of the genotoxic potential of azidothymidine in the Comet, micronucleus, and Pig-a assay. Toxicol. Sci. 2013, 135:309-316.
61. Custer L.L. Impact of new mutagenicity data on marketed drugs: a case study. Annual Meeting of the Genetic Toxicology Association 2012, Available from: http://www.gta-us.org/scimtgs/2012Meeting/speakers2012.html (accessed 22.02.14).
62. Dobo K.L., Fiedler R.D., Gunther W.C., Thiffeault C.J., Cammerer Z., Coffing S.L., Shutsky T., Schuler M. Defining EMS and ENU dose-response relationships using the Pig-a mutation assay in rats. Mutat. Res. 2011, 725:13-21.
63. McDaniel L.P., Ding W., Dobrovolsky V.N., Shaddock J.G., Mittelstaedt R.A., Doerge D.R., Heflich R.H. Genotoxicity of furan in Big Blue rats. Mutat. Res. 2012, 742:72-78.
64. Stankowski L.F., Roberts D.J., Chen H., Lawlor T., McKeon M., Murli H., Thakur A., Xu Y. Integration of Pig-A, micronucleus, chromosome aberration and Comet assay endpoints in rodent toxicity studies with 4-nitro quinolone-1-oxide (4nqo): effect of dose fractionation on genotoxic responses. Environ. Mol. Mutagen. 2011, 52(S1):S60. Available from: http://onlinelibrary.wiley.com/doi/10.1002/em.v52.1s/issuetoc (accessed 20.03.14) (abstract P76).
65. Roberts D.J., Nicotra S., Mack M., Gleason C., Custer L.L. Comparison of Comet, micronucleus, and Pig-a mutation endpoints in a 28-day oral repeat dose study in rats with MNU. Environ. Mol. Mutagen. 2012, 53(S1):S54. Available from: http://onlinelibrary.wiley.com/doi/10.1002/em.v53.1s/issuetoc (accessed 22.02.14) (abstract P85).
66. Stankowski L.F., Aardema M.J., Lawlor T.E., Miller S., Pant K., Roy S., Xu Y., Elbedai R. Integration of Pig-a, micronucleus, chromosome aberration, and Comet assay endpoints in a 28-day rodent toxicity study with urethane. Environ. Mol. Mutagen. 2013, 54(S1):S47. Available from: http://onlinelibrary.wiley.com/doi/10.1002/em.v54.S1/issuetoc (accessed 22.02.14) (abstract P71).