{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,27]],"date-time":"2026-06-27T13:23:51Z","timestamp":1782566631698,"version":"3.54.5"},"reference-count":78,"publisher":"Wiley","issue":"2","license":[{"start":{"date-parts":[[2023,2,6]],"date-time":"2023-02-06T00:00:00Z","timestamp":1675641600000},"content-version":"vor","delay-in-days":5,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004182","name":"Nordisk Ministerr\u00e5d","doi-asserted-by":"publisher","award":["2021\u2010022,2021\u2010101"],"award-info":[{"award-number":["2021\u2010022,2021\u2010101"]}],"id":[{"id":"10.13039\/501100004182","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Environ and Mol Mutagen"],"published-print":{"date-parts":[[2023,2]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The <jats:italic>in vivo<\/jats:italic> comet assay is widely used to measure genotoxicity; however, the current OECD test guideline (TG 489) does not recommend using the assay to assess testicular germ cells, due to the presence of testicular somatic cells. An adapted approach to specifically assess testicular germ cells within the comet assay is certainly warranted, considering regulatory needs for germ cell\u2010specific genotoxicity data in relation to the increasing global production of and exposure to potentially hazardous chemicals. Here, we provide a proof\u2010of\u2010concept to selectively analyze round spermatids and primary spermatocytes, distinguishing them from other cells of the testicle. Utilizing the comet assay recordings of DNA content (total fluorescence intensity) and DNA damage (% tail intensity) of individual comets, we developed a framework to distinguish testicular cell populations based on differences in DNA content\/ploidy and appearance. Haploid round spermatid comets are identified through (1) visual inspection of DNA content distributions, (2) setting DNA content thresholds, and (3) modeling DNA content distributions using a normal mixture distribution function. We also describe an approach to distinguish primary spermatocytes during comet scoring, based on their high DNA content and large physical size. Our concept allows both somatic and germ cells to be analyzed in the same animal, adding a versatile, sensitive, rapid, and resource\u2010efficient assay to the limited genotoxicity assessment toolbox for germ cells. An adaptation of TG 489 facilitates accumulation of valuable information regarding distribution of substances to germ cells and their potential for inducing germ cell gene mutations and structural chromosomal aberrations.<\/jats:p>","DOI":"10.1002\/em.22527","type":"journal-article","created":{"date-parts":[[2023,1,11]],"date-time":"2023-01-11T15:14:22Z","timestamp":1673450062000},"page":"88-104","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Assessing testicular germ cell <scp>DNA<\/scp> damage in the comet assay; introduction of a proof\u2010of\u2010concept"],"prefix":"10.1002","volume":"64","author":[{"given":"Yvette","family":"Dirven","sequence":"first","affiliation":[{"name":"Norwegian Institute of Public Health Division of Climate and Environmental Health  Oslo Norway"},{"name":"Centre for Environmental Radioactivity (CERAD, Centre of Excellence of the Norwegian Research Council)  Oslo Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dag Markus","family":"Eide","sequence":"additional","affiliation":[{"name":"Norwegian Institute of Public Health Division of Climate and Environmental Health  Oslo Norway"},{"name":"Centre for Environmental Radioactivity (CERAD, Centre of Excellence of the Norwegian Research Council)  Oslo Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erika Witasp","family":"Henriksson","sequence":"additional","affiliation":[{"name":"Swedish Chemicals Agency, Department of Development of Legislation and Other Instruments Unit of Proposals for Classification and Restriction  Sundbyberg Sweden"},{"name":"Swedish Chemicals Agency, Department of Development of Legislation and Other Instruments Unit of Evaluation of Substances  Sundbyberg Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rune","family":"Hjorth","sequence":"additional","affiliation":[{"name":"The Danish Environmental Protection Agency  Odense Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anoop Kumar","family":"Sharma","sequence":"additional","affiliation":[{"name":"Technical University of Denmark National Food Institute  Lyngby Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anne","family":"Graupner","sequence":"additional","affiliation":[{"name":"Norwegian Institute of Public Health Division of Climate and Environmental Health  Oslo Norway"},{"name":"Centre for Environmental Radioactivity (CERAD, Centre of Excellence of the Norwegian Research Council)  Oslo Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gunnar","family":"Brunborg","sequence":"additional","affiliation":[{"name":"Norwegian Institute of Public Health Division of Climate and Environmental Health  Oslo Norway"},{"name":"Centre for Environmental Radioactivity (CERAD, Centre of Excellence of the Norwegian Research Council)  Oslo Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jarle","family":"Ballangby","sequence":"additional","affiliation":[{"name":"Norwegian Institute of Public Health Division of Climate and Environmental Health  Oslo Norway"},{"name":"Centre for Environmental Radioactivity (CERAD, Centre of Excellence of the Norwegian Research Council)  Oslo Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anne Mette Zenner","family":"Boisen","sequence":"additional","affiliation":[{"name":"Technical University of Denmark National Food Institute  Lyngby Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stellan","family":"Swedmark","sequence":"additional","affiliation":[{"name":"Swedish Chemicals Agency, Department of Development of Legislation and Other Instruments Unit of Evaluation of Substances  Sundbyberg Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristine Bjerve","family":"G\u00fctzkow","sequence":"additional","affiliation":[{"name":"Norwegian Institute of Public Health Division of Climate and Environmental Health  Oslo Norway"},{"name":"Centre for Environmental Radioactivity (CERAD, Centre of Excellence of the Norwegian Research Council)  Oslo Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ann\u2010Karin","family":"Olsen","sequence":"additional","affiliation":[{"name":"Norwegian Institute of Public Health Division of Climate and Environmental Health  Oslo Norway"},{"name":"Centre for Environmental Radioactivity (CERAD, Centre of Excellence of the Norwegian Research Council)  Oslo Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2023,2,6]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrfmmm.2009.10.011"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms161226214"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mad.2008.01.007"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.3109\/17435390.2015.1071443"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00204-013-1070-0"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/0890-6238(95)00038-C"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/0890-6238(95)02018-7"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1093\/mutage\/gei033"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1093\/toxsci\/kfs187"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1002\/pst.530"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.3389\/fgene.2015.00061"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1124\/pr.110.002790"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1385\/MB:26:3:249"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.3389\/fgene.2014.00359"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1093\/toxres\/tfaa093"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1383-5718(02)00236-X"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10565-008-9072-z"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1093\/mutage\/gep014"},{"key":"e_1_2_8_20_1","unstructured":"ECHA. (2012)ECHA Report on Survey of Worldwide Cros: Costs and Practicalities of Two New OECD Guidelines For Testing Chemical Substances OECD 443 Extended One\u2010Generation Reproductive Toxicity Study and OECD 488 Transgenic Rodent Somatic and Germ Cell Mutation Assay."},{"key":"e_1_2_8_21_1","unstructured":"ECHA. (2017)Guidance on information requirements and chemical safety assessment chapter R.7a: endpoint specific guidance. Version 6.0. AGENCY E. C. (ed.)."},{"key":"e_1_2_8_22_1","first-page":"2379","article-title":"Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment","volume":"9","author":"EFSA, S. C","year":"2011","journal-title":"EFSA Journal"},{"key":"e_1_2_8_23_1","doi-asserted-by":"publisher","DOI":"10.1093\/mutage\/ges032"},{"key":"e_1_2_8_24_1","doi-asserted-by":"publisher","DOI":"10.1093\/mutage\/gep048"},{"key":"e_1_2_8_25_1","doi-asserted-by":"publisher","DOI":"10.1093\/mutage\/geu069"},{"key":"e_1_2_8_26_1","unstructured":"GHS2009.Globally Harmonized System of Classification and Labelling of Chemicals (GHS). vi 559."},{"key":"e_1_2_8_27_1","doi-asserted-by":"publisher","DOI":"10.1186\/2051-4190-23-11"},{"key":"e_1_2_8_28_1","doi-asserted-by":"publisher","DOI":"10.1002\/em.22121"},{"key":"e_1_2_8_29_1","doi-asserted-by":"publisher","DOI":"10.1093\/mutage\/geu059"},{"key":"e_1_2_8_30_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrgentox.2014.07.010"},{"key":"e_1_2_8_31_1","volume-title":"An introduction to genetic analysis","author":"Griffiths A.","year":"2000"},{"key":"e_1_2_8_32_1","doi-asserted-by":"publisher","DOI":"10.1093\/mutage\/geab035"},{"key":"e_1_2_8_33_1","doi-asserted-by":"publisher","DOI":"10.1111\/andr.12233"},{"key":"e_1_2_8_34_1","doi-asserted-by":"publisher","DOI":"10.1093\/mutage\/get012"},{"key":"e_1_2_8_35_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrgentox.2014.08.006"},{"key":"e_1_2_8_36_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrgentox.2009.12.012"},{"key":"e_1_2_8_37_1","doi-asserted-by":"publisher","DOI":"10.1093\/mutage\/gex036"},{"key":"e_1_2_8_38_1","doi-asserted-by":"publisher","DOI":"10.1186\/1477-7827-1-107"},{"key":"e_1_2_8_39_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/29.8.1791"},{"key":"e_1_2_8_40_1","doi-asserted-by":"publisher","DOI":"10.1095\/biolreprod61.4.927"},{"key":"e_1_2_8_41_1","doi-asserted-by":"publisher","DOI":"10.4061\/2010\/541050"},{"key":"e_1_2_8_42_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrgentox.2019.01.007"},{"key":"e_1_2_8_43_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrgentox.2008.05.002"},{"key":"e_1_2_8_44_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrgentox.2019.03.008"},{"key":"e_1_2_8_45_1","doi-asserted-by":"publisher","DOI":"10.1093\/mutage\/gex002"},{"key":"e_1_2_8_46_1","doi-asserted-by":"publisher","DOI":"10.3389\/fgene.2015.00266"},{"key":"e_1_2_8_47_1","doi-asserted-by":"publisher","DOI":"10.1002\/em.22327"},{"key":"e_1_2_8_48_1","doi-asserted-by":"publisher","DOI":"10.1111\/andr.12753"},{"key":"e_1_2_8_49_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41596-020-0398-1"},{"key":"e_1_2_8_50_1","doi-asserted-by":"publisher","DOI":"10.1210\/er.2014-1101"},{"key":"e_1_2_8_51_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.fct.2020.111865"},{"key":"e_1_2_8_52_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkz1077"},{"key":"e_1_2_8_53_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0094904"},{"key":"e_1_2_8_54_1","unstructured":"OECD. (2016)Test No. 489: In Vivo Mammalian Alkaline Comet Assay."},{"key":"e_1_2_8_55_1","unstructured":"OECD. (2020)Test No. 488: Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays."},{"key":"e_1_2_8_56_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2006.5"},{"key":"e_1_2_8_57_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.990160404"},{"key":"e_1_2_8_58_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/29.8.1781"},{"key":"e_1_2_8_59_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkg216"},{"key":"e_1_2_8_60_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.taap.2005.01.060"},{"key":"e_1_2_8_61_1","article-title":"Simple and efficient technique for the preparation of testicular cell suspensions","volume":"78","author":"Rodriguez\u2010Casuriaga R.","year":"2013","journal-title":"Journal of Visualized Experiments"},{"key":"e_1_2_8_62_1","doi-asserted-by":"publisher","DOI":"10.1080\/10408440008951123"},{"key":"e_1_2_8_63_1","doi-asserted-by":"publisher","DOI":"10.1080\/02772248.2018.1529236"},{"key":"e_1_2_8_64_1","doi-asserted-by":"publisher","DOI":"10.1002\/em.20613"},{"key":"e_1_2_8_65_1","doi-asserted-by":"publisher","DOI":"10.1016\/0014-4827(88)90265-0"},{"key":"e_1_2_8_66_1","doi-asserted-by":"publisher","DOI":"10.1093\/mutage\/gen008"},{"key":"e_1_2_8_67_1","doi-asserted-by":"publisher","DOI":"10.1385\/1-59259-973-7:275"},{"key":"e_1_2_8_68_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrgentox.2014.09.006"},{"key":"e_1_2_8_69_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrrev.2008.03.005"},{"key":"e_1_2_8_70_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrgentox.2020.503171"},{"key":"e_1_2_8_71_1","first-page":"207","article-title":"Drug transporters, the blood\u2010testis barrier, and spermatogenesis","volume":"208","author":"Su L.","year":"2011","journal-title":"The Journal of Endocrinology"},{"key":"e_1_2_8_72_1","unstructured":"UN ENVIRONMENT PROGRAMME. (2019)UN Environment Global Chemicals Outlook II\u2010From Legacies to Innovative Solutions: Implementing the 2030 Agenda for Sustainable Development."},{"key":"e_1_2_8_73_1","unstructured":"UNECE. (2020)Proposal to create an informal working group on clarification of the criteria for classification for germ cell mutagenicity in category 1B and set up its terms of reference. Committee of Experts on the Transport of Dangerous Goods and on the Globally Harmonized System of Classification and Labelling of Chemicals."},{"key":"e_1_2_8_74_1","unstructured":"UNECE. (2021)Status of the work of the informal working group on clarification of the criteria for classification for germ cell mutagenicity. Committee of Experts on the Transport of Dangerous Goods and on the Globally Harmonized System of Classification and Labelling of Chemicals."},{"key":"e_1_2_8_75_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrgentox.2012.05.002"},{"key":"e_1_2_8_76_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrgentox.2015.01.008"},{"key":"e_1_2_8_77_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkp826"},{"key":"e_1_2_8_78_1","doi-asserted-by":"publisher","DOI":"10.1001\/jamanetworkopen.2020.2159"},{"key":"e_1_2_8_79_1","doi-asserted-by":"publisher","DOI":"10.1080\/095530097144157"}],"container-title":["Environmental and Molecular Mutagenesis"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/em.22527","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/em.22527","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/em.22527","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,8,17]],"date-time":"2023-08-17T23:15:30Z","timestamp":1692314130000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/em.22527"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,2]]},"references-count":78,"journal-issue":{"issue":"2","published-print":{"date-parts":[[2023,2]]}},"alternative-id":["10.1002\/em.22527"],"URL":"https:\/\/doi.org\/10.1002\/em.22527","archive":["Portico"],"relation":{},"ISSN":["0893-6692","1098-2280"],"issn-type":[{"value":"0893-6692","type":"print"},{"value":"1098-2280","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,2]]},"assertion":[{"value":"2022-08-19","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2023-01-06","order":1,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2023-02-06","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}]}}