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DISSERTATIONS | KAJAL KUMARI | IN VIVO AND IN VITRO STUDIES ON THE HEALTH EFFECTS OF... | No 320
KAJAL KUMARI
IN VIVO AND IN VITRO STUDIES ON THE HEALTH EFFECTS OF INTERMEDIATE FREQUENCY MAGNETIC FIELDS
PUBLICATIONS OFTHE UNIVERSITY OF EASTERN FINLAND
Human exposure to electromagnetic fields has rapidly increased due to numerous electrical
appliances in households and at work. New and emerging technologies have brought about a need to produce adequate data for assessing possible health risks of intermediate
frequency (IF) magnetic fields (MF). This thesis aimed at producing basic data on possible adverse biological effects of IF MFs
to support health risk assessment. Possible biological effects of IF MFs were investigated on many selected endpoints such as indicators
of male fertility, behavioural tests on adult mice, measures of behavioural teratology, and
genotoxicity in vivo and in vitro.
KAJAL KUMARI
IN VIVO AND IN VITRO STUDIES ON THE HEALTH EFFECTS OF INTERMEDIATE FRE-
QUENCY MAGNETIC FIELDS
Kajal Kumari
IN VIVO AND IN VITRO STUDIES ON THE HEALTH EFFECTS OF INTERMEDIATE FRE-
QUENCY MAGNETIC FIELDS
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences
No 320
University of Eastern Finland Kuopio
2018
Academic dissertation
To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium CA102 Canthia Building at the University of East-
ern Finland, Kuopio, on November, 22, 2018, at 12 o’clock noon
Juvenes Print Tampere, 2018
Editors: Pertti Pasanen, Matti Vornanen, Jukka Tuomela, Matti Tedre
Distribution: University of Eastern Finland / Sales of publications www.uef.fi/kirjasto
ISBN: 978-952-61-2921-1 (nid.) ISBN: 978-952-61-2922-8 (PDF)
ISSNL: 1798-5668 ISSN: 1798-5668 ISSN: 1798-5676 (PDF)
Author’s address: Kajal Kumari
University of Eastern Finland
Depart. of Environmental and Biological Sciences
P.O. Box 1627
70211 KUOPIO, FINLAND
email: kajal.kumari@uef.fi
Supervisors: Professor Jukka Juutilainen, Ph.D.
University of Eastern Finland
Depart. of Environmental and Biological Sciences
P.O. Box 1627
70211 KUOPIO, FINLAND
email: jukka.juutilainen@uef.fi
Associate professor Jonne Naarala, Ph.D.
University of Eastern Finland
Depart. of Environmental and Biological Sciences
P.O. Box 1627
70211 KUOPIO, FINLAND
email: jonne.naarala@uef.fi
Reviewers: Dr. René de Seze, Ph.D.
National Institute for Industrial Environment and Risks
Depart. of Experimental Toxicology Verneuil-en-Halatte
Picardie, France
email: rene.de-seze@ineris.fr
Dr. Zenon Sienkiewicz, Ph.D.
Centre for Radiation, Chemical and Environ- mental Hazards, Public Health England Chilton, Oxfordshire, OX11 ORQ United Kingdom
email: zenon.sienkiewicz@phe.gov.uk
Opponent: Dr. Maria Rosaria Scarfì, Ph.D.
National Research Council of Italy-Institute for Electromagnetic Sensing of the Environment Via Diocleziano 328 - 80124, Naples, Italy email: scarfi.mr@irea.cnr.it
Kumari, Kajal
Title of the thesis. In vivo and in vitro studies on the health effects of intermediate frequency magnetic fields
Kuopio: University of Eastern Finland, 2018 Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences 2018; 320 ISBN: 978-952-61-2921-1 (print)
ISSNL: 1798-5668 ISSN: 1798-5668
ISBN: 978-952-61-2922-8 (PDF) ISSN: 1798-5676 (PDF)
ABSTRACT
In the last few decades, manmade appliances emitting electromagnetic fields have become increasingly common, leading to changes in the natural environment. This thesis focuses particularly on intermediate frequency (IF) magnetic fields (MF), defined as MFs with frequencies from 300 Hz to 100 kHz (or up to 10 MHz according to some definitions). Human exposure to IF MFs is increasing due to new applications such as induction heaters, wireless power transfer (at work or at home), and electronic article surveillance systems (used in, e.g., supermarkets to prevent shoplifting). Assessment of any health risk of IF MFs is therefore important;
when the number of exposed persons is high, even a small increase in individual risk may have major impacts on public health. At the same time, only limited data are available on possible adverse health effects.
The aim of the study was to produce basic data for adequate assessment of the health risks of IF MFs. The study included experiments to assess effects of IF MFs on reproductive health, cognition and behavior, and prenatal development in mice, as well as genotoxicity in mice and in vitro using rat primary astrocytes.The frequency of the MF was 7.5 kHz (used in electronic article surveillance systems) and the field strengths were 12 and 120 μT in the animal experiments, and 30 and 300 μT in the in vitro experiments. The highest exposure levels were higher than the reference level (100 μT) suggested for occupational exposure by the International Commission on Non-Ionizing Radiation Protection. The in vitro study included combined exposure of genotoxic chemicals and IF MFs to reveal possible co- genotoxicity. The chemicals used were menadione (an agent that induces mitochondrial superoxide production and DNA damage) and methyl methanesulfonate (an alkylating agent).
Measurements of fertility-related indicators in male mice exposed to 7.5 kHz MFs did not show adverse effects on reproductive organ weights, sperm counts, sperm head morphology or sperm motility. However, increased sperm motility was observed in high-exposure group.
In studies on cognition and behavior in adult mice, no effects were observed on body weight, spontaneous activity, motor coordination, level of anxiety or aggression. In the Morris swim task, mice in the 120 μT group showed less steep learning curve than the other groups but did not differ from controls in their search bias in the probe test. The passive avoidance task indicated a clear impairment of memory over 48 h in the 120 μT group. No effects on astroglial activation or neurogenesis were observed in the hippocampus. The mRNA expression of brain- derived neurotrophic factor did not change but expression of the proinflammatory cytokine tumor necrosis factor alpha mRNA was significantly increased in the 120 μT group. These findings suggest that exposure to a 7.5 kHz, 120 μT MF may lead to mild impairment of learning and memory, possibly through an inflammatory reaction in the hippocampus.
A behavioural teratology study was conducted to assess adverse effects on the developing brain from prenatal and early postnatal exposure to IF MFs. No exposure-related differences were observed in body weight development, spontaneous motor activity, anxiety, spatial learning, or memory. Furthermore, histopathological analysis did not reveal any effects on astroglial activation or hippocampal neurogenesis.
A genotoxicity study was conducted to assess the effects of IF MFs. The results did not support genotoxicity or co-genotoxicity of 7.5 kHz MFs at magnetic flux densities up to 300 μT in vitro or in vivo. On the contrary, there was some evidence that exposure to 7.5 kHz MFs might reduce the level of genetic damage. Strongest evidence for biological effects was obtained from counting relative cell number, which was significantly and consistently increased after MF exposure in all in vitro experiments.
In conclusion, exposure to 7.5 kHz MFs up to 300 μT did not show adverse effects on male fertility indicators, prenatal and early postnatal development of the brain, or genotoxicity in vivo or in vitro. However, mild impairment of learning and memory may result from exposure of adult animals. Interesting findings indicating favourable rather than adverse biological effects included increased sperm motility, increased relative cell number (indicating increased proliferation or reduced cell death) and suggestive decrease in the level of genetic damage.
Universal Decimal Classification: 614.875
CAB Thesaurus: magnetic field, radiation exposure, reproductive health, male fertility, brain, mental ability, memory, learning, animal behavior, genotoxicity, mice
TIIVISTELMÄ
Viime vuosikymmeninä erilaisia sähkömagneettisia kenttiä hyödyntävät laitteet ovat yleistyneet, mikä on johtanut muutoksiin luonnollisessa ympäristössämme.
Tämä väitöskirja keskittyy erityisesti välitaajuisiin magneettikenttiin, joiden taa- juusalueeksi määritellään 300 Hz - 100 kHz (tai joidenkin määritelmien mukaan 10 MHz saakka). Ihmisten altistuminen välitaajuisille kentille on kasvussa uusien lait- teiden kuten induktioliesien, langattoman tehonsiirron sekä esimerkiksi kaupoissa käytettävien varashälytinlaitteiden myötä. Välitaajuisten magneettikenttien tervey- dellisten riskien arviointi on tärkeää, koska altistuneiden ihmisten määrä on suuri ja pienikin riski yksilölle voi olla merkittävä väestötasolla. Tällä hetkellä välitaajuis- ten magneettikenttien mahdollisista haitallisista terveysvaikutuksista on vain vä- hän tietoa.
Tämän tutkimuksen tavoitteena oli tuottaa perustietoa välitaajuisten magneetti- kenttien terveysriskien arviointiin. Tämä tutkimus sisälsi hiirikokeita välitaajuisten magneettikenttien vaikutuksista lisääntymisterveyteen, kognitioon ja käyttäytymi- seen sekä sikiön kehitykseen. Perimämyrkyllisyyttä tutkittiin hiirillä ja viljellyillä rotan primääriastrosyyteillä. Tutkimuksissa käytettiin 7.5 kHz magneettikenttää, joka vastaa joidenkin varashälytinlaitteiden käyttämää taajuutta. Magneettivuon tiheys oli 12 tai 120 μT eläinkokeissa ja 30 tai 300 μT soluviljelmissä. Korkeimmat altistustasot ylittivät Kansainvälisen ionisoimattoman säteilyn komission (ICNIRP) työntekijöille asettaman viitearvon (100 μT). Soluviljelmillä tehdyt tutkimukset sisälsivät myös yhteisaltistuksen tunnetuille perimämyrkyllisille kemikaaleille mahdollisten yhteisvaikutusten selvittämiseksi. Käytetyt kemikaalit olivat me- nadioni, joka lisää solun sisäistä superoksidituotantoa ja DNA-vaurioita, sekä me- tyylimetaanisulfonaatti, joka on alkyloiva kemikaali.
Hedelmällisyysindikaattoreiden mittaukset 7.5 kHz magneettikentille altiste- tuissa hiirissä eivät osoittaneet haitallisia vaikutuksia lisääntymiselinten painoon, siittiöiden määrään, siittiöiden morfologiaan tai siittiöiden liikkuvuuteen. Kuiten- kin voimakkaammalle magneettikentälle altistetussa ryhmässä havaittiin lisäänty- nyttä siittiöiden liikkuvuutta.
Tutkittaessa kognitiota ja käyttäytymistä aikuisissa hiirissä ei havaittu vaikutuk- sia ruumiin painoon, oma-aloitteiseen aktiivisuuteen, koordinaatioon tai ahdistus- ja aggressiotasoihin. Hiirillä tehdyssä Morrisin uintitestissä 120 μT ryhmällä oli loivempi oppimiskäyrä kuin muilla ryhmillä. Passiivinen välttelykoe viittasi sel- vään muistin häiriöön 120 μT ryhmässä. Astrogliojen aktivaatiota tai vaikutusta neurogeneesiin ei havaittu hippokampuksessa. Geenien ilmenemisen mittaukset eivät osoittaneet muutoksia BDNF:n mRNA-tasossa, mutta TNFα:n mRNA-taso suureni 120 μT ryhmässä. Nämä tulokset viittaavat siihen, että altistuminen 7.5 kHz, 120 μT magneettikentälle saattaa johtaa lievään oppimisen ja muistin heikke- nemiseen, mahdollisesti hippokampuksen tulehdusreaktion kautta.
Käyttäytymisteratologisessa tutkimuksessa selvitettiin raskaudenaikaisen ja syn- tymän jälkeisen magneettikenttäaltistuksen vaikutuksia kehittyviin aivoihin. Altis-
tuksen ei todettu vaikuttavan ruumiin painon kehittymiseen, oma-aloitteiseen ak- tiivisuuteen, ahdistuneisuuteen, avaruudelliseen oppimiseen tai muistiin. Histopa- tologisissa tutkimuksissa ei havaittu vaikutuksia astrogliojen aktiivisuuteen tai neurogeneesiin hippokampuksessa.
Vaikutuksia perimämyrkyllisyyteen tai yhteisvaikutuksia perimämyrkyllisten kemikaalien kanssa ei havaittu viljellyissä soluissa eikä hiirissä. Näytti päinvastoin siltä, että altistuminen 7.5 kHz magneettikentälle saattaa vähentää perimään koh- distuvia vaurioita. Vahvin osoitus biologisista vaikutuksista saatiin lisääntyneestä suhteellisesta solumäärästä, joka oli toistuvasti ja tilastollisesti merkitsevästi suu- rentunut magneettikenttäaltistuksen jälkeen kaikissa soluviljelmillä tehdyissä ko- keissa.
Yhteen vedettynä altistus 7.5 kHz magneettikentälle 300 μT tasolle asti ei vaikut- tanut haitallisesti hedelmällisyysindikaattoreihin, sikiöaikaiseen ja syntymän jälkei- seen aivojen kehittymiseen tai perimämyrkyllisyyteen eläimissä tai soluviljelmissä.
Aikuisissa eläimissä altistus saattaa kuitenkin aiheuttaa lievää oppimisen ja muistin heikkenemistä. Mielenkiintoisia löydöksiä, jotka viittaavat pikemminkin hyödylli- siin kuin haitallisiin biologisiin vaikutuksiin, havaittiin siittiöiden liikkuvuudessa, lisääntyneessä suhteellisessa solumäärässä (joka viittaa lisääntyneeseen solukas- vuun tai vähentyneeseen solukuolemaan) sekä mahdollisesti vähentyneissä peri- mävaurioissa.
“I love to travel, but hate to arrive”- Albert Einstein
ACKNOWLEDGEMENTS
This study was carried out during the years 2014-2018 in the department of Environmental and Biological Sciences, University of Eastern Finland (UEF). This study was supported by the funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under Grant agreement number 603794 - the GERONIMO project, from the UEF under PhD student position, Alfred Kordelin foundation and EPHB doctoral programme, UEF.
One of my happiest moments was getting the email reply from my current supervisor, Prof. Jukka Juutilainen, telling me I could apply for a PhD studentship position at the UEF. Through the process of making my research plan and application and helping me I still feel thankful and deeply grateful, I cannot thank him enough for the great help he provided throughout my project. Surprisingly, I got the position I moved here and survived the Finnish weather and now I am writing my PhD book and preparing for my PhD degree these all have been possible after his unbelievable help. I want to thank Prof. Juutilainen for taking the time to answer every small questions, which came up from a gap in my knowledge about my topic. I appreciate his extraordinary help in improving my writing skills from zero, which also has made me realize, he has the patience that is required in a good leader. I just always feel that you have been a perfect supervisor for my PhD.
I am also deeply grateful to my supervisor associate professor Jonne Naarala. I am fortunate to have such an outstanding supervisor who has such an exceptional attitude, always willing to compliment each small bit of progress I have made along my PhD journey. I think this great attitude from my supervisors has helped me a lot towards completing my PhD studies and has helped me to remain very positive about myself, and my research.
I am also deeply grateful to Prof. Heikki Tanila for being exceptionally supportive during the whole PhD work. You were the one who always send me first comment on my manuscripts or even first comment I got on my thesis was from you, which always motivated me to work and work. I am really thankful to you for planning all the animal studies earlier than needed which allowed me to have enough publications made me today to finish my PhD.
I sincerely thank the official reviewers of this thesis Dr. Zenon Sienkiewicz and Dr. René de Seze, who helped me a lot with improving my thesis.
I would like to thank all my co-authors Hennariikka Koivisto, Matti Viluksela, Kaisa M. A. Paldanius, Mikael Marttinen, Mikko Hiltunen, Myles Capstick, Antonino Mario Cassara, Mikko Herrala, and Jukka Luukkonen, Heikki Tanila, Jonne Naarala, and Jukka Juutilainen, it makes me proud to have articles with you all. Mikko thanks a lot for teaching me to play floorball. Henna thanks a lot for teaching me all the behavioural tests as well as teaching me winter sports and being a co-author with me as well as my friend out of the university. Most importantly, I will never forget your great help that you shared your office with me when I was sick in Snellmania.
Many thanks to Hanne Vainikainen and Pasi Miettinen to help with all laboratory work, whenever I needed. I thank you Jukka Luukkonen, for teaching me how to read the comet assay in a perfect way.
Now I would like to give my special thanks to my parents who, even though, it made their lives difficult, provided me with everything to reach this grateful day of my life. You have been such broad-minded parents to educate me and send me out of the family and village alone to study in the university. What I am today is just your reflection.
Thankyou my dear siblings Gautam (babu), Niju (babi) and Guriya (Guda) for making me laugh on phone whenever I feel alone in Kuopio and motivated towards work.
What can I express to you is not enough my dear “Ram”, you are the one who has been my best friend and husband and given me the confidence that I can do research and be away from you to follow my passion. I think you are just an exceptional husband who always discuss research only. Thanks for understanding me in every situation more than myself.
Arja and Seppo thank you very much for making me feel at home whenever I visit you and taking care of me as your daughter. Special thanks goes to my dear friend Stralina for listening to me and also for understanding always as well as inviting me to every event or gathering that helped me to survive in Kuopio in the beginning stage of Kuopio life, when I did not know anybody here. I would like to thank my friend Finlay Smith for language check of Acknowledgments of this thesis.
Sincerely Kajal Kumari
Kuopio, 17th September 2018
LIST OF ABBREVIATIONS
μT Micro tesla, the unit of magnetic flux density A/m Ampere per meter
ANOVA-RM Analysis of variance for repeated measures BDNF Brain-derived neurotrophic factor
CE Cauda epididymis
cDNA Complementary deoxyribonucleic acid
DCX Doublecortin
DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid
EAS Electronic article surveillance EMF Electromagnetic fields
ELF Extremely low frequency (0-300 Hz) EMA Ethidium monoazide bromide FBS Fetal bovine serum
GAPDH Glyceraldehyde 3-phosphate dehydrogenase GD Gestational day
GFAP Glial fibrillary acidic protein
h Hour
Hz Hertz, the unit of frequency
HPRT hypoxanthine-guanine-phosphoribosyl transferase ICNIRP International Commission on Non-Ionising Radiation
Protection
IF Intermediate frequency (300 Hz- 100 kHz or 10 MHz) IH Induction heater
INMA Environment and childhood (from INfancia y Medio Ambiente) cohort
MF Magnetic field
MMS Methyl methanesulfonate
MnPCE Micronucleated plychromatic erythrocytes MnNCE Micronucleated normochromatic erythrocytes
MQ Menadione
MRI Magnetic resonance imaging mRNA Messenger ribonucleic acid OD Optical density OTM Olive Tail Moment PBS Phosphate buffer saline PND Postnatal days
qPCR Quantitative polymerase chain reaction
RF Radiofrequency (100 kHz or 10 MHz - 300 GHz) RNA Ribonucleic acid
RT PCR Reverse transcription polymerase chain reaction
SCENIHR Scientific Committee on Emerging and Newly Identified Health Risks (European Commission)
SEM Standard error of mean SSB Single strand breaks
TNFα Tumor necrosis factor alpha VDTs Video display terminals V/m Volt per meter
WHO World Health Organization
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following original publications that are referred to in the text by the Roman numbers studies I-IV.
I Kumari K, Capstick M, Cassara AM, Herrala M, Koivisto H, Naarala J, Tanila H, Viluksela M, Juutilainen J. (2017). Effects of intermediate frequency magnetic fields on male fertility indicators in mice. Environmental Research, 157:64-70.
II
Kumari K, Koivisto H, Viluksela M, Paldanius KMA, Marttinen M, Hiltunen M, Naarala J, Tanila H, Juutilainen J. (2017). Behavioral testing of mice exposed to intermediate frequency magnetic fields indicates mild memory impairment. PLoS ONE, 12(12).
III
Kumari K, Koivisto H, Capstick M, Naarala J, Viluksela M, Tanila H,
Juutilainen J. (2018). Behavioural phenotypes in mice after prenatal and early postnatal exposure to intermediate frequency magnetic fields. Environmental Research, 162:27-34.
IV Herrala M*, Kumari K*, Koivisto H, Luukkonen J, Tanila H, Naarala J and Juutilainen J. (2018). Genotoxicity of intermediate frequency magnetic fields in vitro and in vivo. Environmental Research, 167:759-769.
*Equal first authors
The original publications have been reproduced with permission of the copyright holders.
AUTHOR’S CONTRIBUTION
I) The author designed the study with the help of Heikki Tanila, Jonne Naarala, Matti Viluksela and Jukka Juutilainen. Practical work was done and the first draft of the manuscript was written by the author. Myles Capstick and Antonino Mario Cassara performed dosimetry. Mikko Herrala took video for motility counting and helped with setup of the exposure system. Hennariikka Koivisto helped in animal care. All co-authors edited and reviewed the manuscript.
II) The author designed the study with the help of Heikki Tanila, Jonne Naarala, Matti Viluksela and Jukka Juutilainen. Kaisa M. A. Paldanius, Mikael Marttinen and Mikko Hiltunen analyzed, and discussed the RNA data. The author did all behavioural tests, histological staining and analysis, and drafted the manuscript. Hennariikka Koivisto collected the histology samples. All authors commented on the manuscript.
III) The author designed the study with the help of Heikki Tanila, Jonne Naarala, Matti Viluksela and Jukka Juutilainen. The author did all behavioural tests, histological staining and analysis, and drafted the manuscript. Hennariikka Koivisto helped in animal care and collected the histology samples. Myles Capstick performed the dosimetry and wrote the dosimetry section of the manuscript. All co-authors commented and edited the manuscript.
IV) The author designed the in-vivo part of the study with the help of Heikki Ta- nila, Jonne Naarala and Jukka Juutilainen. The author performed all practical work related to the in-vivo study and wrote the first draft of in-vivo part of the manuscript. Mikko Herrala designed and performed the in vitro part of the study with the help of Jonne Naarala, Jukka Juutilainen and Jukka Luukko- nen. Hennariikka Koivisto helped in animal care and collected the blood samples for in-vivo study. All co-authors commented and reviewed the man- uscript.
CONTENTS
1 Introduction ... 21
2 Literature review ... 23
2.1 Electromagnetic fields ... 23
2.2 Intermediate frequency magnetic field ... 24
2.2.1 Sources and exposure levels ... 25
2.3 Interaction of magnetic fields with the body ... 28
2.3.1 Penetration of MF into the body ... 28
2.3.2 Induction of electric fields and currents ... 28
2.4 Mechanisms of biological effects ... 29
2.4.1 Stimulation ... 29
2.4.2 Radical pair mechanism ... 30
2.4.3 Other mechanisms ... 31
2.5 Biological and Health effects of IF MFs ... 32
2.5.1 In vitro and in vivo studies ... 32
2.5.2 Epidemiological studies ... 33
3 Aims of the study ... 39
4 Materials and methods ... 41
4.1 Animals ... 41
4.2 Cell culture ... 41
4.3 Exposure systems ... 41
4.3.1 In vivo ... 41
4.3.2 In vitro ... 47
4.4 Exposure ... 48
4.5 Behavioural testing ... 48
4.5.1 Exploratory activity ... 48
4.5.2 Rotarod ... 49
4.5.3 Marble burying test ... 49
4.5.4 Novelty suppressed feeding test ... 49
4.5.5 Isolation induced aggression ... 50
4.5.6 Morris water maze ... 50
4.5.7 Passive avoidance test ... 51
4.6 Histology ... 51
4.7 RNA extraction and quantitative PCR (qPCR) analysis... 52
4.8 Fertility indicators ... 53
4.8.1 Reproductive tissue weight ... 53
4.8.2 Sperm motility and total sperm counts ... 53
4.8.3 Morphology of sperm heads ... 53
4.8.4 Testicular spermatid counts ... 53
4.9 Genotoxicity testing ... 54
4.9.1 DNA damage level and DNA repair rate (Comet assay) ... 54
4.9.2 Micronucleus assay ... 54
4.10 Statistical analysis ... 55
5 Results ... 57
5.1 Effects of IF MF on fertility related indicators of adult male mice (study І) 57 5.2 Effects of IF MF on learning and memory of adult mice (study II) ... 59 5.3 Effects of IF MF on the developing brain (study III) ... 61 5.4 Genotoxic effects of IF MFs (study IV) ... 64 5.4.1 Effects on blood cells of adult mice ... 64 5.4.2 Effects on rat primary astrocytes exposed in vitro ... 64
6 Discussion ... 67
6.1 Dosimetry of IF MFs (studies I-IV) ... 67 6.2 Fertility of mice and IF MF (study І) ... 67 6.3 Behaviour of mice and IF MF (studies II & III) ... 68 6.4 Genotoxicity of IF MFs in mice and rat primary astrocytes (study IV) ... 69 6.5 Future perspectives ... 70 6.6 Methodological considerations ... 70
7 Conclusions ... 73
8 References... 75
21
1 INTRODUCTION
Electromagnetic fields (EMF) are ubiquitous in industrialized societies. Human exposure to EMF has rapidly increased due to numerous electrical appliances (con- sidered almost indispensable) in households and at work. New and emerging tech- nologies have brought about a need to produce adequate data for assessing possible health risks of intermediate frequency (IF) magnetic fields (MF). Health risks of extremely low frequency (ELF; ≤300 Hz) MFs produced by the generation, distribu- tion, and utilization of electricity as well as those of radio frequency (RF; 100 kHz – 300 GHz) EMFs from, e.g., wireless communication devices, have been investigated extensively over four decades. The frequencies of IF MFs lie between the ELF and RF ranges and are usually considered to cover frequencies from 300 Hz to 100 kHz (or up to 10 MHz; the upper limit depends on how RF is defined). Human exposure to IF MFs is increasing due to increased number of devices emitting fields in this frequency range, such as electronic article surveillance (EAS) systems, wireless power transfer systems, various medical equipment, industrial or domestic induc- tion heaters, industrial welding machines, compact fluorescent lighting, liquid crys- tal display screens and proximity readers. A few studies have reported exposure levels near these sources and observed that IF MF strength may exceed exposure guidelines in the close proximity of some devices (Christ et al. 2012; Alanko et al.
2011).
This study attempted to produce basic data on possible adverse biological ef- fects of IF MFs to support future health risk assessment. Possible biological effects of IF MFs were investigated on many selected endpoints such as indicators of male fertility, behavioural tests on adult mice, measures of behavioural teratology, and genotoxicity in vivo and in vitro.
The MF frequency used in this study was 7.5 kHz, which corresponds to the IF MFs emitted by one of the EAS technologies commonly used in supermarkets, other stores and libraries to combat theft. Four magnetic flux densities (12, 30, 120 or 300 μT) were used in this study. The highest flux densities exceeded the references val- ues (27 μT for the general public and 100 μT for occupational exposure) recom- mended by the International Commission on Non-Ionizing Radiation Protection (ICNIRP).
22
23
2 LITERATURE REVIEW
2.1 ELECTROMAGNETIC FIELDS
Electromagnetic fields consist of electric and magnetic fields. An electric field sur- rounds any electric charge and exerts a force on other charges. Magnetic fields are produced by moving electric charges (or electric currents), and exert a force on moving charges. The unit of electric field strength (E) is volt per meter (V/m) and that for magnetic field strength (H) ampere per meter (A/m). Alternatively, the magnitude of a magnetic field is often expressed as magnetic flux density (B;
amount of magnetic flux passing through a unit cross section area) in teslas (T).
Electromagnetic radiation refers to oscillating electromagnetic waves propagating through space with the speed of light, consisting of coupled electric and magnetic fields. The electric and magnetic fields are coupled only in the far field, which in most cases means a distance of at least one wavelength from the source.
The spectrum of non-ionizing electromagnetic radiation with wavelengths and frequencies is described below in Figure 1. The spectrum of electromagnetic radia- tion is usually divided into non-ionizing and ionizing radiation. The non-ionizing radiation spectrum is composed of static electric and magnetic fields, extremely low frequency fields, intermediate frequency fields, radio frequency fields (microwaves as a part of the radiofrequency range), infrared radiation, visible light and part of the ultraviolet radiation. The ionizing radiation spectrum (not shown in Figure 1) is composed of part of the ultraviolet radiation, gamma-rays and x-rays.
The frequency (f) and wavelength (λ) of electromagnetic waves are inversely proportional to each other according to the equation c = fλ, where c is the speed of light. Photon energy E increases with increasing frequency (or decreasing wave- length) according to the equation E = hf, where h is the Planck constant.
24
Figure 1. The spectrum of non-ionizing radiation. Source of the figure: Jukka Juutilainen’s radiation biology course materials 2015.
2.2 INTERMEDIATE FREQUENCY MAGNETIC FIELD
Table 1. Sources of IF electromagnetic fields.
Occupational Domestic Medical equipment Military
Dielectric heater sealers, induction heaters, plasma heaters, broadcast and communications transmitters, spot resistance welders, anti-theft devices
Induction cookers or hobs, plasma balls, digital smart- boards, touch screens, portable hearing units, induction furnaces, toys including electric engines, computer monitors, televi- sion sets, proximity read- ers, compact fluorescent lamps, wireless inductive charging of batteries or charging systems for elec- tric cars, high power acous- tic systems (amplifiers, loudspeakers etc.)
MRI systems, elec- tromagnetic nerve stimulators, electro- surgical units, wearable insulin pump
Auxiliary power units in most aircraft, anten- nas, power units, subma- rine communi- cation trans- mitters
MRI- magnetic resonance imaging
25 2.2.1 Sources and exposure levels
Many sources emitting IF EMFs exist at workplaces and homes (Table 1), and new sources are appearing with the development of new technologies. However, the field strengths and frequencies of the IF EMFs from domestic and occupational appliances are not well known (World Health Organization; WHO 2007), and need to be characterized better. Most measurements of human exposure to electric and magnetic fields have focused on the ELF and RF ranges. Studies that have meas- ured IF electric and magnetic fields in residential and occupational environments are described in Table 2.
Kurokawa et al. (2004) measured the exposure to IF MF in residential environ- ments and observed that the background levels were below 50 nT.
Gallastegi et al. (2017) characterized the IF exposure of children in a Spanish co- hort. The measurements were carried out extensively in 104 homes, 26 schools and their playgrounds, and 105 parks. The interquartile range for magnetic fields was from 0.02 to 0.23 μT and that for electric fields was from 0.2 to 0.5 V/m. The maxi- mum values observed in homes were 0.03 μT and 1.51 V/m.
Bakos et al. (2010) measured electric fields in the 1.2-100 kHz frequency range at about 15 cm from compact fluorescent lamps. The field strength was >42 V/mfor all tested lamps. In nine cases out of 17, the field strength exceeded 87 V/m and the highest measured value was 216 V/m.
Kos et al. (2011) measured MFs produced by domestic induction cooking hobs.
This study included measurements using pots with diameters of 15, 20 and 25 cm on the same (21 cm) cooking area. The measured values with the 15, 20 and 25 cm pots were 4.5, 2.4 and 0.9 μT respectively at a 5 cm horizontal distance from the appliance.
A Finnish study assessed the electric field strength from a recreational plasma ball (Alanko et al. 2011). The frequency was 20 kHz and the measured electric field strength was 677 V/m at 35 cm. The electric fields at 1.2 m from the source exceeded the ICNIRP (2010) reference level for the general public exposure, and that for oc- cupational exposure was exceeded at 1 m.
Van Den Bossche et al. (2014) performed in situ measurements for many IF sources. They found that the ICNIRP (2010) reference values were exceeded for many appliances such as touchscreens (44 kHz: up to 155.7 V/m at 5 cm), energy- saving bulbs (38-52 kHz: up to 117.3 V/m), fluorescent lamps (52 kHz: up to 471 V/mat 5 cm) and electrosurgical units (920 kHz: up to 792 V/mat 0.5 cm). Magnetic field strengths up to 1.8 and 10.5 A/m were measured close to the electrosurgical units and portable hearing units respectively. Large differences of measured field values were found between the various operating modes of the measured equip- ment. Compliance distances for the general public ranged from 15.3 cm (touchscreen) to 25 cm (fluorescent lamps).
Aerts et al. (2017) performed an extensive measurement survey of electric and magnetic fields in residential environments in three European countries - Belgium,
26
Slovenia, and the United Kingdom - in 42 residences and for 279 appliances. The maximum peak field strengths observed were 41.5 V/m and 2.7 A/m at 20 cm from induction cookers. However, the emissions of no other appliances exceeded the ICNIRP reference levels.
The measured electric and magnetic fields from non-directional beacons were 881.6 V/m and 9.1 A/m, respectively. The maximum electric fields exceeded ICNIRP reference levels for occupational exposure at seven non-directional beacons sites, and the magnetic fields at two of the seven sites (Joseph et al. 2012a).
Trulsson et al. (2007) measured the MFs around the EAS systems in Swedish shops and libraries in real life conditions. Measurements were performed at 45 points then arithmetic mean was calculated for each EAS system. The measured MFs exceeded the ICNIRP’s reference levels for several EAS systems. The arithme- tic mean was 0.02-47 A/m and maximum measured MFs were 0.25-118 A/m.
Joseph et al. (2012b) measured in situ MF exposure of EAS systems. The meas- ured spatially averaged fields exceeded the reference levels for five of the six inves- tigated systems. For the (de)activators, the spatially averaged fields did not exceed the reference levels. Maximal fields up to 148.0 A/m were measured at distances less than 20 cm.
In Finnish stores, measurements were performed for EAS systems. At the cash- ier’s seat, peak magnetic flux density did not exceed the ICNIRP reference levels for IF MFs, and was found much lower, varying from 0.2 to 4 μT. However, the refer- ence levels were exceeded with a maximum of 189 μT when cashiers walked through the EAS gates for short time during work. The corresponding reference value for the general public (38 μT) was exceeded in many cases between the gates (Roivainen et al. 2014).
In the studies discussed above, measurements were generally performed at fixed distances from the sources. As field strength changes rapidly with distance, such measurements do not necessarily adequately describe human whole body exposure.
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Table 2. Measurement of magnetic and electric fields from intermediate frequency field sources. Sources and frequency ranges (kHz) Distance (cm) Magnetic fieldElectric field (V/m) Reference Near power lines, railroads, elec- tric appliances (10-1000)----- Below 50 nT----- Kurokawa et al. 2004 Children of the Spanish INMA cohort in 104 homes, 26 schools and their playgrounds and 105 parks 110 cm above thefloor in home and classrooms, inthe center of the corresponding space (geographical center or center ofthe children's play area)
0.02- 0.23, maximum 0.03 μT in homes0.2 - 0.5, maximum in homes 1.51Gallastegi et al. 2017 Compact fluorescent lamps (1.2- 100)0, 10----- 39-168 and maxi- mum 216Bakos et al. 2010 Plasma ball (20) 35 ----- 677 Alanko et al. 2011 42 residences for 279 appliances 20 0.01 - 3.7 A/m0.18- 42.7Aerts et al. 2017 Non-directional beacons----- 9.1 A/m882 Joseph et al. 2012a EAS systems (5-8600) 5 0.02 - 47 and maximum peak 0.25 - 118 A/m----- Trulsson et al. 2007 EAS systems20 0.01 -127, maximum148 A/m----- Joseph et al. 2012b EAS systems (5-7.5) At cashier’s seat, between the gates0.2 -4 μT at cashier’s seat, maximum 189μT when cashiers walked through the EAS gates ----- Roivainen et al. 2014 EAS - electronic article surveillance; INMA - Environment and childhood (from INfancia y Medio Ambiente) cohort; THD, total harmonic distortion
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2.3 INTERACTION OF MAGNETIC FIELDS WITH THE BODY
To understand the biological effects of electric and magnetic fields, it is important to consider how fields interact with the animal and human body as well as various tissues and organs in the body. The first stage of MF interaction with the body is physical interaction, i.e., coupling of the MF to the body and induction of electric fields and currents inside the body. The area of science that describes the relation between external fields and induced electric fields and current density is called dosimetry. The basic principles of magnetic field interactions at frequencies lower than 100 kHz (where the electric and magnetic fields are “quasi-static” and interac- tions of electric and magnetic fields with the body are independent) are briefly de- scribed below.
Besides direct coupling of MFs with the body, MF coupling with any conductor such as wire fence can cause electric currents to pass through a body in contact with it.
2.3.1 Penetration of MF into the body
Low frequency magnetic fields penetrate the human or animal body easily because the relative permeability of tissues is similar to that of air. As the human or animal body does not perturb the MF distribution, the MF is similar inside and outside the body. The quantities of the magnetic materials are generally very low in biological tissues, and they can be neglected in macroscopic dosimetry. However, magnetic phenomena should be taken into account when considering interaction at a micro- scopic scale, as many cell types have been shown to contain, e.g., magnetite parti- cles (see 2.4.3).
2.3.2 Induction of electric fields and currents
Alternating MFs induce circulating currents in a plane perpendicular to the direc- tion of the magnetic field. The main interaction of MF with the animal or human body is the Faraday induction of electric fields and associated currents into conduc- tive tissues. The distribution of induced electric fields depends on conductivity of various tissues and organs.
Induced internal electric fields and currents are also affected by the direction of the MF in relation to the body. If the direction of the MF is along the axis of the body then the induced electric fields are weakest, and the highest induced fields are produced when the external MF orientation is along the front-to-back axis of the body. Thus, coupling is maximized when the MF is perpendicular to the frontal cross section of the body. The induced electric fields and current densities also de- pend on the shape, size and posture of the body. The induced fields are greatest in a large body because of a large conduction loop around the body. The maximum induced fields are present at the surface of the body and decrease towards center.
29 When frequency increases, the magnetically induced electric fields and current densities increase linearly as a function of frequency. Overall, assessment of inter- nal electric field is difficult. Electric fields and currents induced in the human body cannot not be measured easily. Measurements in animals have been performed, but data is limited, and accuracy of the measurement is relatively poor. Information about the internal fields can be obtained by dosimetric modeling (Kos et al. 2011).
This requires knowledge of the geometry of the source, size and shape of the ex- posed animal or human, its position and orientation in relation to the source, as detailed information about the shapes, locations and conductivities of various tis- sues. Dosimetric calculations have been done in a few studies for humans exposed to IF field sources. Kos et al. (2011) measured both magnetic field flux density and did computational dosimetry. They reported that the MF produced by domestic induction cooking hobs did not induce internal electric fields that would exceed the ICNIRP basic restrictions (2010) in adults, children and fetuses. The maximum computed whole body electric fields were 0.11 and 0.66 V/m in pregnant women at 26 and 30 weeks, and 0.28 and 2.28 V/m in 6 and 11 years old children, while the ICNIRP basic restriction is 4.25 V/m. The maximum computed current densities were 46 and 42 mA/m2 in pregnant women at 26 and 30 weeks, and 27 and 16 mA/m2 in the 6 and 11 years old children, while the ICNIRP basic restriction is 70 mA/m2.
2.4 MECHANISMS OF BIOLOGICAL EFFECTS
2.4.1 Stimulation
Stimulation is an established mechanism for effects of strong MFs. Exposure to MFs induces electric fields in tissues, and these can affect electrically excitable neurons and muscle cells. Induction of magnetophosphenes (visual sensations caused by an alternating magnetic field) is a well-established phenomenon that results from stimulation of retinal cells (Reilly 2002; Saunders and Jefferys 2007; ICNIRP 2010).
The orientation-dependent stimulus thresholds for large diameter myelinated nerve fibers of human peripheral nervous system have been estimated to be about 6 V/m (Reilly 1998, 2002) using a nerve model. However, the threshold for perception was suggested to be 2 V/m in peripheral nerve simulation induced during exposure to switched gradient MFs of a magnetic resonance system (Nyenhuis et al. 2001), in calculations that were done using a homogenous human phantom. An accurate estimation of electric fields in tissues using a heterogeneous human model was carried by So et al. (2004), who estimated that the threshold for peripheral nerve simulation was 4-6 V/m. This calculation was based on assumption that simulation took place in skin or subcutaneous fat.
Inactive (resting phase) mammalian nerve cells are negatively charged inside the cells in comparison to the outside, and the membrane potentials are 60-75 mV.
Thresholds of action potential for many axons have been determined, and are about 50-55 mV, 10-15 mV above the resting potential. As described in the previous para-
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graph, stimulation is biophysically plausible when the internal field strength (in- duced by an external field) exceeds a few volts per meter. Even much weaker fields can affect synaptic transmission in neuronal networks as opposed to single cells.
Neuronal networks have very complex non-linear dynamics which can be very sensitive to a small applied voltage. The neuronal network sensitivity has been studied theoretically by Adair (1998, 2001). They describe exceptional sensitivity to weak electric fields in sharks and other elasmobranch fish species. They can re- spond behaviorally to weak fields in seawater as low as 0.5 μV/m.
Muscle cells are less sensitive to the direct stimulation than nerve cells (Reilly 1998). The guideline by ICNIRP (2010) suggests more attention on cardiac muscle effects because any changes in the cardiac function could be life-threatening. The threshold for diffuse stimulation of cardiac tissue has been estimated to be 12 V/m based on experimental data (Reilly 1993, 1998). The threshold increases above 120 Hz because of longer time constant of muscle fibers in comparison to myelinated neurons.
Exposure to fields below the threshold for direct nerve or muscle excitation can induce magnetophosphenes. Phosphenes, described as perception of faint flickering light in the periphery of the visual field, have been observed during exposure to low frequency MFs (ICNIRP 2010). This phenomenon is believed to result from interaction of induced electric fields with electrically excitable retinal cells. The es- timated phosphene threshold for induced electric fields in human retina is 50-100 mV/m at 20 Hz, and the threshold increases at higher or lower frequency (Saunders and Jefferys 2007; ICNIRP 2010). In the Environmental Health Criteria document (WHO 2007) the threshold was estimated to lie between 10 and 100 mV/m.
Some experimental and epidemiological studies suggest possible biological ef- fects below the threshold for stimulation of excitable cells, but there is currently no generally accepted mechanistic explanation for biological effects of such weak magnetic fields. However, a few mechanisms have been proposed, and are de- scribed below.
2.4.2 Radical pair mechanism
The radical-pair mechanism (RPM) is considered one of the most plausible biophys- ical mechanisms for explaining the biological effects of weak MFs. The RPM is well established theoretically as well as experimentally in biochemical systems (Eveson et al. 2000). However, its biological significance is not fully understood. The RPM describes MF effects on the life time of radical pairs, which can increase or decrease the cellular concentration of free radicals.
Many cellular processes and external agents (such as UV radiation) generate radical pairs, formed in a singlet or triplet state. A quantum mechanical intercon- version occurs between the singlet and triplet states, driven by internal magnetic interactions within the radicals. This interconversion is sensitive to external mag- netic fields. A relatively well-established biological role for the RPM is magnetore- ception in birds. Many bird species are known to use the earth’s magnetic field for
31 navigation, and magnetically sensitive radical pair reactions in cryptochrome (CRY) proteins have been proposed to be the basis of avian magnetoreception (Hore and Mouritsen, 2016). The CRYs have been found in several retinal cell types (Bolte et al. 2016), but exact location of the avian magnetoreceptors has not been identified.
CRY are blue light-sensitive proteins involved in the regulation of circadian rhythms.
It is unclear whether the RPM can result in other significant biological effects than animal magnetoreception. In principle, the RPM can lead to changes in cellular free radical concentration, and this can modify biological events either by causing radical-induced damage or through the important role of radicals as signalling molecules (Juutilainen et al. 2018). It should be noted that the direction of the MF effect on radical pairs depends on magnetic flux density. For a radical pair formed in a singlet state, the concentration of free radicals increases in low fields (the low field effect) and decreases in high fields (the high field effect) (Brocklehurst et al.
1996; Timmel et al. 1998). The low field effect occurs below about 1 mT, and is therefore more relevant for possible adverse biological effects of weak environmen- tal magnetic fields.
2.4.3 Other mechanisms
Several other mechanisms have been proposed for direct biophysical interaction of MFs with human or animal body. Magnetite particles offer another plausible mech- anism for direct interaction with weak MFs. A MF exerts a moment (turning force) on anything that has a magnetic moment. If any ferro- or ferrimagnetic particles are present in the body, a MF could produce a moment on them and cause them to vibrate in an alternating MF. Crystals of the iron oxide magnetite are ferrimagnetic particles well known to be present in biological systems, including animal and hu- man tissues. For a long time, these particles have been discussed as the basis of the magnetic field sense of animals (Shaw et al. 2015), such as migratory birds, and the current understanding is that the avian magnetoreception system may include both a magnetite-based and a RPM-based component (Hore and Mouritsen, 2016). Alt- hough magnetite has been found in the human brain, human reception of the geo- magnetic field has not been found (Kirschvink et al. 1992).
Other mechanisms for direct biophysical interaction of MFs with organisms have been proposed. These include ionization and breaking of chemical bonds, forces on charged particles, effects with narrow bandwidth such as cyclotron reso- nance, Larmor precession and quantum mechanical resonance phenomena, as well as stochastic resonance. However, these mechanisms are not considered to provide a plausible explanation for effects at typical human exposure levels (WHO 2007).
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2.5 BIOLOGICAL AND HEALTH EFFECTS OF IF MFs
2.5.1 In vitro and in vivo studies
The biological effects of IF MFs have not been extensively studied. The 27 studies described in Table 3 have used 2-90 kHz MFs with magnetic flux densities ranging from 6.5 μT to 3.9 mT. Most studies have been conducted using either 20 kHz MFs, similar to those emitted by cathode ray tubes that were previously used as a com- puter displays, or 21 kHz or 23 kHz MFs similar to those generated by induction heaters. Two studies used 60 and 90 kHz MFs, as these frequencies are also used in induction heaters.
Although most in vivo studies detected no adverse effects after exposure to IF MFs, a few studies reported positive findings. Win-Shwe et al. (2015) exposed C57BL/6J male mice to sinusoidal 21 kHz, 3.8 mT MFs for 1 h/d during organogene- sis on days 7-17 of pregnancy or on postnatal days 27-48. They observed changes in memory related genes, but those changes were transient and were not observed in the group allowed to recover for one day.
A significant increase in the incidence of resorptions was reported by Frölen et al. (1993) in CBA/S mice. They exposed animals to a 20 kHz MF with sawtooth waveform and peak-to-peak flux density of 15 μT on gestational days 0-18, 1-18, 4- 18 or 6-18 for 24 h/d. The number of resorptions was statistically significantly in- creased, and this finding was replicated in 4 experiments. A significantly reduced weight and length of the exposed fetuses was also detected when the MF treatment began on day 7 post-conception. However, no increase of resorptions was reported by Huuskonen et al. (1998b) in CBA/Ca mice, and other developmental outcomes were not affected. Furthermore, Huuskonen et al. (1998a) failed to replicate the findings of Frölen et al. (1993) in CBA/S mice. Several studies (but not all) have reported increased incidence of minor skeletal anomalies in mice and rats exposed to 18-20 kHz magnetic fields (Huuskonen et al. 1993; Huuskonen et al. 1998a, b;
Kim et al. 2004). Overall, the evidence for an association between IF MF and repro- ductive and developmental effects is weak.
Biochemical and hematological analysis was performed and immune functions (cytotoxic or phagocytic activity, populations of T-cells) were measured in a study using male Sprague-Dawley rats at the age of 4-5 weeks after exposure to sinusoi- dal 21 kHz, 3.8 mT for 1 h/d for 14 days (Ushiyama et al. 2014). A significant change in inorganic phosphorus level in blood was observed, but other parameters did not show any detectable change.
Possible genotoxicity of IF MFs was evaluated in five in vitro studies, and in one in vivo study. No effects of IF MFs exposure on DNA damage, micronucleus for- mation or mutations were observed in these studies.
The in vivo study reported by Huuskonen et al. (1998a) found no effects of IF MF exposure (sawtooth, vertical 20-kHz, 15 μT peak-to-peak flux density, 24 h/d for 18 days) on micronuclei in the bone marrow of CBA/S mice.
33 Miyakoshi et al. (2007) exposed Chinese hamster ovary cells (CHO-K1) to 23 kHz MFs at 532 μT for 2 h. No effects were observed on single or double DNA strand breaks or micronucleus formation. This study also included measurements of mutagenicity using Salmonella typhimurium and Escherichia coli bacterial mutagen- icity tests, and the hypoxanthine-guanine-phosphoribosyl transferase (HPRT) gene mutation test in V-79 cells. No increase in mutation rates were observed. Nakasono et al. (2008) used sinusoidal 2 kHz MFs at 0.91 mT, 20 kHz MFs at 1.1 mT, and 60 kHz at 0.11 mT. Exposure to these fields for 48 h did not affect mutagenicity or co- mutagenicity in Salmonella typhimurium and Escherichia coli bacterial tests, or yeast strain Saccharomyces cerevisiae XD83 Gene Conversion Test. Ikehata et al. (2011) ex- posed the Chinese hamster lung cell line (CHL/IU) to 21 kHz MFs at 2, 3 or 3.9 mT for 24 h, and reported no effects on cell growth or micronucleus formation. Yoshie et al. (2011) studied the effects of 21 kHz MFs (2 or 3.9 mT for 24, 48 or 72 h) on cell growth using Chinese hamster ovary cells (CHO-K1) and its DNA repair deficient derivatives. No exposure related statistically significant changes were observed.
Furthermore, no effects on HPRT mutation frequency were observed in the CHO- K1 cell line after exposure to 21 kHz MFs at 2, 3 or 3.9 mT for 24 h. Shi et al. (2014) exposed human lens epithelial cells (HLECs) to sinusoidal 90 kHz MFs at 93.36 μT for 2 or 4 h. No effects were reported on proliferation, apoptosis or DNA single and double strand breaks.
2.5.2 Epidemiological studies
2.5.2.1 Video display terminals and reproductive risks
Several epidemiological studies have investigated possible health risks among women working with video display terminals (VDTs). Concerns about such health risks were raised in the 1980s after finding that the risk of adverse pregnancy out- comes was increased among several clusters of women who used VDTs. These studies investigated spontaneous abortion, intrauterine growth retardation, pre- term birth, stillbirth, birth weight, congenital malformations and development.
Windham et al. (1990) observed that the use of VDTs was associated with spon- taneous abortion and intrauterine growth retardation. They also found a stronger association of the use of VDTs with spontaneous abortions at earlier gestational days (≤ 12 weeks) as compared to later days. The risk of low birth weight was not increased. In contrast to the findings of Windham et al. (1990), several studies have detected no association between adverse pregnancy outcomes and use of VDTs (Landrigan et al. 1983; Kurppa et al.1985; Ericson and Källén 1986; Nielsen and Brandt 1990, 1992; Schnorr et al. 1991; Roman et al.1992; Grajewski et al. 1997;
Grasso et al. 1997). In all these studies, “exposure” was just use of VDTs, and no measurements of MFs exposure were done. As the MF emissions of VDTs are gen- erally very low, these studies are not informative for assessment of possible effects associated with higher exposures.
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Only three studies measured the EMFs of the VDTs. Schnorr et al. (1991) con- ducted a cohort study among female telephone operators. In this study, the meas- urements of MF emissions were conducted at 30 cm from each side of the VDT unit.
The measurements were also performed for the face, chest and abdomen while operators were seated. The abdominal exposure to IF (15 kHz) MFs was higher among VDT operators than among those who did not use VDTs. No association was detected between spontaneous abortion and VDT use during the first trimester.
Using the same cohorts of telephone operators, Grajewski et al. (1997) showed no association between use of VDT and risk of reduced birthweight and preterm birth.
Lindbohm et al. (1992) carried out a retrospective study of miscarriages among women employed in three companies in Finland during years 1975-85. The MF exposure from VDTs was assessed based on information on the VDTs model used by each study subject and laboratory measurements of IF and ELF MFs at a fixed location around these VDT models. Although the risk of miscarriages was in- creased among those women who used VDTs with exceptionally high ELF MF emissions, no association of miscarriage risk with IF MF exposure was observed.
2.5.2.2 Electronic article surveillance systems and reproductive risks
Recently, a register-based cohort study was carried out by Khan et al. (2018). They investigated the association of reproductive risks with 8.2 MHz MF exposure among cashiers working near EAS systems. In this study including 4157 women, those who had worked as cashiers in supermarkets with EAS systems were consid- ered as exposed, and workers of grocery stores without EAS systems were consid- ered as unexposed. No adverse effects were detected on the risk of miscarriage, reduced birth weight, gestational age, or preterm birth. Low exposure level was a limitation of the study. The women were found to be exposed to IF MFs (up to 1.38 μT) only when they passed by the gates at near distance; the IF MFs at the cashier’s seat varied from 0.004 to 0.055 μT and did not differ between the two store types.
