Chlordioxid (CDL) gegen Coronavirus - Sammlung wissenschaftliche Studien

Chlordioxid gegen Coronavirus Ein revolutionärer, einfacher und effektiver Ansatz Sammlung wissenschaftliche Studien

3. überarbeitete Auflage 09. Juli 2021 Zusammengestellt von Reinhard Wirth Kontakt: wirth-reinhard@t-online.de

Inhaltsverzeichnis Vorwort………………………………………………………………………………………………………………………………………..

1

Petition an den Bundesgesundheitsminister Jens Spahn………………………………………………………………

2

Was Politiker und Ärzte jetzt über Chlordioxid wissen müssen – Die wichtige Reise von Andreas Kalcker durch Südamerika……………………………………………………………………………………………………………

3

COVID19 – Angst und Arroganz; das unheilige Geschwisterpaar………………………………………………….

4

Chlordioxid gegen Coronavirus: ein revolutionärer, einfacher und effektiver Ansatz…………………..

5

Eine neue Perspektive zur Vorbeugung und Heilung von COVID-19-Patienten…………………………….

6

Journal of Molecular and Genetic Medicine, 2021 - Determination of the Effectiveness of Chlorine Dioxide in the Treatment of COVID 19…………………………………………………………………………..

7

Can chlorine dioxide prevent the spreading of coronavirus or other viral infections? Medical hypotheses…………………………………………………………………………………………………………………………………..

8

Biological Efficacy of Chlorine Dioxide………………………………………………………………………………………….

9

Internationales Patent: A method to treat blood (Chlordioxid in Blutkonserven) ……………………….

10

Investigation on virucidal activity of chlorine dioxide. Experimental data on Feline calicivirus, HAV and Coxsackie B5…………………………………………………………………………………………………………………

11

Ungewöhnliche Merkmale des SARS-CoV-2-Genoms, die auf eine ausgeklügelte Labormodifikation hindeuten………………………………………………………………………………………………………

12

Chlorine Dioxide Is a Size-Selective Antimicrobial Agent……………………………………………………………..

13

Efficacy and Safety Evaluation of a Chlorine Dioxide Solution……………………………………………………..

14

Kontrollierte klinische Evaluierungen von Chlordioxid, Chlorit und Chlorat am Menschen………….

15

Controlled Clinical Evaluations of Chlorine Dioxide, Chlorite and Chlorate in Man………………………

16

Behandlung mit Medikamenten, die noch nicht zugelassen sind…………………………………………………

17

WMA Deklaration von Helsinki – Ethische Grundsätze für die medizinische Forschung am Menschen……………………………………………………………………………………………………………………………………

18

Verordnung über das Inverkehrbringen von Arzneimitteln ohne Genehmigung oder ohne Zulassung in Härtefällen (Arzneimittel Härtefall-Verordnung - AMHV)…………………………………………

19

EXPERIMENTELLE FORSCHUNGSARBEITEN DER ECUADORIANISCHEN VEREINIGUNG VON FACHÄRZTEN FÜR INTEGRATIVE MEDIZIN…………………………………………………………………………………..

20

Vorwort Wir möchten hier in einem kurzen Vorwort von einer Begebenheit berichten, welche wir selber im Jahr 2020 erlebt haben und kaum fassen können, was wir mit einem Virus erlebt haben. Die Corona Krise war doch schon schlimm genug für uns alle. Glücklicherweise leben wir als Schleswig-Holsteiner in einem Land zwischen den Meeren und haben immer frische Luft. Corona war hier nicht so stark verbreitet wie in Ballungsgebieten. Die täglichen Mund-Nasenbedeckungen gaben uns allerdings schon Sorgen auf, denn wir wissen ja alle, dass die Gewebestruktur viel zu großmaschig ist um das Virus aufhalten zu können. Sowohl beim Einatmen, als auch beim Ausatmen kann das Virus wie eine Fliege durch einen Maschendrahtzaun schlüpfen. Viele Menschen schauen uns jetzt anders an als früher oder gar nicht mehr. Alle haben Angst.

Besondere Sorgen machen uns aber die Enkelkinder, denn die müssen sogar schon im Unterricht mit dem „Schnutenpulli“ rumsitzen.

Wird Impfen die Lösung bringen? Sind 750.000 im Voraus geschätzte Impfschäden (O-Ton Bill Gates) weltweit vertretbar? Immer wieder ertappen wir uns bei solchen Zahlenspielen und wischten mit den täglichen Nachrichten aus allen Kanälen unserer Medienwelt solche Bedenken einfach weg. Was aber ist, wenn das Virus mutiert und der Impfstoff dann wirkungslos ist? Was ist, wenn 2021 oder 2022 ein ganz neues Virus auftaucht, das noch schlimmer ist als SARS-CoV-2? Fängt dann wieder alles von vorne an und wir haben immer noch kein Heilmittel für die Erkrankten? Impfen ist ja nur Vorbeugung. Was aber haben wir als Therapie anzubieten? Wir standen alle unter Stress und hatten Angst.

In dieser Situation griff uns, bzw. meine Frau Ende August ein anderes Virus an. Am Freitagabend klagte sie über Schmerzen am rechten Hals. Die Lymphkoten waren heftig angeschwollen und über der rechten Augenbraue auf der Stirn, in der Augenbraue selbst und auf dem Augenlid bildeten sich schmerzhafte rote Pocken. Über das Wochenende blühten die Pocken in voller Pracht, platzen auf und nässten. Am Montag diagnostizierte der Hausarzt eine Gesichtsrose, deren Ursache das Herpes Zoster Virus ist. Meine Frau fühlte sich echt elend und hatte große Schmerzen. Das Herpes Zoster Virus verursacht auch Gürtelrose und Windpocken.

Der Arzt verschrieb ihr vier Medikamente. Das erste Medikament war gegen das Virus, das zweite Medikament gegen die Nebenwirkungen des ersten, denn das würde Epilepsie auslösen. Das dritte sei gegen die Schmerzen und das vierte eine Augensalbe gegen Erblindung. Er erklärte eine Zeit von 6-8 Wochen für den Behandlungszeitraum und verbot ihr das Autofahren, da die Medikamente sie ausknocken würden. In dieser Situation klingelte es bei uns im Kopf und ein etwas verdrängtes Wissen meldete sich an der Oberfläche. Da gab es doch so eine Chemikalie namens Chlordioxid.

Schnell wurde in den Recherchen klar, dass es seit Jahren ein unglaubliches Gezerre um das Thema gibt.

Hauptsächlich richten sich die Aussagen der Verbraucherzentralen, des Bundesinstitutes für Risikobewertung und des Bundesamtes für Verbraucherschutz und Lebensmittelsicherheit und auch die USamerikanische Lebensmittelüberwachungs- und Arzneimittelbehörde (FDA) gegen MMS, welches auch Chlordioxid enthält. Andererseits stießen wir auch auf Heilungszeugnisse. Nach einigem Hin und Her entschied sich meine Frau das mit dem Chlordioxid einmal auszuprobieren. Falls das nicht funktioniere, könne sie ja immer noch die Medikamente nehmen, sagte sie.

Noch am selben Tag besorgten wir eine 0,3%ige wässrige Lösung Chlordioxid (CDL) und mischten 30ml dieses Trinkwasserdesinfektionsmittels zu 3 Liter Leitungswasser hinzu. Mein Frau trank also jeden Tag 3 Liter Wasser mit CDL. Die Verdünnung war so groß, dass man das nicht wirklich riechen oder schmecken konnte. Am zweiten Tag waren die Schmerzen verschwunden und der Hals etwas dünner. Sie war jetzt nicht mehr schläfrig, sondern putzmunter. Am dritten Tag war der Hals abgeschwollen und die roten Pocken auf der rechten Gesichtshälfte flach und nässten nicht mehr. Am Ende des vierten Tages waren die Pocken nur noch rosa blasse Flecken. Da beschlossen wir die Therapie zu beenden. Sie war vollkommen genesen. Von den Medikamenten des Arztes hatte sie nichts angeführt. Kannte der Arzt CDL etwa gar nicht?

Das hat uns dermaßen verblüfft, dass wir weitere Recherchen zu dem Mittel unternahmen und stießen auf so kernige Aussagen wie: „Chlordioxid bekämpft auch Corona!“. Jetzt wollten wir es genauer wissen und konnte kaum glauben, dass dies sogar der Donald Trump öffentlich in einer Pressekonferenz gesagt hatte.

Wir wissen heute genau was dann geschah. Die öffentlichen Medien verspotteten den Präsidenten in Grund und Boden. Bleichmittel sollen wir trinken! Das ginge ja gar nicht!

Aber unsere Recherchen gingen weiter. Wir stießen auf eine Gruppe von Ärzten in Südamerika, die in Ermangelung von Medikamenten und aufgrund einer sehr hohen Mortalität in ihrem Land Experimente mit Chlordioxid machten und tatsächlich Erfolge erzielten. Angeblich sollen hochgradig erkrankte Menschen buchstäblich in letzter Minute durch eine intravenöse Gabe des Mittels vor dem Tod gerettet worden sein.

Das musste weiter recherchiert werden. Uns hatte man in Deutschland gebetsmühlenartig erzählt, dass es keine Heilmittel geben würde und wir auf einen Impfstoff warten müssten. Bis es so weit sei, müssen drastische Maßnahmen ergriffen werden.

Dann kam eine wirklich aussagestarke Botschaft aus Bolivien.

Mehr als 4000 Ärzte haben sich angeblich davon überzeugen lassen und gehen jetzt diesen Weg mit CDL. Sie laufen in ihren Krankenhäusern jetzt teilweise ohne Mundschutz herum und behandeln ihre Patienten mit Chlordioxid, hieß es.

Gut organisierte Gruppen von Ärzten gingen auf dem Land von Haus zu Haus und reichten Chlordioxid in Wasserflaschen in die Häuser, wo Menschen mit Symptomen lebten. Die Kurve der Neuinfizierten sank seit September 2020 bis heute dramatisch ab. Jeder Neuinfizierte kann in Bolivien CDL bekommen, wenn er es wünscht. Die Regierung hat dieses Vorgehen legalisiert. Natürlich haben sich die Ärzte und Mitarbeiter ebenfalls mit CDL abgesichert und stecken sich nicht an, weil sie es prophylaktisch einnehmen. Hammer! Was für eine Kontroverse gegenüber Europa!

Während In USA und Deutschland von behördlicher Seite massiv vor Chlordioxid gewarnt wird, werden auf der anderen Seite der Erde Menschen von COVID19 geheilt. Während man hierzulande Chlordioxid als grobe Verfehlung gegen COVID19 vom Tisch gewischt hat und auf Lockdown, AHA und Impfstoffe baut, kommt ein Drittweltland daher und zeigt uns wie man es anders machen kann. Während man hierzulande von Bleichmittel und schädlichen Nebenwirkungen spricht, wird völlig außer Acht gelassen, dass es eine USamerikanische klinische Doppelblind-Studie aus dem Jahr 1982 gibt, die eine Unbedenklichkeit bescheinigte (hier in der Dokumentensammlung). Während hierzulande die Menschen der schädlichen Wirkung eines Virus je länger, desto schlimmer mit Langzeitauswirkungen ausgesetzt werden bis hin zur Inkaufnahme des Todes, werden in Südamerika die Patienten innerhalb von vier Tagen symptomfrei, und zwar ambulant!

Irgendetwas stimmt hier nicht mit der Kommunikation. Wir möchten den Bundesgesundheitsminister mit einer Petition, die deutsche Regierung als Ganzes und die Ministerien dazu auffordern, ein Untersuchungsteam nach Bolivien zu entsenden und/oder eigene wissenschaftliche Studien zu beginnen, bzw. die schon vorhandenen Studien in dieser vorliegenden Sammlung gewissenhaft auszuwerten.

Wissenschaft ist nicht Internet, sondern messen und wiegen vor Ort. Die oben erwähnten Ärzte in Südamerika, über 4000 an der Zahl, müssen doch zu sprechen sein, ebenso die geheilten Patienten. Wir möchten die Menschen finden, welche sich in der aktuellen Pandemielage einen Augenblick lang geistig auf die andere Seite stellen können um das Gefühl kennenzulernen, welches man hat, wenn man ein 100%iges Heilmittel hätte. Pandemie, Maske und Lockdown adieu!

Wir als Familie haben dieses Gefühl. Wir leben vollkommen angstfrei, denn wir haben seit August 2020 Chlordioxid in unserer Hausapotheke. Viren haben bei uns keine Chance mehr.

Reinhard Wirth Struxdorf, 10.11.2020

Therapieansatz bei Herpes Zoster Virus (Windpocken, Gürtelrose, Gesichtsrose)

Bild oben: die Medikamente Bild links: Gesichtsrose (Herpes Zoster Virus), Bildquelle: www.netdoktor.de

Zum Vergleich: Therapie bei Gesichtsrose Herpes Zoster Virus Medikamente vom Arzt: Chlordioxid (CDL): Knockout, heftige Nebenwirkungen, Gefahr von Schmerzfrei schon nach dem 1. Tag, nach 4 Epilepsie, 6-8 Wochen Krankheit mit Schmerzen Tagen geheilt, Wunden geschlossen, null u. offene Wunden, ansteckend, Gefahr von Nebenwirkungen, Cent-Beträge Erblindung, teure Medikamente

An den Deutschen Bundestag Petitionsausschuss Platz der Republik 1 11011 Berlin

-

Für Ihre Unterlagen

Petition an den Deutschen Bundestag (mit der Bitte um Veröffentlichung)

Persönliche Daten des Hauptpetenten Anrede

Herr

Name

Wirth

Vorname

Reinhard

Titel

Anschrift Wohnort

Struxdorf

Postleitzahl

24891

Straße und Hausnr.

Aruper Straße, 30

Land/Bundesland.

Deutschland

Telefonnummer

04623183973

E-Mail-Adresse

wirth-reinhard@t-online.de

Petition 117186 - 20. October 2020

-

Petition an den Deutschen Bundestag (mit der Bitte um Veröffentlichung)

Seite 2

Wortlaut der Petition Wissenschaftliche Studie zu Chlordioxid zur Bekämpfung des COVID-19 Virus.

Begründung Buchstäblich hunderte Patienten bezeugen vor allem in Nord- und Südamerika, aber auch in Deutschland und den europäischen Nachbarländern die rasche Genesung von einer COVID-19 Infektion nach einer Behandlung mit Chlordioxid in wässriger Lösung und geringer Dosierung. Die Chemikalie wird von zahlreichen Experten eher als giftig und gefährlich eingestuft. Da die Genesungen hingegen aber Fakten sind und gut belegt werden können und zur großen Überraschung keinerlei unerwünschte Nebenwirkungen bekannt sind, fordern wir den medizinische Dienst auf, er möge eine Studie in Auftrag geben, welche den Wirkmechanismus von Chlordioxid im menschlichen Körper erklärt und als Option für einen Behandlung bewertet bzw. ggf. zulässt.

Chlordioxid ist ein völlig neuer antiviraler Ansatz im Kampf gegen COVID-19. Bekannt ist, dass Chlordioxid ein Oxidationsmittel ist und durch Verbrennung jede Unterart oder mutierte Variante eines Virus zuverlässig eliminieren kann. Aus diesem Grund wird derzeit Chlordioxid obligatorisch jeder Blutkonserve beigesetzt um ein mögliches HIV-Virus in der Konserve zu eliminieren. Bei einer Transfusion gelangt Chlordioxid in den menschlichen Blutkreislauf. Chlordioxid ist auch zur Desinfektion von für den Verzehr bestimmtes Trinkwasser offiziell zugelassen.

Angesichts der Notsituation, in der wir uns derzeit mit COVID-19 befinden, ist die orale Verwendung von Chloroxid für viele Menschen schon heute eine Option nach der sie greifen. Sie ist im Handel schnell verfügbar und wird ausnahmslos ohne ärztliche Anleitung auf eigene Verantwortung angewendet. Die Ablehnung diese Studie würde die orale Einnahme von Betroffenen nicht verhindern, wohingegen ein Forschungsauftrag bzw. eine offizielle Studie dazu führen würde, dass die missbräuchliche Anwendung von angstgetriebenen Menschen verhindert werden kann und es nicht zu Überdosierungen und Vergiftungen kommt. Die Dosis macht das Gift, heißt es. Eine wissenschaftliche Studie kann Ärzten, Kliniken, Apotheken die Legalität und die korrekte Dosierung an die Hand geben, ja im günstigsten Fall sogar ggf. zu einer Beherrschung der Pandemie führen.

Anregungen für die Forendiskussion Mehr als 50 Video-Zeugnisse von Patienten bestätigen die Wirksamkeit von Chlordioxid bei ihrer Infektion mit COVID-19. Zahlreiche Menschen in Südamerika sind buchstäblich in letzter Minute dem sicheren Tod entronnen, nachdem sie Chlordioxid oral oder intravenös bekamen. Wäre das auch eine Option für für Deutschland? https://lbry.tv/@Forbiddenhealth:1/Covid-endlich-besiegt:6

Petition 117186 - 20. October 2020

Petition an den Deutschen Bundestag (mit der Bitte um Veröffentlichung)

Seite 3

Soweit Sie es für wichtig halten, senden Sie bitte ergänzende Unterlagen in Kopie (z.B. Entscheidungen der betroffenen Behörde, Klageschriften, Urteile) nach Erhalt des Aktenzeichens auf dem Postweg an folgende Kontaktadresse:

Deutscher Bundestag Sekretariat des Petitionsausschusses Platz der Republik 1 11011 Berlin Tel: (030)227 35257

Petition 117186 - 20. October 2020

Was Politiker und Ärzte jetzt über Chlordioxid wissen müssen EHRENDOKTORWÜRDE FÜR ANDREAS KALCKER - CHLORDIOXID AUF DEM VORMARSCH - DIE UNGLAUBLICHE REISE DURCH SÜDAMERIKA.

Öffentliche Vorträge über Chlordioxid.

An jedem Ort seiner Vorträge das gleiche Bild.

Hunderte Menschen kamen um die Vorträge von Dr. Andreas Kalcker über Chlordioxid anzuhören.

Warum sie kamen? Praktisch jeder hatte Familienmitglieder oder Freunde, die an Corona erkrankt sind. Viel zu viele sind gestorben. In Lateinamerika wütet Corona viel heftiger, als in Deutschland. Offensichtlich wirkt der Krankheitserreger dort etwas anders als bei uns. Von der Infektion bis zum Tod vergehen oftmals nur wenige Tage. Doch nachdem man Chlordioxid in wässriger Lösung (CDL / CDS) entdeckt und eingesetzt hatte, entgingen diese Patienten dem Tod. Das sprach sich rasend schnell in der Bevölkerung herum und so kamen sie scharenweise zu seinen Vorträgen.

Quelle mit Video: https://gehtanders.de/was-politiker-und-aerzte-jetzt-ueber-chlordioxid-wissenmuessen/

Bis zu sechs Interviews am Tag. Der deutschstämmige Andreas Kalcker war ein gefragter Mann. Er bereiste vornehmlich Bolivien und Mexiko, informierte auf Vorträgen in fließend spanischer Sprache von der Wirkungsweise von CDS und verwies auf die Heilungserfolge von Menschen, die er persönlich kannte. Zahlreiche TV-Sender und Radiostationen luden ihn als Gast zu einem Interview ein. Die Menschen feierten ihn teilweise wie einen Popstar. Statt Passkontrolle wollten die Beamten am Flughafen ein Selfie mit ihm machen. Nicht wenige wollten ihn umarmen, da durch Anwendung von CDS sie selbst oder ihre Kinder gerettet wurden, andere sprachen ihn auf der Straße an und bedanken sich überschwänglich.

Das Militär und CDS. Insbesondere Ärzte und Militärangehörige wussten um den Wert von CDS, denn sie gehörten zu den ersten Menschen, die auf das Mittel zurückgegriffen hatten, als sie selbst schwer erkrankten. Sie könnten die Folgen ihrer Erkrankung am besten einschätzen und so wurden sie teilweise buchstäblich in ihrer letzten Stunde durch intravenöse Gabe von CDS gerettet und geben heute kräftige Zeugnisse ab. Jetzt weht dort ein anderer Wind. Andreas Kalcker wurde von der staatlichen Universität UNAM (Universidad Nacional Autónoma de México) in Mexiko-Stadt einen Ehrendoktortitel verliehen und ein Dutzend weiter Auszeichnungen, die in dem Video unten gezeigt werden.

Krankenhäuser und Apotheken machen mit.

Die Dokumentation zeigt, dass viele Krankenhäuser und Apotheken mitmachen und CDS selber herstellen. In Bolivien stellt die ehrwürdige Universität UPEA sogar selbst CDS her, wie wir bereits in unserem Beitrag Chlordioxid Massenproduktion in Universität El Alto (UPEA) Bolivien berichtet haben. Sie verteilen es an die Bevölkerung oder verkaufen es in Apotheken. Nebenbei probierten einige Ärzte aus der COMUSAV.com Bewegung CDS auch bei anderen Krankheiten mit großem Erfolg aus. Das Kind Elias z.B. hatte Leukämie im Endstadium und sollte zum Sterben nach Hause geschickt werden.

Doch der Arzt gab ihm CDS und nun ist der Junge vollkommen gesund wieder zu Hause.

Fachmagazin veröffentlicht wissenschaftliche Studie zu CDS. Nicht überall wird CDS wohlwollend entgegengenommen. In Kolumbien z. B. wird derzeit auf Chlordioxid noch eine regelrechte Hexenjagd gemacht. Nicht anders ist es in Deutschland und USA. Menschen werden verklagt, wenn sie MMS oder CDS gegen Corona anpreisen, die Verbraucherzentralen und selbsternannten Faktencheckern bis hin zu den offiziellen Gesundheitsbehörden und bezahlten YouTube-Influenzern jagen diese Idee mit falschen Killerargumenten und sind bis zur Stunde nicht bereit die Wahrheiten zu prüfen. Nun hat ein angesehenes zitierfähiges Fachmagazin (Journal of Molecular and Genetic Medicine) eine sehr erfolgreiche CDS-Studie an Menschen mit COVID-19 veröffentlicht. Jetzt kann kein MMS / CDS – Kritiker mehr behaupten es gäbe keine wissenschaftlichen Studien. Auch wir haben uns mit unserem Blog sehr bemüht solche Studien zur Verfügung zu stellen. Politiker und Ärzte müssen das eben mal lesen. Unser Blog fordert seit Monaten dazu auf und scheibt die Regierungsvertreter immer wieder an, denn wir sagen: Pandemie besiegen ist möglich und das geht anders!

Was COVID-19 im Körper macht und wie CDS Viren bekämpft.

Im folgenden Bericht hier unten berichtet Andreas Kalcker von den Ergebnissen der medizinischen Forschung der Ärzte. So wissen sie heute, dass die Menschen an der Koagulierung des Blutes in den Arterien sterben. Die roten Blutkörperchen kleben zusammen. Sie können in diesem Zustand keinen Sauerstoff mehr transportieren und verstopfen feine Arterien. Chlordioxid löst diese Verklumpung sehr rasch auf und liefert Sauerstoff direkt ins Blut und erhöht den PH-Wert. Es handelt sich also nicht vernehmlich um eine Lungenkrankheit, sondern um eine Blutkrankheit, weshalb eine künstliche Beatmung mehr schadet als nützt.

Was man bei der Behandlung mit Chlordioxid wissen muss.

Je früher ein infizierter Körper Chlordioxid bekommt, desto geringer sind auch die Langzeitschäden durch das Virus. Mutationen spielen keine Rolle. Bei einer Sauerstoffsättigung im Blut unter 60% hilft jedoch meistens nur noch die intravenöse Gabe, so die Erfahrung der Ärzte. Übrigens, Nebenwirkungen oder Resistenzen wurden nicht festgestellt. Wechselwirkungen mit anderen Substanzen hingegen durchaus. Da Chlordioxid durch Oxidation seine Wirkung entfaltet, dürfen während der Behandlung z. B. keine Antioxidantien wie Vitamin C gegeben werden. Auch blutverdünnende Medikamente müssen in der Zeit abgesetzt werden, da CDL wie oben beschrieben selbst blutverdünnend ist. Unsere Politiker und auch zahlreiche Ärzte in Deutschland sind alle informiert worden. Eine Petition wurde eingereicht.

Jetzt liegt unser Schicksal in deren Händen oder eben bei uns selbst.

COVID19 – Angst und Arroganz; das unheilige Geschwisterpaar.

GAMECHANGER - WARUM IN BOLIVIEN CORONA BESIEGT WORDEN IST.

Als COVID19 in Bolivien angekommen war.

Vielleicht ist es auch nur pure Dummheit, Engstirnigkeit, Unbelehrbarkeit, doch egal wie wir es auch bezeichnen, letztlich sind es solche Gründe, warum wir Europäer seit Monaten fest in dem Klammergriff der COVID19 Pandemie stecken und die Maßnahmen der Regierungen unser Leben zermürben. Was macht Bolivien anders?

Dieses kleine Land im Herzen von Südamerika mit etwas mehr als 11 Millionen Einwohnern hatte auch Angst vor dem Tod durch COVID19. Völlig berechtigt, denn das Virus dort war von der besonders schlimmen Sorte. Irgendwann trauten sich die Leute nicht mehr ins Krankenhaus, weil sie beobachteten, dass die Verwandten von dort nicht mehr zurückkommen.

Eine wichtige Erkenntnis setzte sich durch.

Dann hörten immer mehr Ärzte von Chlordioxid in wässriger Lösung und dass ein paar Menschen sich damit behandeln ließen.

Zunächst war dies nur eine Art Geheimtipp, der im Untergrund weiterverbreitet wurde, denn es herrschte dieselbe Borniertheit wie auch hierzulande in Deutschland. Man sprach von Bleichmittel und von Gefährlichkeit.

Mittlerweile aber sollen mehr als viertausend Ärzte in Bolivien einen Heileffekt dieses seltsamen Mittels erkannt haben. Glaubt man den Recherchen in Bolivien und zieht man ein Fazit aus den oft widersprüchlichen Beiträgen im Internet, so dauert es wohl noch einige Zeit, bis sich auch die Wissenschaftler des Landes einig sind.

Die klinische Doppelblindstudie aus USA. Immerhin gab es vor fast vierzig Jahren schon einmal das Bedürfnis nach Klarheit bzgl. der Wirkungen von Chlordioxid im menschlichen Körper, weil man sich um die Qualität des Trinkwassers sorgte. Bekanntermaßen wird Trinkwasser sehr häufig aus Stauseen entnommen und durch Rohrleitungen gepumpt. Wer schon einmal in Italien geduscht hat, vernimmt einen leicht chlorhaltigen Geruch im Badezimmer. US-amerikanische Die US-amerikanische Unbedenklichkeitsstudie Wissenschaftler gaben im Jahr 1982 eine Studie in Auftrag, denn man musste herausfinden wie Desinfektionsmittel (Chlor, Chlorat und Chlordioxid) im menschlichen Organismus wirkt, wenn man es dem Trinkwasser zusetzt. Die klinische Doppelblindstudie brachte zutage, dass es keine negativen Auswirkungen gäbe und so wurde Chlordioxid auch in Deutschland zur Trinkwasserdesinfektion als unbedenklich zugelassen. Die US-Studie liegt heute sogar in Deutsch vor.

Was in Bolivien gemacht wird.

Indem man Natriumchlorit und 4%ige Salzsäure zu gleichen Teilen zusammenführt, entsteht in einer chemischen Reaktion gasförmiges Chlordioxid. In einem geschlossenen Gefäß mit Wasser kann das Gas kondensieren und man erhält CDL (Chlordioxidlösung). Praktisch jeder Apotheker kann diese Lösung herstellen und so geschieht es derzeit in Bolivien. Doch ungefährlich ist es dennoch nicht, denn es kommt auf die richtige Auch in Ecuador benutzt man Chlordioxid Dosierung an. Es gehört daher in die Hände von Ärzten und Kliniken und selbstverständlich muss deshalb von behördlicher Stelle gewarnt werden. Die Ärzte in Bolivien fanden heraus, dass eine Gabe von 30ml einer 3%igen wässrigen Lösung Chlordioxid pro Tag genügen, um COVID19 zuverlässig zu eliminieren. Als Zugabe in 3 Liter Trinkwasser genüge diese äußerst geringe Konzentration anscheinend vollkommen um das Virus SARS-CoV-2 und auch „normale“ Grippe-Viren oder Herpes Zoster Viren im menschlichen Körper abzutöten. Sogar intravenös soll es gegeben worden sein, so die Aussage der Bolivianischen Mediziner.

Die kognitive Dissonanz.

Drei Liter Wasser täglich? Nur vier Tage lang?

Ist Chlordioxid der Gamechanger? Was bedeutet nun dieser Vorstoß in Bolivien für uns? Da gibt es im Internet z. B. die Seite correctiv.org/faktencheck mit dem Titel „Irreführende Behauptungen über Einsatz von Chlordioxid gegen Corona in Bolivien“ von Kathrin Wesolowski. Einseitig akribisch legte sie sich bereits in der Überschrift auf die Kontraseite fest und listet alle bekannten Links USA und Deutschland warnen vor, Bolivien heilt mit zu den deutschen Gesundheitsbehörden wie Chlordioxid die Verbraucherzentrale, das Bundesinstitut für Risikobewertung und das Bundesamt für Verbraucherschutz und

Lebensmittelsicherheit und auch die US-amerikanische Lebensmittelüberwachungs- und Arzneimittelbehörde (FDA) auf, um Chlordioxid als schlimme Verfehlung bei COVID19 darzustellen. Seriös recherchiert in Bolivien hat sie nicht. Sie schrieb einfach ab, was man im Internet so findet. Man bezieht sich bei den angegebenen Links im Wesentlichen auf das artverwandte MMS, das im Grunde auch Chlordioxid enthält.

Fake News oder Fakten?

Nun haben wir den Salat. Auf der einen Seite die Warnungen der Behörden und auf der anderen Seite die geheilten Patienten. Wir können davon ausgehen, dass den ehemaligen Patienten diese Warnungen egal waren, denn sie gingen die Behandlung auf eigenes Risiko ein. Es ist zu vermuten, dass sie vor der Behandlung die Folgen der Corona Krankheit mit den Risiken von Chlordioxid abgewogen hatten. Laut Zeugnisse der Betroffenen waren sie jedoch selbst von der raschen Genesung angenehm überrascht und beklagten keinerlei Nebenwirkungen. Es ist praktisch dieselbe Erfahrung, die auch schon die Probanden im Jahr 1982 anlässlich der US-Studie machten. Jüngsten Berichten zufolge, gehen in Bolivien jetzt über viertausend Ärzte diesen Weg. Und bei uns? Im Gegensatz zum Rest der Welt – einschließlich Deutschland – fällt in Bolivien die statistische Kurve der tatsächlich Corona Kranken gegen null. Die Krankenhäuser sind leer. Das sind Fakten und keine Fake News.

RKI, Jens Spahn, Petition – Bollwerke und Verhinderer.

Was soll man davon halten? Wer heilt hat Recht, oder? Der Autor dieses Artikels hat eine Petition an den Bundesgesundheitsminister Jens Spahn verfasst, in der er den Minister auffordert eine deutsche Studie in Auftrag zu geben, um das Phänomen Bolivien, bzw. die Wirkung von Chlordioxid im Zusammenhang mit COVID19 zu erforschen. Im nachfolgenden Video wird in einem Interview mit Andreas Kalcker die Lage in Bolivien dargestellt. Nach unserer Auffassung können die Demonstrationen auf der Straße nicht die Wende bringen, wohingegen Chlordioxid das Potenzial besitzt ein echter Gamechanger zu sein. Andreas Kalcker schildert die Situation und berichtet von den Aktivitäten, die in naher Zukunft aller Welt vorgestellt werden.

LIVE – Interview mit Andreas Kalcker – Corona ist besiegt worden

Video: https://www.mediarebell.com/watch/IaMXbLAcKTJOSUx Dauer: 49:56 Min. Thema: Die jüngste Entwicklung in Bolivien.

Chlordioxid gegen Coronavirus: ein revolutionärer, einfacher und effektiver Ansatz.

March 2020 DOI: 10.13140/RG.2.2.23856.71680 License CC BY-NC-SA 4.0 Project: Toxicity study of chlorine dioxide in solution (CDS) ingested orally, Andreas Ludwig Kalcker co. : Liechtensteiner Verein für Wissenschaft und Gesundheit LI-9491 Ruggel www.lvwg.org E-mail alk@lvwg.org

Chlordioxid ​(ClO​2​) wird seit über 100 Jahren zur Bekämpfung aller Arten von Bakterien, Viren und Pilzen eingesetzt. Es ist als Desinfektionsmittel anerkannt und wirkt durch Oxidation. ​[1 # BiologicalEfficacyList ] Es ist der Funktionsweise unseres eigenen Körpers sehr ähnlich, beispielsweise bei der Phagozytose, bei der ein Oxidationsprozess verwendet wird, um alle Arten von Krankheitserregern zu eliminieren.

Chlordioxid ​(ClO​2​) ​ist ein gelbliches Gas, das bisher als Wirkstoff nicht Eingang in die herkömmlichen Arzneibücher gefunden hat, obwohl es zur Desinfektion und Aufbewahrung von Blutbeuteln für Transfusionen vielerorts obligatorisch ist. ​[2# Alcide studies on blood disinfection] E ​ s wird auch in den meisten zum Verzehr geeigneten Mineralwässern in Flaschen verwendet, da es keine toxischen Rückstände hinterlässt. Es ist ein in Wasser sehr gut lösliches Gas, das ab 11 °C verdunstet.

Die jüngste Covid-19-Coronavirus-Pandemie erfordert dringende Lösungen mit neuen Ansätzen. Aus diesem Grund verspricht Chlordioxid (ClO​2​) in wässriger Lösung in niedrigen Dosen eine ideale, schnelle und wirksame Lösung zur Eliminierung dieses Virus zu sein. Einerseits wissen wir, dass Viren absolut oxidationsempfindlich sind, und andererseits, wenn sie in menschlichen Blutkonserven gegen Viren wie HIV und andere Krankheitserreger wirken, warum sollten sie dann nicht auch gegen das Coronavirus wirken?

1. Chlordioxid entfernt Viren durch einen selektiven Oxidationsprozess in sehr kurzer Zeit.

Dies geschieht durch Denaturierung der Kapsidproteine und anschließend durch Oxidation des genetischen Materials des Virus, wodurch dieses deaktiviert wird.

Die Anwendung von Chlordioxid (ClO​2 ​orale oder sogar intravenös) ist ein Ansatz, der von Andreas Ludwig Kalcker seit über dreizehn Jahren mit dem Ergebnis von drei pharmazeutischen Patenten zur parenteralen Anwendung untersucht wurde. Es kann von jeder Apotheke als ​Magistralrezeptur ​hergestellt werden und wurde in ähnlicher Form als (DAC N-055) im alten Deutschen Arzneimittelkodex als “Natrium Chlorosum” seit 1990

verwendet.Soweit bekannt, wurden zur Bekämpfung des Corona Virus bisher nur auf Impfstoffen basierende Lösungen vorgeschlagen, die zu extrem langsamen und auch riskanten Prozessen im menschlichen Körper führen, da sie vom jeweiligen menschlichen Körper immer ausreichende Energiereserven erfordern, die ein von der Krankheit betroffener Körper nicht bereitstellen kann. Der große Vorteil von Chlordioxid (ClO​2​) besteht dagegen darin, dass es im Gegensatz dazu die Zellfunktion und damit das Immunsystem des menschlichen Körpers unterstützt und somit zusätzliche Energien schafft, statt diese aufzubrauchen.

Davon abgesehen funktioniert Chlordioxid (ClO​2​) für alle viralen Unterarten. Auch gibt es keine möglichen Resistenzen gegen diese Art der Oxidation. [#3 Investigation on virucidal activity of chlorine dioxide] E ​ s ist zu beachten, dass diese Substanz seit 100 Jahren im Abwasser verwendet wird, ohne irgendeine Art von Resistenz zu erzeugen.

2. Es gibt bereits wissenschaftliche Beweise dafür, dass Chlordioxid sowohl gegen das Coronavirus ​SARS-CoV-2, dem ​Stammvirus von COVID-19 ​[SARS Fact Sheet, National Agricultural Biosecurity Center, Kansas State University] , ​und in der Coronavirus-Familie im Allgemeinen wirksam ist. ​· [​Chlorine Dioxide, Part 1 A Versatile, High-Value Sterilant for the Biopharmaceutical Industry, Barry Wintner, Anthony Contino, Gary O'Neill. BioProcess International DECEMBER 2005.] ​Es wurde ​gezeigt, dass es beim menschlichen Coronavirus wirksam ist ​[# 4 BASF Aseptrol-Dokument] ​, aber auch bei Tieren wie Hunden, bei denen es als canines respiratorisches Coronavirus bekannt ist, oder auch Katzen, einschließlich des enterischen Katzen-Coronavirus (FECV) und des bekanntesten katzenartigen infektiösen Peritonitis-Virus (FIPV), da es die Kapside durch Oxidation denaturiert und das Virus in kurzer Zeit inaktiviert ​[2-log 4.2 / 4-log 25.1 Source USEPA 2003 WHO Guidelines for drinking water Quality]​.

Pharmacology. 2016;97(5-6):301-6. doi: 10.1159/000444503. Epub 2016 Mar 1.​Inactivation of Airborne Bacteria

and

Viruses

Using

Extremely

Low

Concentrations

of

Chlorine

Dioxide

Gas.

Zu beachten ist weiters, dass die Aufnahme von Chlordioxid ein antiviraler Ansatz ist, da es ein Oxidationsmittel ist und durch Verbrennung jegliche Unterarten oder Virusvariationen eliminieren kann. ​[6#ClO2 is a size selective biocide] ​Angesichts der Notsituation, in der wir uns derzeit mit Covid-19 befinden, wird daher die orale Anwendung von ClO2 als bereits bekanntes und verwendetes Protokoll vorgeschlagen.

3. Toxizität​: Die größten Probleme, die bei Medikamenten im Allgemeinen auftreten, sind auf ihre Toxizität und Nebenwirkungen zurückzuführen. Vorliegende Toxizitätsstudien belegen, dass Chlordioxid problemlos angewendet werden kann. ​[7#New Clo2 safety evaluation 2017] O ​ bwohl die Toxizität von Chlordioxid bei Inhalation hoher Dosen bekannt ist, hat es selbst bei oraler Einnahme in hohen Dosen seit Bekanntsein dieser Substanz vor über hundert Jahren keinen einzigen, darauf zurückführbaren klinisch nachgewiesenen Todesfall gegeben. ​[8#Controlled Clinical Evaluations of Clo2 in Man] A ​ ls letale Dosis (LD50, akutes Toxizitätsverhältnis) wird erst die Einnahme von 292 mg pro Kilogramm pro Tag über einen Zeitraum von 14 Tagen angesehen. Umgerechnet ergibt dies bei einem 50 kg schweren Erwachsenen eine Dosis von 15‘000 mg Gas, welche in Wasser gelöst während zwei Wochen jeden Tag verabreicht werden müssten. Das ist fast unmöglich, denn entweder wäre die Flüssigkeit damit so konzentriert, dass sie nicht eingenommen werden könnte, oder wir würden hier von einer Menge von ca. 30 Liter Wasser pro Tag sprechen, die nicht getrunken werden könnte. ​[9# toxicity of ClO2 and clorite ions] Die von uns verwendeten oralen Dosen betragen dagegen nur etwa 50 mg in 100 ml Wasser gelöst, bis zu 10-mal täglich, was einer Tagesdosis von max. 0,5 g Chlordioxid ClO2 in 1 Liter Wasser pro Tag entspricht (und daher nur 1/30 der oben genannten LD50 Dosis von 15 g ClO2 pro Tag).

Da Chlordioxid dissoziiert, zerfällt es im menschlichen Körper in wenigen Stunden in eine unbedeutende Menge an Kochsalz (NaCL) und Sauerstoff (O2). Darüber hinaus haben Messungen von venösen Blutgasen gezeigt, dass es in der Lage ist, die Sauerstoffversorgung der Lunge des betroffenen Patienten wesentlich zu verbessern.

Beispiel 1: IV-Anwendung von 500 ml NaCl bei einer Konzentration von 50 ppm ClO2

Beispiel 2: IV-Anwendung von 500 ml NaCl bei einer Konzentration von 50 ppm ClO2

Beispiel 3: IV-Anwendung von 250 ml NaCl bei einer Konzentration von 50 ppm ClO2

FUNKTIONSMECHANISMUS VON CHLORDIOXID GEGEN VIREN In der Regel verhalten sich die meisten Viren ähnlich: Sobald sie sich an entsprechende Bakterien oder Zellen binden, injizieren sie über Nukleinsäure ihre genetische Information in die infizierte Zelle und übernehmen dann den Proteinsynthesevorgang der infizierten Zelle.

Bestimmte Segmente der viralen Nukleinsäure sind auch für die Vervielfältigung des genetischen Materials des Kapsids verantwortlich. In Gegenwart dieser Nukleinsäuren wird das CLO2-Molekül instabil und dissoziiert, wobei der entstehende Sauerstoff an das umgebende Medium abgegeben wird, was wiederum dazu beiträgt, das umgebende Gewebe mit Sauerstoff zu versorgen. Aufgrund dieser höheren Sauerstoffversorgung wird die mitochondriale Aktivität und die Wirksamkeit des Immunsystems erhöht und damit die Kapside denaturiert. Dadurch wird die Andockfähigkeit des Virus blockiert. ​[6#ClO2 is a size selective biocide] Nukleinsäuren, DNA-RNA, bestehen aus einer Kette von Purin- und Pyrimidinbasen: Guanin (G), Cytosin (C), Adenin (A) und Thymin (T). Es ist die Abfolge dieser vier Einheiten entlang der Kette, die ein Segment von einem anderen unterscheidet. Die Guaninbase, die sowohl in RNA als auch in DNA vorkommt, ist sehr oxidationsempfindlich und bildet 8-Oxoguanin als Nebenprodukt. Wenn das CLO2-Molekül mit Guanin in Kontakt kommt und es oxidiert, führt dies zur Bildung von 8-Oxoguanin, wodurch die Vervielfältigung viraler Nukleinsäuren durch Basenpaarung blockiert wird. Obwohl die Vervielfältigung des Proteinkapsids weitergehen könnte, wird die Bildung des voll funktionsfähigen Virus dank CLO2 durch Oxidation blockiert.

Zusammenfassend kann man festhalten, dass das CLO2-Molekül Eigenschaften aufweist, die es zu einem idealen Kandidaten für die Behandlung im klinischen Umfeld machen, da es ein Produkt mit einer hohen selektiven Oxidationskraft und einer großen Fähigkeit zur Verringerung der Azidose und zur Erhöhung des Sauerstoffs in Geweben und Mitochondrien ist. Dies erleichtert die schnelle Genesung von Patienten mit Lungenerkrankungen.

VORSICHTSMASSNAHMEN UND KONTRAINDIKATIONEN Chlordioxid reagiert mit Antioxidantien und verschiedenen Säuren. Die Verwendung von Vitamin C ​oder Ascorbinsäure während der Behandlung wird ​nicht empfohlen​, da dies die Wirksamkeit von Chlordioxid bei der Beseitigung von Krankheitserregern annulliert. Die antioxidative Wirkung des einen verhindert die selektive Oxidation des anderen. Daher ist es nicht ratsam, während der Behandlungstage Antioxidantien einzunehmen. Es wurde gezeigt, dass Magensäure die Wirksamkeit nicht beeinträchtigt. Da beobachtet wurde, dass Chlordioxid die Durchblutung verbessert, sollten bei Patienten mit Warfarin-Behandlung die Werte ständig überprüft werden, um Fälle von Überdosierung zu vermeiden.

Obwohl Chlordioxid in Wasser sehr gut löslich ist, hat es den Vorteil, dass es nicht hydrolysiert, so dass keine toxischen krebserzeugenden THM (Trihalogenmethane) wie bei Chlor erzeugt werden. Es verursacht auch keine genetischen Mutationen oder Missbildungen. ​Es wurde ein Protokoll entwickelt, mit dem eine Lösung dieser Verbindung oral und intravenös eingenommen werden kann.

Rechtsgrundlage für die sofortige Anwendung: * Die jeweilige nationale Gesetzgebung ist dabei jedenfalls zu beachten und insbesondere deren Verwendungsbestimmungen im Katastrophenfall.

HELSINKI WORLD MEDICAL ASSOCIATION STATEMENT Auszug: Ethische Grundsätze für die medizinische Forschung am Menschen, verabschiedet von der 18. Generalversammlung der WMA, Helsinki, Finnland, Juni 1964, geändert von der Ausschuss:

64. WMA-Generalversammlung, Fortaleza, Brasilien, Oktober 2013

Allgemeine Grundsätze 3. Die Genfer Erklärung der World Medical Association verpflichtet den Arzt mit dem Grundsatz "fürsorglich und vor allem auf die Gesundheit meines Patienten achten". Im International Code of Medical Ethics heißt es: "Der Arzt muss bei der medizinischen Versorgung berücksichtigen, was für den Patienten am besten ist."

4. Der Arzt hat die Aufgabe, die Gesundheit, das Wohlbefinden und die Rechte der Patienten, einschließlich derjenigen, die an der medizinischen Forschung beteiligt sind, zu fördern und sicherzustellen. Das Wissen und Gewissen des Arztes muss der Erfüllung dieser Pflicht untergeordnet sein.

5. Der Fortschritt der Medizin basiert auf Forschungen, die letztendlich Studien am Menschen umfassen müssen.

Unbekannte Interventionen in der klinischen Praxis 37.

Bei der Behandlung eines einzelnen Patienten, bei dem es keine nachgewiesenen Heilverfahren gibt oder andere bekannte Heilverfahren unwirksam waren, kann der Arzt nach Einholung eines Expertenrats mit der informierten Zustimmung des Patienten oder eines gesetzlich bevollmächtigten Vertreters ein unbestätigtes Heilverfahren anwenden, wenn sich nach dem Urteil des Arztes die Hoffnung auf die Rettung von Leben, die Wiederherstellung der Gesundheit oder die Linderung von Leiden bietet. Diese Intervention sollte anschließend zum Gegenstand von Forschungsarbeiten gemacht werden, um ihre Sicherheit und Wirksamkeit zu bewerten. In allen Fällen müssen neue Informationen aufgezeichnet und gegebenenfalls öffentlich zugänglich gemacht werden. .

Quelle: 8/9 © World Medical Association, Inc.

Liste der Pathogenwirksamkeit (Referenzen)

Virus Adenovirus Type 40 6 Calicivirus 42 Canine Parvovirus 8 Coronavirus​ 3 ​ Feline Calici Virus 3 Foot and Mouth disease 8 Hantavirus 8 Hepatitis A, B & C Virus 3,8 Human coronavirus​ 8 ​ Human Immunodeficiency Virus 3 Human Rotavirus type 2 (HRV)15 Influenza A22 Minute Virus of Mouse (MVM-i)8 Mouse Hepatitis Virus spp.8 Mouse Parvovirus type 1 (MPV-1)8 Murine Parainfluenza Virus Type 1 (Sendai)8 Newcastle Disease Virus 8 Norwalk Virus 8 Poliovirus 20 Rotavirus 3 Severe Acute Respiratory Syndrome (SARS) ​coronavirus ​43 Sialodscryoadenitis Virus 8 Simian rotavirus SA-11 15 Theiler’s Mouse Encephalomyelitis Virus 8 Vaccinia Virus 10

Bacterien Blakeslea trispora 28 Bordetella bronchiseptica 8 Brucella suis 30 Burkholderia spp.36 Campylobacter jejuni 39 Clostridium botulinum 32 Clostridium dificile 44 Corynebacterium bovis 8 Coxiella burneti (Q-fever) 35 E. coli spp .1,3,13

Erwinia carotovora (soft rot) 21 Franscicella tularensis 30 Fusarium sambucinum (dry rot) 21 Helicobacter pylori 8 Helminthosporium solani (silver scurf) 21 Klebsiella pneumoniae 3 Lactobacillus spp .1,5 Legionella spp. 38,42 Leuconostoc spp.1,5 Listeria spp. 1,19 Methicillin-resistant Staphylococcus aureus 3 Mycobacterium spp.8,42 Pediococcus acidilactici PH31 Pseudomonas aeruginosa 3,8 Salmonella spp.1,2,4,8,13 Shigella 38 Staphylococcus spp.1,23 Tuberculosis 3 Vancomycin-resistant Enterococcus faecalis 3 Vibrio spp.37 Multi-Drug Resistant Salmonella typhimurium 3 Yersinia spp.30,31,40

Bakteriensporen Alicyclobacillus acidoterrestris 17 Bacillus spp.10,11,12,14,30,31 Clostridium. sporogenes ATCC 1940412 Geobacillus stearothermophilus spp.11,31 Bacillus thuringiensis 18 OTHER Beta Lactams 29 Amplicons 46 Volatile organic compounds (VOCs)47 PROTOZOA Chironomid larvae 27 Cryptosporidium 34 Cryptosporidium parvum Oocysts 9 Cyclospora cayetanensis Oocysts 41 Giardia 34 Alternaria alternata 26 Aspergillus spp.12,28 Botrytis species 3 Candida spp.5, 28 Chaetomium globosum 7 Cladosporium cladosporioides 7 Debaryomyces etchellsii 28

Eurotium spp.5 Fusarium solani 3 Lodderomyces elongisporus28 Mucor spp.28 Penicillium spp.3,5,7,28 Phormidium boneri3 Pichia pastoris 3 Poitrasia circinans 28 Rhizopus oryzae 28 Roridin A33 Saccharomyces cerevisiae 3 Stachybotrys chartarum 7 Verrucarin A 33 Biofilms 4 5

REFERENZEN 1. Selecting Surrogate Microorganism for Evaluation of Pathogens on Chlorine Dioxide Gas Treatment, Jeongmok Kim, Somi Koh, Arpan Bhagat, Arun K Bhunia and Richard H. Linton.Purdue University Center for Food Safety 2007 Annual Meeting October 30 - 31, 2007 at Forestry Center, West Lafayette, IN. 2. Decontamination of produce using chlorine dioxide gas treatment, Richard Linton, Philip Nelson, Bruce Applegate, David Gerrard, Yingchang Han and Travis Selby.

3. Chlorine Dioxide, Part 1 A Versatile, High-Value Sterilant for the Biopharmaceutical Industry, Barry Wintner, Anthony Contino, Gary O’Neill. BioProcess International DECEMBER 2005.

4. Chlorine Dioxide Gas Decontamination of Large Animal Hospital Intensive and Neonatal Care Units, Henry S. Luftman, Michael A. Regits, Paul Lorcheim, Mark A. Czarneski, Thomas Boyle, Helen Aceto, Barbara Dallap, Donald Munro, and Kym Faylor. Applied Biosafety, 11(3) pp. 144-154 © ABSA 2006 5. Efficacy of chlorine dioxide gas as a sanitizer for tanks used for aseptic juice storage, Y. Han, A. M. Guentert*, R. S. Smith, R. H. Linton and P. E. Nelson. Food Microbiology, 1999, 16, 53]61 6. Inactivation of Enteric Adenovirus and Feline Calicivirus by Chlorine Dioxide, Thurston-Enriquez, J.A., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2005, p. 3100–3105.

7. Effect of Chlorine Dioxide Gas on Fungi and Mycotoxins Associated with Sick Building Syndrome, S. C. Wilson,* C. Wu, L. A. Andriychuk, J. M. Martin, ... D. C. Straus. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2005, p. 5399–5403.

8. BASF Aseptrol Label EPA Registration Number: 70060-19 9. Effects of Ozone, Chlorine Dioxide, Chlorine, and Monochloramine on Cryptosporidium parvum Oocyst Viability, D. G. KORICH, J. R. MEAD, M. S. MADORE, N. A. SINCLAIR, AND C. R. STERLING. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1990, p. 1423-1428.

10. NHSRC’s Systematic Decontamination Studies, Shawn P. Ryan, Joe Wood, G. Blair Martin, Vipin K. Rastogi (ECBC), Harry Stone (Battelle). 2007 Workshop on Decontamination, Cleanup, and Associated Issues for Sites Contaminated with Chemical, Biological, or Radiological Materials Sheraton Imperial Hotel, Research Triangle Park, North Carolina June 21, 2007.

11. Validation of Pharmaceutical Processes 3rd edition, edited by Aalloco James, Carleton Frederick J. Informa Healthcare USA, Inc., 2008, p267

12. Chlorine dioxide gas sterilization under square-wave conditions. Appl. Environ. Microbiol. 56: 514-519 1990. Jeng, D. K. and Woodworth, A. G. 13. Inactivation kinetics of inoculated Escherichia coli O157:H7 and Salmonella enterica on lettuce by chlorine dioxide gas. Food Microbiology Volume 25, Issue 2, February 2008, Pages 244-252, Barakat S. M. Mahmoud and R. H. Linton.

14. Determination of the Efficacy of Two Building Decontamination Strategies by Surface Sampling with Culture and Quantitative PCR Analysis. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2004, p. 4740–4747. Mark P. Buttner, Patricia Cruz, Linda D. Stetzenbach, Amy K. Klima-Comba, Vanessa L. Stevens, and Tracy D. Cronin 15. Inactivation of Human and Simian Rotaviruses by Chlorine Dioxide. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1990, p. 1363-1366. YU-SHIAW CHEN AND JAMES M. VAUGHN 16. Information obtained from CSI internal testing with Pharmaceutical customer.May 2006 Pages 364-368 17. Efficacy of chlorine dioxide gas against Alicyclobacillus acidoterrestris spores on apple surfaces, Sun-Young Lee, Genisis Iris Dancer, Su-sen Chang, Min-Suk Rhee and Dong-Hyun Kang, International Journal of Food Microbiology, Volume 108, issue 3, May 2006 Pages 364-368 18. Decontamination of Bacillus thuringiensis spores on selected surfaces by chlorine dioxide gas, Han Y, Applegate B, Linton RH, Nelson PE. J Environ Health. 2003 Nov;66(4):16-21.

19. Decontamination of Strawberries Using Batch and Continuous Chlorine Dioxide Gas Treatments, Y Han, T.L. Selby, K.K.Schultze, PE Nelson, RH Linton. Journal of Food Protection, Vol 67, NO 12, 2004.

20. Mechanisms of Inactivation of Poliovirus by Chlorine Dioxide and Iodine, MARIA E. ALVAREZ AND R. T. O'BRIEN, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1982, p. 1064-1071 21. The Use of Chlorine Dioxide in potato storage, NORA OLSEN, GALE KLEINKOPF, GARY SECOR, LYNN WOODELL, AND PHIL NOLTE, University of Idaho, BUL 825.

22. Protective effect of low-concentration chlorine dioxide gas against influenza A virus infection Norio Ogata and Takashi Shibata Journal of General Virology (2008), 89, 60–67 23. Preparation and evaluation of novel solid chlorine dioxide-based disinfectant powder in single-pack Zhu M, Zhang LS, Pei XF, Xu X. Biomed Environ Sci. 2008 Apr;21(2):157-62.

24. Chlorine dioxide oxidation of dihydronicotinamide adenine dinucleotide (NADH), Bakhmutova-Albert EV, et al. Inorg Chem. 2008 Mar 17;47(6):2205-11. Epub 2008 Feb 16.

25. Oxidative elimination of cyanotoxins: comparison of ozone, chlorine, chlorine dioxide and permanganate, Rodríguez E, Water Res. 2007 Aug;41(15):3381-93. Epub 2007 Jun 20.

26. Inhibition of hyphal growth of the fungus Alternaria alternata by chlorine dioxide gas at very low concentrations, Morino H, Matsubara A, ...Yakugaku Zasshi. 2007 Apr;127(4):773-7. Japanese.

27. Inactivation of Chironomid larvae with chlorine dioxide, Sun XB, Cui FY, Zhang JS, Xu F, Liu LJ., J Hazard Mater. 2007 Apr 2;142(1-2):348-53. Epub 2006 Aug 18.

28. Information obtained from CSI decontamination at Pharmaceutical facility.

29. Information obtained from CSI beta-lactam inactivation at Pharmaceutical facility.

30. Decontamination of Surfaces Contaminated with Biological Agents using Fumigant Technologies, S Ryan, J Wood, 2008 Workshop on Decontamination, Cleanup, and Associated Issues for Sites Contaminated with Chemical, Biological, or Radiological Materials Sheraton Imperial Hotel, Research Triangle Park, North Carolina September 24, 2008.

31. Sporicidal Action of CD and VPHP Against Avirulent Bacillus anthracis – Effect of Organic Bio-Burden and Titer Challenge Level, Vipin K. Rastogi, Lanie Wallace & Lisa Smith, 2008 Workshop on Decontamination, Cleanup, and Associated Issues for Sites Contaminated with Chemical, Biological, or Radiological Materials Sheraton Imperial Hotel, Research Triangle Park, NC 2008 Sept 25.

32. Clostridium Botulinum, ESR Ltd, May 2001.

33. Efficacy of Chlorine Dioxide as a Gas and in Solution in the Inactivation of Two Trichothecene Mycotoxins, S. C. Wilson, T. L. Brasel, J. M. Martin, C. Wu, L. Andriychuk, D. R. Douglas, L. Cobos, D. C. Straus, International Journal of Toxicology, Volume 24, Issue 3 May 2005 , pages 181 – 186.

34. Guidelines for Drinking-water Quality, World Health Organization, pg 140.

35. Division of Animal Resources Agent Summary Sheet, M. Huerkamp, June 30, 2003.

36. NRT Quick Reference Guide: Glanders and Melioidosis 37. Seasonal Occurrence of the Pathogenic Vibrio sp. of the Disease of Sea Urchin Strongylocentrotus intermedius Occurring at Low Water Temperatures and the Prevention Methods of the Disease, K. TAJIMA, K. TAKEUCHI, M. TAKAHATA, M. HASEGAWA, S. WATANABE, M. IQBAL, Y.EZURA, Nippon Suisan Gakkaishi VOL.66;NO.5;PAGE.799-804(2000).

38. Biocidal Efficacy of Chlorine Dioxide, TF-249, Nalco Company, 2008.

39. Sensitivity Of Listeria Monocytogenes, Campylobacter Jejuni And Escherichia Coli Stec To Sublethal Bactericidal Treatments And Development Of Increased Resistance After Repetitive Cycles Of Inactivation, N. Smigic, A. Rajkovic, H. Medic, M. Uyttendaele, F. Devlieghere, Oral presentation.

FoodMicro 2008, September 1st – September 4th, 2008, Aberdeen, Scotland.

40. Susceptibility of chemostat-grown Yersinia enterocolitica and Klebsiella pneumoniae to chlorine dioxide, M S Harakeh, J D Berg, J C Hoff, and A Matin, Appl Environ Microbiol. 1985 January; 49(1): 69– 72.

41. Efficacy of Gaseous Chlorine Dioxide as a Sanitizer against Cryptosporidium parvum, Cyclospora cayetanensis, and Encephalitozoon intestinalis on Produce, Y. Ortega, A. Mann, M. Torres, V. Cama, Journal of Food Protection, Volume 71, Number 12, December 2008 , pp. 2410-2414.

42. Inactivation of Waterborne Emerging Pathogens by Selected Disinfectants, J. Jacangelo, pg 23.

43. SARS Fact Sheet, National Agricultural Biosecurity Center, Kansas State University.

44. High sporocidal activity using dissolved chlorine dioxide (SanDes) on different surface materials contaminated by Clostridium difficile spores, Andersson J., Sjöberg M., Sjöberg L., Unemo M., Noren T. Oral presentation. 19th European Congress of Clinical Microbiology and Infectious Diseases, Helsinki, Finland, 16 - 19 May 2009.

45. Inactiviation of Listeria monocytogenes on ready-to-eat food processing equipment by chlorine dioxide gas, Trinetta, V., et al. Food Control, Vol 26, 2012 46. Exposure to chlorine dioxide gas for 4 hours renders Syphacia ova nonviable, Czarra, J.A., et al.

Journal of the American Association for Laboratory Animal Science. 2014 4 Jul: 53(4): 364-367 47. Hu, Cheng (2017). Modeling reaction kinetics of chlorine dioxide and volatile organic compounds with artificial neural networks, December 2003.

Eine neue Perspektive zur Vorbeugung und Heilung von COVID-19-Patienten: Ermutigung der Ärzteteams, Kontakt zu mit Chlordioxidlösung (CDS) behandelten Heilpraktikern aufzunehmen.

Mediterranean BioMedical Journals Integratives Journal für medizinische Wissenschaften 2020, Band 7, 229 DOI: 10.15342 / ijms.7.229 Enrique A. Martínez Universität Católica del Norte, Coquimbo, Chile

ZUSAMMENFASSUNG Dieser Artikel soll Ärzteteams auf der ganzen Welt dazu ermutigen, COVID19-Patienten zu kontaktieren, die bereits mit Chlordioxidlösung (CDS), einem wasserlöslichen Gas, behandelt wurden. Kontaktaufnahme mit den Ärzteteams, die die Studienfälle begleiten, um den tatsächlichen Gesundheitszustand der Patienten zu überprüfen. Schließlich wird die Frage gestellt, ob CDS in den jeweiligen örtlichen Gesundheitseinrichtungen getestet werden sollte, da es kostengünstig ist, gegen alle Virusinfektionen hochwirksam erscheint und fast keine Nebenwirkungen hat. Schlüsselwörter: CDS; Chlordioxidlösung; COVID-19; Erklärung von Helsinki. Korrespondenz: Dr. Martinez Enrique, Foyer der Charité de Provence, BP63, Lambesc, 13410, Frankreich.

Email: enrique.a.martinez@ceaza.cl Copyright © 2020 Martinez EA. Dies ist ein Open-Access-Artikel, der unter Creative Commons Attribution 4.0 International vertrieben wird und die uneingeschränkte Verwendung, Verbreitung und Reproduktion in jedem Medium ermöglicht, sofern das Originalwerk ordnungsgemäß zitiert wird. Bis Juni 2020 hat die COVID-19-Pandemie weltweit mehr als 4,3 Millionen bestätigte Fälle und mehr als 290.000 Todesfälle verursacht. Die Pandemie hat die Welt für immer verändert. Soziale, wirtschaftliche, ökologische und gesundheitliche Veränderungen [1] haben stark in Frage gestellt, wie die Menschheit so schnell auf diese und andere invasive Bedrohungen reagieren kann. Dieser Artikel soll Ärzteteams ermutigen, zu verfolgen, was in einigen Ländern getan wurde, in denen COVID19-Patienten erfolgreich mit CDS (Chlordioxidlösung) behandelt wurden. Beutel gegen Viren wie HIV und andere Krankheitserreger [3], warum nicht bei Patienten mit COVID-19 versuchen, die dann so wenige Behandlungsalternativen haben? Diese Studien führten zur Wiederherstellung aller Patente, die in nur vier Behandlungstagen behandelt wurden [2]. Unter Berücksichtigung der Erklärung der Helsinki World Medical Association - Abschnitt Nr. 37, in der es heißt, dass "jeder Arzt berechtigt ist, neue oder unbewiesene präventive, diagnostische und therapeutische Verfahren anzuwenden", und der Tatsache, dass Tausende von Patienten an diesem COVI litten -19 Infektionen starben, im Mai 2020 beschlossen ecuadorianische Ärzte, Chlordioxid in Lösung zu testen, um 104 Patienten zu behandeln.

Diese Patienten unterzeichneten notariell eine Genehmigung und Entlassung, um diese Behandlung zu erhalten [2].

Sie verwendeten das CDS, um zu erkennen, dass Viren einerseits absolut oxidationsempfindlich sind und andererseits im menschlichen Blut wirken.

Was ist die CDS? Dieses Gasmolekül (ClO2) ist in Wasser gut löslich. Einmal in Lösung, ergibt das Gas durch physikalische Brechung eine gelbliche Farbe. Bisher wurde es nicht als Wirkstoff in das herkömmliche Arzneibuch aufgenommen, obwohl es seit 1994 nach seiner patentierten Verwendung als antivirales Mittel im Jahr 1991 obligatorisch zur Desinfektion und Aufbewahrung von Blutbeuteln für Transfusionen verwendet wird [3]. Es wird auch in den meisten zum Verzehr geeigneten Flaschenwässern verwendet, da es keine giftigen Rückstände hinterlässt. Darüber hinaus verdampft das Gas bei Temperaturen über 11 ° C. Wie funktioniert CDS bei Viren (und Bakterien)? Die geringe Größe von Viren (und Bakterien) ermöglicht es Sauerstoff, diese sehr schnell zu oxidieren, ohne die größeren Zellen lebender Organismen zu beeinträchtigen, die mit Viren oder Bakterien infiziert sind [4]. Die genaue Wirkung liegt auf Nukleinsäuren. Die Nukleinsäuren DNARNA bestehen aus einer Kette von Purin- und Pyrimidinbasen, Guanin (G), Cytosin (C), Adenin (A) und Thymin / Uracil. 2020; 7: 2 p 1 Integr J Med SciMartinez EA Perspective zur Vorbeugung und Heilung von COVID-19 (T / U) -Patienten.

Guaninbasen, die sowohl in RNA als auch in DNA vorkommen, sind sehr oxidationsempfindlich und bilden 8oxoguanin als Nebenprodukt. Wenn das ClO2-Molekül mit Guanin in Kontakt kommt und es oxidiert, was zur Bildung von 8-Oxoguanin führt, blockiert es daher die Replikation der viralen Nukleinsäure durch Basenpaarung. Obwohl die Replikation des Proteinkapsids fortgesetzt werden kann, wird die Bildung des voll funktionsfähigen Virus dank ClO2 durch Oxidation blockiert.

Der Sauerstoff in ClO2 scheint sich im Blutkreislauf schneller zu bewegen, da kein Hämoglobin transportiert werden muss. Diese Hypothese würde nach den aufgezeichneten Daten erklären, warum Patienten, die mit CDS behandelt wurden und an COVID-19 leiden, ihre Sauerstoffsättigung so schnell wiedererlangten [2]. In der Vergangenheit wurden dieses Gas und verwandte Moleküle gegen verschiedene Viren, Bakterien, aber auch gegen andere größere Parasiten wie Pilze und Hefen getestet [5]. Darüber hinaus weist CDS kurz- und langfristig eine äußerst geringe Toxizität gegenüber Menschen auf (5,6,7,8,9,10). Diese Studien zeigten keine schädlichen Auswirkungen auf physiologische Eigenschaften wie Puls, Atemfrequenz und Temperatur oder auf biochemische Parameter wie Glucose, Harnstoffstickstoff, Phosphate, alkalische Phosphatase, Transaminasen oder Schilddrüsenausscheidungen. Bei Dosen von 24 ppm pro Tag wurden zwei Wochen lang weder Hämolyse noch Anämie festgestellt. Chlordioxid war sogar für Trinkwasser unbedenklich, wie eine Studie an 198 Personen über 115 Tage zeigte [11]. Die globale Überreaktion auf Menschen wie die soziale Blockade scheint mehr Probleme zu verursachen als das Virus selbst [12]. Die wenigen noch verfügbaren Behandlungen, die hohen Kosten.

LITERATUR [1] Nicola M., Alsafi Z., Sohrabi C., Kerwan A., Al-Jabir A., Iosifidis C., Agha M., Agha R. Die sozioökonomischen Auswirkungen der Coronavirus-Pandemie (COVID-19): Eine Übersicht. Int J Surg. 2020; 78: 185 & ndash; 193. Verfügbar um: https://doi.org/10.1016/j.ijsu.2020.04.018 (besucht am 17. September 2020).

[2] Über 100 [Personen] mit CDS von Ärzten der ecuadorianischen Vereinigung für Integrierte Medizin (AEMEMI) nach dem spanischen Akronym aus Covid-19 (Asociación Ecuatoriana de Médicos Expertos en Medicina Integrativa) geborgen. Verfügbar um: https://lbry.tv/@Kalcker: 7 / 100Recovered-Aememi-1: 7 und www.andreaskalcker.com (besucht am 17. September 2020). Die Daten dieser Studie zu 104 Patienten befinden sich auf der Website des Journals und sind auf Anfrage des Autors auch verfügbar. [3] Kross RD, Scheer DI. Zusammensetzung und Verfahren zur Desinfektion von Blut und Blutbestandteilen. Verfügbar um: https://patents.google.com/patent/US5019402A/en. 1991 (besucht am 17. September 2020).

[4] Noszticzius Z., Wittmann M., Kály-Kullai K., Beregvári Z., Kiss I., Rosivall L. et al. Chlordioxid ist ein größenselektives antimikrobielles Mittel. 2013. PLoS ONE 8 (11): e79157. Verfügbar um: https://doi.org/10.1371/journal.pone.0079157 (besucht am 17. September 2020).

[5] Barry Wintner B, Contino A, O'Neill G. Chlordioxid, Teil 1: Ein vielseitiges, hochwertiges Sterilisationsmittel für die biopharmazeutische Industrie. BioProcess International Technical Dezember 2005. S. 42-46. Verfügbar um: https://www.academia.edu/3829889/Chlorine_Dioxide_Pa rt_1_A_Versatile_High_Value_Sterilan t_for_the_Biophar maceutical_Industry (besucht am 17. September 2020). und niedrige Geschwindigkeit der Impfstoffentwicklung scheinen alle darauf hinzudeuten, dass alternative Behandlungen und sogar Ernährungsfaktoren [6] Patel Y, Wong D. Toxikologische Überprüfung von Chlordioxid und Chlorit. EPA / 635 / R-00/007.

2000. Verfügbar unter: https://cfpub.epa.gov/ncea/iris/iris_documents/documents/ toxreviews / 0648tr.pdf (besucht am 17. September 2020).

[7] Lubbers, JR; Chauhan, S; Bianchine, JR. Kontrollierte klinische Bewertungen von Chlordioxid, Chlorit und Chlorat beim Menschen. Fundam Appl Toxicol 1: 334 & ndash; 338. (1981) [8] Lubbers, JR; Chauhan, S; Bianchine, JR. Kontrollierte klinische Bewertungen von Chlordioxid, Chlorit und Chlorat beim Menschen. Environ Health Perspect 46: 57-62. (1982) [9] Lubbers, JR; Chauhan, S; Miller, JK, Bianchine, JR Die Auswirkungen der chronischen Verabreichung von

Chlordioxid, Chlorit und Chlorat an normale gesunde erwachsene männliche Freiwillige. J Environ Pathol Toxicol Oncol 5: 229 & ndash; 238. (1984a).

[10] Lubbers, JR; Chauhan, S; Miller, JK; Bianchine, JR. Die Auswirkungen der chronischen Verabreichung von Chlorit an Glucose-6-Phosphat-Dehydrogenase-Mangel gesunde erwachsene männliche Freiwillige. J Environ Pathol Toxicol Oncol 5: 239 & ndash; 242. (1984b).

[11] Michael, GE, Miday, RK, Bercz, JP, Miller, RG, Greathouse, DG, Kraemer, DF und Lucas, JB. Chlordioxid-Wasserdesinfektion: Eine vorausschauende epidemiologische Studie, Archiv für Umweltgesundheit: An International Journal, 36: 1, 20-27, DOI: 10.1080 / 00039896.1981.10667601 (1981) [12] Boretti, A. Nachhaltige Lockdown-Strategie nach Covid19 durch evidenzbasierte Politik: Analyse der Todesfälle in Covid19 in ganz Europa. Integr J Med Sci 2020, Band 7, ID 172 DOI: 10.15342 / ijms.7.172 [13] Zhang Y., Chen, G. Ernährungsüberlegungen bei COVID19-Pandemie. Integrative Journal of Medical Sciences 2020, Band 7, ID 198. DOI: 10.15342 / ijms.7.198.

Volume 15:S1, 2021

Journal of Molecular and Genetic Medicine

ISSN: 1747-0862

Open Access

Review Article

Determination of the Effectiveness of Chlorine Dioxide in the Treatment of COVID 19 Insignares-Carrione Eduardo1*, Bolano Gómez Blanca2, Andrade Yohanny3, Callisperis Patricia4, Suxo Ana Maria5, Arturo Bernardo Ajata San Martín6 and Camila Ostria Gonzales7 LVWG Global Research Director, Liechtensteiner Verein für Wissenschaft und Gesundheit, Liechtenstein, Switzerland (https: //orcid.org/0000-0001-9337-0884) Director of the Research Department, Genesis Foundation, Colombia 3 Specialist in Medical Bioethics, Oncology Palliative Care Specialist, Spain 4 Director, Orthopedic and Traumatology Specialist, South clinic, La Paz, Bolivia, Spain 5 MSc, Training and Research Center - Bolivia Today Association, Epidemiologist, South Clinic, La Paz, Bolivia, Spain 6 Internal Medicine specialist, C Sorata 1146 V Victory, La Paz, Bolivia, Spain 7 La Paz, Bolivia, Spain (linkedin.com/in/camila-ostria-gonzales-96bb0514a) 1 2

Abstract Introduction: The aim of this review is to determine the Effectiveness of Oral Chlorine Dioxide in the Treatment of COVID-19.

Methods: Research on the mechanism of action of chlorine dioxide on viruses, on the oral consumption of water-solubilized ClO2 and on its toxicity was reviewed; a quasi-experimental investigation was conducted on the use of oral water-soluble chlorine dioxide in the treatment of 20 patients with active COVID-19 infection, compared to a control group of 20 patients not treated with chlorine dioxide.

Results: To compare the effect in the experimental group versus the control group, a paired test was performed using the Wilcoxon-Mann-Whitney test (α: 95%). Within the experimental group, a significant difference was observed in all general symptoms when comparing day 0 to day 07.

Some even disappeared after day 7. Those values ​​that did not disappear completely on day 7 did not differ from those on days 14 and 21 (p>0.05).

As for headache, it decreased very significantly (p<0.0001) from day 0 with respect to subsequent days. When comparing the experimental group with the control group on the seventh day post manifestation of symptoms, a significant difference was found in the experimental group with respect to the control group for the symptoms Fever (p: 0000), Cough (p: 0.0001), Chills (p: 0.0015) and Dyspnea (p: 0.0047). When performing the visual analogous comparison of pain in the control group and in the experimental group, it was found that in all the items that make up the scale decreased significantly in this group with respect to the Control group. On day 14 post manifestation the difference was greater (p<0.001).

When evaluating both groups (Control and Experimental) in the laboratories, a difference was found for the values ​​for the parameters PC Reactive (p: 0.0398) and DH Lactate (0.0422), with higher scores for the experimental group; no difference was found in the rest of the parameters. The results in general (p<0.05) demonstrate the hypothesis that chlorine dioxide is effective in the treatment of COVID-19. It was found that in all the items that make up the scale decreased significantly in this group with respect to the Control group. On day 14 post manifestation the difference was greater (p<0.001).

Discussion: When evaluating both groups (Control and Experimental) in the laboratories, a difference was found for the values ​​for the parameters PC Reactive (p: 0.0398) and DH Lactate (0.0422), with higher scores for the experimental group; no difference was found in the rest of the parameters. The results in general (p<0.05) demonstrate the hypothesis that chlorine dioxide is effective in the treatment of COVID-19. It was found that in all the items that make up the scale decreased significantly in this group with respect to the Control group. On day 14 post manifestation the difference was greater (p<0.001). When evaluating both groups (Control and Experimental) in the laboratories, a difference was found for the values ​​for the parameters PC Reactive (p: 0.0398) and DH Lactate (0.0422), with higher scores for the experimental group; no difference was found in the rest of the parameters. The results in general (p<0.05) demonstrate the hypothesis that chlorine dioxide is effective in the treatment of COVID-19. a difference was found for the values ​​for the parameters PC Reactive (p: 0.0398) and DH Lactate (0.0422), with higher scores for the experimental group; no difference was found in the rest of the parameters. The results in general (p<0.05) demonstrate the hypothesis that chlorine dioxide is effective in the treatment of COVID-19. A difference was found for the values ​​for the parameters PC Reactive (p: 0.0398) and DH Lactate (0.0422), with higher scores for the experimental group; no difference was found in the rest of the parameters. The results in general (p<0.05) demonstrate the hypothesis that chlorine dioxide is effective in the treatment of COVID-19.

Conclusion: Chlorine dioxide is effective in the treatment of COVID-19 and the mechanisms of action by which it acts to achieve it are proposed in this work. We recommend doing more research. We recommend conducting double-blind studies and delving into studies of toxicological safety and therapeutic efficacy of chlorine dioxide in pathologies of epidemiological impact in the near future.

Keywords: SARS-CoV-2 • COVID-19 • Effectiveness • Chlorine Dioxide *Address for Correspondence: Dr. Eduardo Insignares-Carrione, LVWG Global Research Director, Liechtensteiner Verein für Wissenschaft und Gesundheit, Switzerland-Liechtenstein; E-mail: eduardoinsignarescarrione@gmail.com / eic@lvwg.org Copyright: © 2021 Insignares - Carrione, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Received 22 February 2021; Accepted 01 March 2021; Published 08 March 2021

Eduardo IC, et al.

Introduction In December 2019, a new respiratory illness emerged in Wuhan, China.

The source of this infection was identified as a new coronavirus, related to other coronaviruses that had previously caused outbreaks of Severe Acute Respiratory Syndrome (SARS) between 2002 and 2004 and Middle East Respiratory Syndrome (MERS) in 2012 (National Institutes of Health, 2020).

This virus was called "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2) and the disease resulting from infection by this virus was called "COVID-19". On March 11, 2020, the World Health Organization (WHO) declared COVID-19 a pandemic.

Coronaviruses are a group of enveloped RNA viruses that can damage multiple organ systems. Like other coronaviruses, the SARS-CoV-2 virus is a spherical particle with glycoprotein spikes on its surface. Coronaviruses enter host cells when a region of the spike, known as the "receptor-binding domain," binds to angiotensin-converting enzyme 2 (hACE2) in human cells. The viral membrane then fuses with the host cell membrane, allowing the viral genome to enter the host cell.

Chlorine dioxide (ClO2) has been suggested as a potential agent in the fight against COVID-19 and has been proposed three clinical trials to examine the ability of ClO2 to treat COVID-19. However, this agent is not well known, is not understood and is even demonized in the medical community; we assume that by confusion conceptual with sodium hypochlorite. Therefore, we set out to verify the effectiveness of oral use of the dioxide of chlorine in COVID-19 through this study, reviewing in depth the history of ClO2, its safety/ toxicity profile and its possible applications in the fight against COVID-19.

The objective of this research is to determine if chlorine dioxide is effective in managing COVID-19 [1-65].

Physical and chemical properties of Chlorine Dioxide (ClO2) Chlorine dioxide, ClO2, is a gas under standard conditions with a molar mass of 67.45 g/mol. Typically, ClO2 gas can be synthesized from the reaction between NaClO2 (sodium chlorite) and Cl2, NaClO2 and HOCl, NaClO2, and an acid such as HCl or H3PO4. It is easily soluble in water with a solubility of 3 g/100 mL https: //www.vyphidroasesores.com/.

J Mol Genet Med, Volume 15:S1, 2021

The therapeutic action of chlorine dioxide is given by its selectivity for pH. It means that this molecule dissociates and releases oxygen when it comes into contact with another acid. When it reacts, it becomes sodium chloride (common salt) and at the same time releases oxygen, which in turn oxidizes (burns) the pathogens (harmful germs) of acidic pH present, converting them into alkaline oxides (“ashes”). Therefore, when chlorine dioxide dissociates, it releases oxygen into the blood, as do erythrocytes (red blood cells) through the same principle (known as the Bohr Effect), which is to be selective for acidity.

Like blood, chlorine dioxide releases oxygen when it encounters acidity, either from lactic acid or from the acidity of the pathogen. ClO2 is a size-selective antimicrobial agent.

Multicellular tissue has the ability to dissipate this charge and is not affected in the same way. Chlorine dioxide, which is the second strongest disinfectant known after ozone, is much more suitable for use therapeutic since it is also capable of penetrating and eliminating biofilm, which ozone does not do. The great advantage of the therapeutic use of chlorine dioxide is the impossibility of a bacterial resistance to ClO2. Although ozone is stronger in terms antiseptics, its high oxidative potential of 2.07 and its short half-life of only 15 minutes at 25°C with a pH value of 7.0 make it less effective, for in vivo therapeutic applications [11-20].

Chlorine dioxide antiviral efficacy: Mechanism of antiviral action of ClO2 ClO2 exhibits antiviral activity through its oxidative chemistry. A virus consists of an outer shell or shell and an inner nucleic acid core. The viral envelope can be composed of proteins, lipid bilayers, and polysaccharides, and the envelope protects the nucleic acid core, in addition to providing selective binding and receptor cell recognition. Altering or modifying the viral envelope or nucleic acid nucleus will disrupt the modes of viral infection.

In the case of SARS-CoV-2, the carbohydrate envelope encloses it and protects it even more, which makes it more difficult to attack than other viruses.

Approximately 70% of the entire surface of the spike protein is coated with

The reactivity of chlorine dioxide is related to its structure and bond.

Chlorine dioxide is a bent symmetric triatomic molecule (C2v point group) with a Cl-O bond length of 147.3 picometers and a bond angle of 117.6 degrees (Figure 1A). ClO2 has an odd number of electrons with 19 valence electrons and therefore, is a paramagnetic radical. Its electronic structure puzzled chemists for a long time because none of the possible structures and Lewis it is very satisfactory for chlorine dioxide [1-10].

The molecular orbital theory reveals that a single electron occupies the highest unoccupied molecular orbital, the p* orbital, which makes the molecule a free radical and explains its reactive nature. The two resonance structures of ClO2 are shown in Figure 1B. The resonance structures of the ClO2 radical indicate the forms of a double bond between the central Cl atom and an O atom, and a single bond in combination with a 3-electron bond in the other Cl-O bond. The unpaired electron it is shared between the three atoms within the two different resonance structures. Most of the density of a single electron is found in one or another O atom. This unique arrangement provides ClO2 with a single unpaired electron and two reaction centers (O and Cl) on which to react.

When chlorine dioxide dissolves in water, most of it does not hydrolyze: it remains as a dissolved gas in solution. Another part does, and forms both chlorite ion (ClO2–) and chlorate ion (ClO3–). The solubility of chlorine dioxide in water at sea level and at 25°C is close to 3 g/L (≈ 3000 ppm). Its solubility increases at lower temperatures. Therefore, when its concentration is greater than 3 grams per liter, it is common to store it at temperatures close to 5°C. Dissolved in pure water, in a hermetically closed container, in the absence of light, and at a low temperature, it is quite stable although it slowly decomposes into chlorine and oxygen. Chlorides catalyze its decomposition. In biological systems, ClO2 can be expected to play an important role as an antiviral/ microbial agent through oxidative chemistry.

Page 2 of 11

Figure 1. (A). Geometry of the ClO2 molecule with group of points (C2v).

The bond angle a is 117.6 O and the length of bond d is 147.3 pm. (B). Two resonance structures of ClO2 are illustrated that reveal the unpaired electron, centered on the Oxygen atom, shared between the three atoms.

Eduardo IC, et al.

glycans; In the case of SARS-CoV-2, sugars are twice as essential. First of all, because they stabilize the spicule in a conformation that allows it to fit with ACE2 receptors on our cells, the process that initiates infection. By removing some glycans from the surface, the spike protein is destabilized and, furthermore, the binding with these receptors is weakened.

A line of research is being developed in post-COVID syndrome by one of the authors (B. Bolano), focusing attention on the possibility that ClO2 acts at the level of inflammasomes or even NETosis as a possible explanation for the improvement in the post COVID-19 syndrome manifested by hundreds of doctors who have reported cases in the world. The relevance and evidence of these findings to the anti-inflammatory mechanism of action of ClO2 remains unclear at this time.

In order to explore the mechanism of action more deeply, we selected published articles (pubmed, google scholar and other search engines) that describe the action of SARS-CoV-2 in cells, in its interaction with hACE2 and, in In particular, we investigated augmented reality videos or simulation videos based on Silicon, for the three-dimensional representation of the places of action, such as the videos in which the spicular protein and the hACE2 receptor are manipulated, among others, with ChimeraX (UCSF) augmented reality software.

Similarly, we reviewed the structure of the virus spike and drew on the research of Daniel Wrapp and Jason S. McLellan of the University of Texas.

The three-dimensional image of the spicular S glycoprotein of the SARS-CoV-2 betacoronavirus has been seen with electron cryomicroscopy in record time.

Thanks to this image with a resolution of 3.5 Å, it is confirmed that this S protein binds to the hACE2 protein of human cells with a higher affinity than that of the SARS-CoV coronavirus. Protein S is the target of the antibodies that immunize us. Its 3D structure allows us to understand why the monoclonal antibodies published against SARS-CoV are not effective against SARS-CoV-2.

Based on this information, we did a study with a doctor in molecular biology from the University of Tsukuba in Japan, in which we sought to build the hypothesis of how chlorine dioxide acts on SARS-CoV-2. As a result, we saw that there is an excellent correlation between its structure and its transmission mechanism, as well as the determination of the sites of action of promising substances or drugs in the treatment of COVID-19 and, however, we have not had the expected results for its rapid control with none of the drugs tested.

Bioinformatics is a tool that uses biological data to study biological problems on a wide range of scales, such as evolution, structure, the function and regulation of nucleic acids and proteins with the help of computational methods. In our work in Japan, we carry out in silico analysis, which is a comprehensive quantitative approach that not only provides information on the dynamics of intracellular and intramolecular processes, but ultimately allows us to predict biological behavior from molecular interactions. This was done in our work by focusing on conserved domains, structure, function, and biochemical characteristics to understand the mechanism of action of chlorine dioxide on the spike and SARS-CoV-2 RNA, which is a complete quantitative approach that not only provides information on the dynamics of intracellular and intramolecular processes, but ultimately allows prediction of biological behavior from molecular interactions. This was done in our work by focusing on conserved domains, structure, function, and biochemical characteristics to understand the mechanism of action of chlorine dioxide on the spike and SARS-CoV-2 RNA, which is a complete quantitative approach that not only provides information on the dynamics of intracellular and intramolecular processes, but ultimately allows prediction of biological behavior from molecular interactions. This was done in our work by focusing on conserved domains, structure, function, and biochemical characteristics to understand the mechanism of action of chlorine dioxide on the spike and SARS-CoV-2 RNA (Figure 2).

This type of analysis not only provides information on RNA replication, but also simulates the dynamics of the interrelation between the spike and the hACE2 receptor. The investigation began with the nucleotide sequence of the viral RNA, the open reading frames (ORF's) were determined, which once analyzed, revealed the oxidation of guanine to 8-oxoguanine. The other bases were not modified. The amino acids oxidized by chlorine dioxide are found in the spike, located in the helices, S1, S2, RBD and hACE2, which could explain the almost immediate effect of clinical improvements achieved by dioxide.

Page 3 of 11

J Mol Genet Med, Volume 15:S1, 2021

Cysteine, belonging to the group of thiols, is an amino acid up to 50 times more reactive with all microbial systems than the other four amino acids and, therefore, it is impossible for it to create resistance against chlorine dioxide.

The hypothesis proposed by the authors Insignares – Carrione et al., is that the cause of the antiviral effect of chlorine dioxide in SARS-CoV-2 can be explained by its actions on at least five amino acids (Cysteine, tryptophan, tyrosine, proline, hydroxyproline).

Pharmacokinetics The pharmacokinetics of ClO2 varies with the route of exposure. Scatina et al. examined the dermal absorption of a ClO2 preparation applied to the shaved back of rats. The maximum absorption of Cl in plasma was observed after 72 hours and a plasma concentration of 69.4 mcg% Cl was reached. The absorption half-life was calculated as 22.1 hours, which corresponds to a rate constant of 0, 0314 h-1 (Figure 3).

Fridyland and Kagan measured the degree of ClO2 absorption in the oral cavity of human subjects by rinsing the mouth with water containing predetermined concentrations of ClO2, and then measuring the residual concentrations of ClO2 in the washings. They found that up to 30% of the ClO2 was absorbed.

Safety/Toxicity The safety/toxicity of ClO2 depends on several variables, including the route of administration (e.g., inhaled, topical, or oral), the state of the molecule (i.e., gaseous or aqueous), the concentration (usually measured in mg/L or ppm for aqueous and ppm or mg/m3 for gaseous), and the duration of exposure. ClO2 gas is toxic at much lower concentrations than aqueous ClO2.

Toxicity also increases with the duration of exposure. The fact that ClO2 is less toxic in aqueous form allows the use of this molecule as a safe disinfectant in municipal drinking water, as a disinfectant for drinking water at points of use and as a food disinfectant, among other uses (Figure 4).

Below is a review of studies examining the safety and toxicity of ClO2.

ClO2 solubilized in water In vitro studies: Noszticzius et al. examined the differential effects of ClO2 on microbes versus humans or animals. They performed perfusion studies using protein membranes and found that the size of the organism exposed to ClO2 influences the effects of the oxidant. They reported that a low concentration of ClO2 quickly kills micron-sized organisms while causing little

Figure 2. Positions of the amino acid (cysteine) in the structure of the spike, one of the possible sites of action of ClO2.

Figure 3. Effectiveness of chlorine dioxide treatment based on concentration and contact time.

Eduardo IC, et al.

J Mol Genet Med, Volume 15:S1, 2021

which subjects drank 0.1, 1.0, 5.0, 10.0, 18.0, and 24.0 mg/L of a ClO2 solution.

(0.34 mg/kg, assuming a reference body weight of 70 kg). In the second study (Lubbers et al., 1984a) groups of 10 adult males received 500 ml of distilled water containing 0 or 5 mg/L ClO2 (0.04 mg/kg-day assuming a reference body weight of 70 kg) for 12 weeks.

In a comparative study of hyperpure chlorine dioxide with two other irrigants regarding the viability of periodontal ligament stem cells (PLDSCs), cell viability experiments demonstrated that the application of ClO2 does not lead to a significant decrease in the viability of PLDSCs. in the concentrations used to kill microbes. In contrast, traditional irrigants, H2O2 and CHX are highly toxic to PDLSCs. The aging of PLDSC cultures (passages 3 vs. 7) has characteristic effects on their responsiveness to these agents, since the increased expression of the mesenchymal stem cell markers becomes decreased. They conclude that although the active ingredients of mouthwashes (H2O2,

Oral ingestion of ClO2 solubilized in water

Figure 4. Review of experimental levels of chlorine dioxide / chlorite systemic toxicity. In blue, reference levels for disinfection of drinking water (US-EPA); in green, levels with evidence of therapeutic efficacy at zero toxicity (Refs A Kalcker and E. Insignares Carrione); red: minimum levels of toxicity detection reported in different in vivo studies in animals and humans. harm to larger organisms, such as humans. The authors suggested that the reasons for this differential effect are: (1) ClO2 cannot penetrate deeply into the tissues of larger organisms and (2) the circulation of larger organisms provides a constant supply of antioxidants, offering protection against the effects of ClO2 oxidants. In vitro studies found that methemoglobin was not formed unless glutathione concentrations in red blood cells were almost completely depleted.

In vivo animal studies: Daniel et al. exposed rats to ClO2 concentrations of 0, 25, 50, 100 or 200 mg/liter for 90 days. This equates to doses of 11.5 mg/ kg/day in males and 14.9 mg/kg/day in females. No exposure-related deaths were reported.

Abdel-Rahman et al. reported LD50 rates for female rats of 340 to 468 mg/ kg and for male rats of 292 to 424 mg/kg after a single gavage dose. Additional reports of LD50 in rats include 94 mg/kg (WHO, 2002), 140 mg/kg, 292 ppm (National Institute for Occupational Safety and Health, 2014) and> 5,000 mg/ kg (US Environmental Protection Agency, 2008). The LD50 in mice has been reported to be> 10,000 mg/kg (Shi et al. 1999).

In a study carried out at the University of Querétaro (Mexico) in 2020, researchers challenged the hypothesis that ClO2 decreases viral load and virus-induced mortality in a vertebrate model. To do this, they determined the viral load, virus-induced lesions, and mortality in 10-day-old chicken embryos inoculated with 104 EID50/mL in media of attenuated avian coronavirus (IBV) strains from Massachusetts and Connecticut. ClO2 treatment had a marked impact on IBV infection. Specifically, viral titers were 2.4 times lower and mortality was reduced by half in infected embryos that were treated with ClO2.

The infection caused developmental abnormalities, regardless of treatment.

Lesions typical of IBV infections were observed in all inoculated embryos, but severity tended to be significantly less in ClO2-treated embryos. They found no gross or microscopic evidence of toxicity caused by ClO2 at the doses used in this case. The study concludes that ClO2 could be a safe and viable way to treat and mitigate the effects of avian coronavirus infections, and raises the possibility that similar effects may be observed in other organisms.

Water Solubilized Chlorine Dioxide (CDS) in human and animal studies The studies by Lubbers et al. (1982, 1984a) at the Ohio State University School of Medicine evaluated the short-term toxicity of drinking ClO2. In the first study (Lubbers et al. 1982), a group of 10 healthy adult males drank 1,000 ml (divided into two 500 ml portions, 4 hours apart) of a gradually increasing dose of ClO2. The study included a series of six sequences of 3 days each in Page 4 of 11

The only study conducted for the use of water-solubilized ClO2 (CDS) ingested by oral route is our present study carried out in a multicenter clinical trial in various countries from central and South America (https: //clinicaltrials. gov/ct2/show/NCT0434374). Statistics of Bolivia where the use of watersolubilized ClO2 for the prevention and treatment of COVID-19 was approved by law in early August 2020, demonstrate a marked reduction in cases and deaths in that country. The highest peak recorded in Bolivia was epidemiological week 29 (10,939 cases), with cases falling to 670 in epidemiological week 45, (representing a 93% decrease). The case-fatality rate decreased from 8% to 4.5% in epidemiological week 36, coinciding with the massive use of dioxide in the population without it being possible to affirm with certainty that dioxide is responsible for this decrease. (https: //snis.minsalud.gob.bo/). From a peak of 2,031 daily cases on August 20, 2020, cases dropped to 147 daily cases on October 21, 2020, representing a 93% decrease. Daily deaths decreased from a peak of 132 on September 3, 2020 to 24 deaths on October 21, 2020, with an 82% decrease in mortality. (https: //www.COVID-19.onl/country/bolivia).

While there was a rebound in cases from epidemiological week 49 to 53, mortality remained relatively low. Some doctors linked to our research group (in Bolivia more than 220 doctors) provide feedback that the population reduced the consumption of dioxide and that could be a possible explanation for the peak, and the increase in the consumption of chlorine dioxide again reduced the peak to the previous levels, maintaining a reduced comparative mortality (personal communications) (Figure 5).

In summary, while in other countries the plateau was maintained, in Bolivia it fell, attributing this decrease (as a possible explanation) to preventive measures, the consumption of chlorine dioxide and the massive use of traditional medicine as a possible explanation.

Dose In vitro and in vivo studies demonstrate that low doses of water-solubilized ClO2 exhibit potent and rapid antiviral activity against a wide range of viruses. In a study on drinking water in the Netherlands, Jin et al. found that treatment with ClO2 at concentrations of 0.5 ppm for 25 minutes, or 1.5 ppm for 10 minutes, or 2 ppm for 5 minutes achieved at least a 4 log reduction of enterovirus. Schijven et al. demonstrated that municipal drinking water treated with ClO2 (0.1 ppm in summer and 0.05 ppm in winter) significantly reduced adenovirus levels to levels that are considered safe for drinking water. Alvarez and O'Brien found a ClO2 concentration of 40 ppm added to wastewater seeded with SARS-CoV that completely inactivated the SARS-CoV in 30 minutes. The dose used in this research is 30 mg per day for 21 days, which is well below the NOAEL, or in the worst case the same as the NOAEL reported in previous studies in animals, as we discussed in previous lines.

Materials and Methods To make the greatest search for scientific information on chlorine dioxide, sand they searched Google Scholar, PubMed (Medline), LILACS, Cochrane Library, Science, Scielo, MedScape, looking for articles in English

Eduardo IC, et al.

J Mol Genet Med, Volume 15:S1, 2021

quickly worldwide, which is why we opted for a quasi-experimental clinical trial.

The NCBI (National Center for Biotechnology Information) for Medline and Pubmed databases recently included the concept of ECE (Quasi Experimental Studies) under the MeSH term "Non-Randomized Controlled Trials as Topic" (NCBI, 2015).

Figure 5. The death rate of the Coronavirus in Latin America. and Spanish, which would contain search terms chlorine dioxide, virus, SARS-CoV-2, coronavirus, oxidant, water purification, "Chlorine dioxide" OR "Chlorine dioxide protocol" OR Chlorine dioxide AND virus; Chlorine dioxide AND SARS-COV-2; OR "COVID-19 drug treatment" OR "spike glycoprotein, COVID-19 virus" OR "severe acute respiratory syndrome coronavirus 2" or "COVID-19" OR "2019-nCoV" OR "SARS-CoV-2" O "2019 new coronavirus "OR" 2019 coronavirus disease "OR (pneumonia).

Additionally, a Google search was conducted using these same search terms. The articles selected for inclusion consisted of publications related to the potential use of ClO2 as prevention or treatment of COVID-19, on the beneficial aspects and possible effects of maleficence or toxicity of the substance. All retrieved articles were screened and a subset of relevant abstracts was selected for further evaluation. The bibliographies of these articles were then searched for additional references. The final articles selected for inclusion in this review consisted of articles examining historical uses, chemistry, toxicity, antimicrobial and virucid ClO2, previous investigations in animals in vitro and in vivo, and research in humans. The registries of www.clinicaltrials.gov and those of the WHO International Clinical Registry Platform (ICTRP) were reviewed to identify ongoing or unpublished clinical trials.

From the search results, we selected those that referred to the virucidal action of chlorine dioxide on various microorganisms, in particular on viruses and, among them, SARS-CoV-2 or SARS-CoV. On the other hand, we selected articles that describe the action of SARS-CoV-2 in cells, in its interaction with ACE2 and, in particular, we investigated augmented reality videos or simulation videos based on Silico, for three-dimensional representation. De sites of action such as videos in which the spicular protein and the ACE2 receptor (37) are manipulated, among others, with the ChimeraX (UCSF) augmented reality software. Similarly, we reviewed the structure of the virus spike and relied on the research of Daniel Wrapp and Jason S. McLellan of the University of Texas, information with which, in a parallel study, we were able to hypothesize how chlorine dioxide acts on SAR-CoV-2.

With the information needed to conclude that chlorine dioxide was beneficent and that its use in the COVID-19 pandemic is turned in an immense possibility to control it based on the scientific foundations revealed in previous investigations, and above all, that it was not maleficent for patients, we take the decision to carry out the investigation for determine if it was effective orally in the treatment of COVID-19.

We decided to carry out a phase IIa design given the urgency to verify an option effective, fast and economical treatment that could be implemented Page 5 of 11

Once the research protocol was done, it was decided to register it in clinicaltrials.gov which was accepted on April 7, 2020 under the number NCT 04343742. The design was made international multicenter in order to have a faster collection of the sample and in the future, to have a much larger and more representative sample. The same protocol was presented in eleven American countries and in Spain for approval. Unfortunately, drug control entities in all countries generated warnings and even bans on its use for human consumption that made it difficult for ethics committees to approve the protocol. In Bolivia the law was approved No. 1351 of 2020 that authorized the elaboration, commercialization, supply and consented use of the CDS chlorine dioxide solution as prevention and treatment in the face of the coronavirus pandemic (COVID-19); The ethics committee endorsed by the Bolivian Ministry of Health was legally constituted, which approved this multicenter research protocol of international scope, retro-prospective, made up of five (5) universities (Technical University of Oruro, Public University of El Alto, Universidad Mayor de San Simón, Gabriel René Moreno Autonomous University and Yacuiba Technical Institute "Gran Chaco") which in turn, through its scientific and ethical clinical research committees, are doing their own research on chlorine dioxide for use in different applications [20-35].

Data collection and registration Data collection and recording was carried out by recording demographic and symptomatic data and by means of observation scales based on visual analogue scale (VAS) and laboratory parameters organized by Likert scale for better evaluation and comparability. Thus, the instruments or formats were built, which were recorded in software specially designed for research with the following characteristics: An electronic form in a WEB environment was specially designed for the registration of information required in the investigation through PCs, smartphones and tablets.

The application was developed by the company SCORPII SAS in 2020, with the following functionality: • Registration of the contact information and basic profile of the investigating physician, who identifies the location of the medical center where the patient was treated for the first time and records their contact details and socio-cultural profile.

• File of the letter of informed consent for the use of an investigational substance, signed by each patient.

• Registration of the information on the patient's initial diagnostic clinical history.

• Record of the patient's follow-up clinical history in relation to his symptoms, pain scale (VAS) and assessment with the Likert scale of the laboratory reports made on day 0, 7, 14 and 21.

• In relation to the security conditions, the registered information does not have options to make changes or adjustments to the registered information by the investigating physicians, guaranteeing the authenticity, integrity and confidentiality of the information. Each researcher has access to the application through the authentication of their email and password, in such a way that they are solely responsible for the registration of information on each of their patients.

• Generation of information consolidation reports for statistical analysis, a function of access exclusively by the research director, without options for modifying the database of records made by investigating physicians.

That is, the instruments made it possible to collect and record the results from the performance of semiological, clinical and laboratory examinations at

Eduardo IC, et al.

the beginning of the study treatment of the experimental group and the control group, as well as after 7, 14 and 21 days.

In addition to demographic data, the following are measured: Semiological: Red eyes, fever, cough, expectoration, asthenia/adynamia, chills, vomiting, dyspnea, nasal congestion. The VAS criteria (The visual analog scale or VAS) for pain it is a straight line in which one end means no pain and the other end means the worst pain imaginable. The patient marks a point on the line that matches the amount of pain felt) and the variables investigated. They are: sore throat, head, chest, general myalgic type, affectation due to low back pain. Likert criteria (The Likert scale is an ordinal scale using 5 levels) grouped paraclinical exams. The variables investigated are lymphocytes, leukocytes, platelets, C-reactive protein, Lactate dehydrogenase, AST, -D-dimer and lactate in oximetry [36-55].

The instruments made it possible to collect and record the results from the performance of semiological examinations, assessment of painful symptoms (VAS) and laboratory (LIKERT) at the beginning of the treatment under study (or baseline) as well as after 7, 14 and 21 days.

Analysis and interpretation of the data It is carried out according to the established variables, with the support of the IBM - SPSS Statistics software; the data of the experimental and control groups were analyzed, the general symptoms by means of a comparison of proportions test and their confidence intervals, and the VAS and Likert criteria by means of a paired test using the Wilcoxon - Mann - Whitney test α: 95%) (Tables 1-3).

Population The experimental population to which the multicenter study was directed consisted of a group of patients with active infection with COVID-19, in various medical centers in Bolivia (14 patients), most of them in the southern clinic in La Paz, Bolivia, Peru (two patients) and Ecuador (four patients) for a total of twenty (20) patients. The control population consisted of eight (8) patients from Ecuador, seven (7) patients from Bolivia, three (3) from Mexico and two (2) from Peru, for a total of twenty (20) patients.

J Mol Genet Med, Volume 15:S1, 2021

in the control group at the beginning, at 7 and 14 days. Post-protocol clinical follow-ups (day 22 onwards) were not included in the experimental group and neither in the control group. It is important to note that there was clinical followup by the research staff in most of the patients treated and not treated with chlorine dioxide, observing resolution completeness of symptoms in the treated group. As an empirical observation, the researchers’ report that patients who consumed chlorine dioxide as a treatment for COVID-19, substantially reduced post-COVID-19 symptoms compared to patients with the disease, not treated with dioxide [56-65].

Total duration of the study The study began in July 2020 and concluded in December 2020.

Assignment of study medication Each patient received, in order of admission to the study, a consecutive patient number (odd numbers were selected) and the corresponding study medication. The allocation of this medication was made prior to the start of the study, by means of a computer generated list of vials produced for the purpose of the research. The patients received the chlorine dioxide base preparation at 3,000 ppm, produced by electrolysis with ultrapure chlorine dioxide generating equipment, brand medalab www.ClO2.com with precise written instructions on how to prepare, drink and preserve dilutions. The chemists of the research team controlled the standardization of the water used for the base preparation of the dioxide at 3000 ppm, as well as the water used for diluting and drinking; the controlled variables were: conditions and characteristics of the preparation process, standardized characteristics of the container, use of standardized Merck 28% sodium chlorite, standardized sterile double-distilled water, buffered at pH 7 and chlorine dioxide concentration at 3,000 ppm.

The control group received anti-inflammatory treatment (ibuprofen at doses between 200 and 400 mg every 8 hours), antibiotics (azithromycin 500 mg daily for 5 days), antihistamines (hydroxyzine × 5 mg every 12 hours), corticosteroids (generally 40 mg of methylprednisolone every 12 hours for 3 days and then 20 mg every 12 hours for 3 days) and supportive measures. The experimental group did not receive this treatment.

Patients

Dosage and routes of administration

The selection of the treatment or experimental group was made based on patients with active COVID-19 infection, who were with positive RT-PCR, symptomatic in intervals from 3 to 7 days, which were not in the resolution phase, who were proposed to be voluntary research subjects. Control group patients chose not to be chlorine dioxide treatment subjects. Similarly, simultaneity was applied, which means that patients were obtained in the same period of time in which the cases arose. Viral load was not determined due to costs, technical and logistical difficulties of the reference laboratories in Mexico, Bolivia, Peru and Ecuador.

The medication used is chlorine dioxide 3000 ppm in × 120 cc bottles marked with a planned label and administered according to the standard established in the research protocol.

Number of patients Twenty patients (n=20) for the experimental group and 20 patients (n=20) for the control group were included in the study. A one-to-one relationship per center was not maintained and they were presented randomly according to a probabilistic sample design.

Acceptance criteria in the study The following inclusion criteria were used: COVID-19 RT-PCR positive, characteristic symptoms of COVID-19: fever, odynophagia, respiratory distress, aged between 18 and 80 years.

Exclusion criteria COVID-19 RT-PCR negative, IV/VI renal failure, congestive heart failure, patients who consume anticoagulants in particular warfarin sodium.

Duration of treatment per patient The observation period per patient in the experimental group was (21) twenty-one days. In the control group it was fourteen (14) days. A review was carried out at the beginning, at 7, 14 and 21 days in the experimental group; Page 6 of 11

Chlorine dioxide administration protocols used Initial protocol, loading or protocol (F): 10 ml of chlorine dioxide 3000 ppm is added to 1 liter of water, and the liter is taken in two hours, divided into eight (8) equal doses. Then it is administered.

Maintenance protocol: (C) 10 ml of chlorine dioxide 3000 ppm is added to 1 liter of water, and the liter is given to drink divided into ten (10) equal doses, during the day, every hour.

The medication is manages orally dissolved in the mouth, at least 60 minutes before or after a meal. There were no remnants or shortages in the medication delivered and the original protocol were strictly adhered to. There is no abandonment of treatment.

Results Preliminary analysis of the parameters at study entry The characteristics of the patients in the experimental group at the time of admission, after verification of the positive RT-PCR, generated the following data: By sex: 13 male patients (65%) and 7 female (35%). The ages range between 36 years the minimum and 72 years the maximum. The average age for men is 47.6 years and for women 58 years of age. The general average age is 53.2 years.

A parallel instrument was designed to record possible adverse effects of chlorine dioxide where the data of each patient and their adherence process

Eduardo IC, et al.

J Mol Genet Med, Volume 15:S1, 2021

are placed. In two patients, a slight sensation of gastritis was registered, 7 days after treatment, temporary, which resolved without intervention and did not require stopping the medication (Figure 6).

Characteristics of the control group The characteristics of the patients in the control group at the time of admission after verification of the positive RT-PCR generated the following data: By sex: 10 male patients (50%) and 10 female (50%).

The ages range between 44 years the minimum and 69 years the maximum. The average age for men is 55.6 years and for women 52 years of age. The general average age is 54.5 years (Figure 7).

Result of the comparison of the Experimental versus Control Group by means of a comparison of proportions test and their confidence intervals and Wilcoxon – Mann – Whitney test (α: 95%) To evaluate the effectiveness of the ClO₂, the results of each of the items were compared by means of a comparison of proportions test and their confidence intervals (general symptoms) and a Wilcoxon – Mann – Whitney test (α: 95%) (VAS and Likert) of the data obtained from the Experimental Group compared with the Control group in the Cohort points of Onset of Symptoms, 7 and 14 days after the first symptomatic manifestation.

Figure 6. Ages of Patients Experimental Group.

Below we present the comparisons between the groups, for general symptoms, VAS and Likert.

Regarding general symptoms, on the day of onset of general symptoms, both groups (Control and Experimental) were the same for most of the items (p>0.05). However, for the Chills the Experimental group had a higher Average Range (25.5) than that of the Control Group (15.5) (p: 0,0001). These results confirm that in general for general symptoms, both groups had a similar behavior.

Figure 7. Ages of Patients. Control group.

When making the comparison on the seventh day post symptom manifestation, a significant difference was found in the Experimental group with respect to the control group for Symptoms Fever (p: 0000), Cough (p: 0.000), Chills (p: 0.000) and Dyspnea (p: 0.0006). At day 14 in the symptoms that had not disappeared, the difference was very significant, decreasing its manifestations in the Experimental group.

of the patients until approximately two months after the onset of the disease, verifying a satisfactory evolution with very few post-COVID symptoms. Some of the patients noted some mild spinal discomfort and some degree of chronic fatigue.

For the VAS Scale, at the time of symptom manifestation, both groups reported the same subjective assessment of pain. When performing the same visual analogue comparison of pain in the control group and in the experimental group, it was found that all the items that make up the scale decreased significantly in this group with respect to the Control group on day 7 (p: 0,0000 ; p: 0,0017). The only painful symptom that remained similar was low back pain (P: 0,9633). On day 14 post manifestation the difference was greater (p: 0.000 ; P: 0,0043 y P: 0,0067).

This research has focused on studying the effectiveness of the use of chlorine dioxide in patients with COVID-19, by measuring before and after treatment, present clinical symptoms and laboratory variables based on standardized and accepted scales in research (VAS and Likert) of an experimental group compared to a control group.

When evaluating both groups (Control and Experimental), a difference was found for the values ​​for the PC Reactive parameters (p: 0.0398) and Lactate DH (0.0422), the scores being higher for the experimental group; no difference was found in the rest of the parameters. When comparing the groups at 7 days post-initial manifestation, only the Lymphocytes and Leukocytes showed no difference between groups (p> 0.05), the other parameters significantly decreased in the Experimental group with respect to the Control. (p: 0,0001; p: 0,0036 ; p: 0,0403; p: 0,0194; p: 0,0003). At day 14, all the biochemical parameters had decreased significantly in the experimental group with respect to the Control group (p<0,05).

The results overall (p<0.05) demonstrate the hypothesis that chlorine dioxide is effective in the treatment of COVID-19.

Permanence of the intervention As we noted in previous lines, the design of the protocol did not include post-intervention follow-up beyond day 21, which was the limit that we set ourselves from the beginning. As a non-parameterized observational annotation, we can share that the group of researchers did clinical follow-up Page 7 of 11

Discussion

Regarding the research question whether chlorine dioxide could effectively reduce morbidity and mortality in COVID-19, the confirmatory significant statistical validation by the U test of Wilcoxon – Mann – Whitney test (α: 95%) in the experimental and control groups reveal this and confirm the hypothesis that chlorine dioxide is effective in the treatment of COVID-19.

This phase IIa clinical trial is the preliminary phase for the realization of an upcoming phase IIb, double-blind randomized study, where we can have a sample of 400 patients, a purpose in which we are already working.

The main advantage of the design we chose is that it is a simple and inexpensive study to carry out (the authors did not receive any external funding and the costs were paid directly and personally by them) than a clinical trial with a larger sample number, in particular double blind that would increase the cost significantly. On the other hand, it is the only way to carry out a study when there are ethical and feasibility problems to carry out a random assignment of the sample or when it is necessary to carry it out under natural conditions and in this case, due to manifest urgency. given the pandemic.

The disadvantages are the high susceptibility to biases, especially those of selection and confusion, so to reduce it, we used random probability

Eduardo IC, et al.

J Mol Genet Med, Volume 15:S1, 2021

Table 1. Result of the comparison of the Control Group versus the Experimental Group by means of a comparison of proportions test and their confidence intervals for General Symptoms.

Syntom Fever Cough Asthenia Adynamia Expectoration Chills Vomit Dysnea Nasal Congestion

Group Ctrl Exp Ctrl Exp Ctrl Exp Ctrl Exp Ctrl Exp Ctrl Exp Ctrl Exp Ctrl Exp

Day 0 n (%) | P-value 18 (90%) 0,3160 17 (85%) 20 (100%) 0,0680 18 (90%) 18 (90%) 0,1015 15 (75%) 7 (35%) 0,1405 4 (20%) 17 (85%) 0,0001 7 (35%) 1 (5%) 0,1524 0 (0%) 18 (90%) 0,5000 18 (90%) 5 (25%) 0,2438 7 (35%)

Day 7 n (%) | P-value 14 (70%) 0,0000 0 (0%) 18 (90%) 0,0000 6 (30%) 20 (100%) 0,0000 2 (10%) 3 (15%) 0,0301 0 (0%) 10 (50%) 0,0000 0 (0%) 0 (0%) NA 0 (0%) 16 (80%) 0,0006 7 (35%) 2 (10%) 0,0680 0 (0%)

Day 14 n (%) | P-value 12 (60%) 0,0000 0 (0%) 18 (90%) 0,0000 6 (30%) 20 (100%) NA 0 (0%) 0 (0%) NA 0 (0%) 4 (20%) 0,0127 0 (0%) 0 (0%) NA 0 (0%) 12 (60%) 0,0005 3 (15%) 0 (0%) NA 0 (0%)

Table 2. Result of the comparison of the Control Group versus the Experimental Group through a Wilcoxon – Mann – Whitney test (α: 95%) for the VAS Scale.

VAS Scale Throat pain Headache Chest pain General pain Lumbar/affect

Group Ctrl Exp Ctrl Exp Ctrl Exp Ctrl Exp Ctrl Exp

Day 0 median (IC) | P-value 3,1 IC ± 0,6 0,9670 3,2 IC ± 0,9 2,9 IC ± 0,5 0,2665 2,6 IC ± 0,8 0,6 IC ± 0,3 0,9018 0,6 IC ± 0,2 2,1 IC ± 0,4 0,8660 2,1 IC ± 0,7 1,5 IC ± 0,3 0,7827 1,6 IC ± 0,3

Day 7 median (IC) | P-value 2,1 IC ± 0,4 0,0000 0,0 IC ± 0,0 2,9 IC ± 0,4 0,0000 0,5 IC ± 0,2 0,6 IC ± 0,3 0,0017 0,1 IC ± 0,1 2,4 IC ± 0,4 0,0000 0,5 IC ± 0,2 1,6 IC ± 0,2 0,9633 1,7 IC ± 0,3

Day 14 median (IC) | P-value 1,3 IC ± 0,3 0,0000 0,0 IC ± 0,0 2,5 IC ± 0,4 0,0000 0,4 IC ± 0,2 0,4 IC ± 0,2 0,0043 0,0 IC ± 0,0 2,6 IC ± 0,4 0,0000 0,0 IC±0,1 1,6 IC±0,2 0,0067 2,0 IC±0,2

Table 3. Result of the comparison of the Control Group versus the Experimental Group through a Wilcoxon – Mann – Whitney test (α: 95%) for Biochemical Parameters on the Likert Scale.

Parameter lymphocytes leukocytes Platelets PC Reactive Lactate DH AST Dimero D Lactate

Group Ctrl Exp Ctrl Exp Ctrl Exp Ctrl Exp Ctrl Exp Ctrl Exp Ctrl Exp Ctrl Exp

Day 0 median (IC) | P-value 1,5 IC ± 0,3 0,7827 1,6 IC ± 0,3 2,6 IC ± 0,4 0,2582 2,8 IC ± 0,4 2,4 IC ± 0,3 0,0816 2,7 IC ± 0,3 3,2 IC ± 0,2 0,0398 3,0 IC ± 0,0 2,6 IC ± 0,3 0,0422 3,2 IC ± 0,4 3,2 IC ± 0,4 0,5319 3,2 IC ± 0,4 3,1 IC ± 0,4 0,3060 3,4 IC ± 0,5 1,9 IC ± 0,3 0,0145 2,5 IC ± 0,4

sampling, numbering the patients who were diagnosed and treated, selecting odd-numbered patients for inclusion in the research; the sample of control patients were selected in the same way.

On the other hand, we chose not to use existing groups so as not to compromise the external validity and applicability of the results. Another inherent Page 8 of 11

Díay 7 median(IC) | P-value 1,6 IC ± 0,2 0,9633 1,7 IC ± 0,3 3,1 IC ± 0,3 0,4842 2,9 IC ± 0,3 2,7 IC ± 0,2 0,0202 3,3 IC ± 0,4 3,4 IC ± 0,2 0,0001 2,0 IC ± 0,5 2,7 IC ± 0,3 0,0036 1,9 IC ± 0,4 3,2 IC ± 0,3 0,0403 2,5 IC ± 0,6 3,4 IC ± 0,4 0,0194 2,6 IC ± 0,4 2,2 IC ± 0,2 0,0003 1,5 IC ± 0,3

Day 14 median (IC) | P-value 1,6 IC ± 0,2 0,0067 2,0 IC ± 0,2 3,3 IC ± 0,2 0,0002 2,5 IC ± 0,3 3,0 IC ± 0,2 0,0212 2,6 IC ± 0,3 3,5 IC ± 0,2 0,0000 1,4 IC ± 0,2 2,7 IC ± 0,2 0,0000 1,5 IC ± 0,2 3,5 IC ± 0,3 0,0000 1,9 IC ± 0,3 3,5 IC ± 0,3 0,0029 2,6 IC ± 0,4 2,5 IC ± 0,2 0,0000 1,1 IC ± 0,1

difficulty in our study is the risk of presenting a placebo effect and a Hawthorne effect, which we could not minimize given that, given the humanitarian crisis of the pandemic; we did not consider it ethical for the subjects to participate in the study without being informed of the intervention that would be applied to them. In fact, all participants signed an informed consent for the use of a drug

Eduardo IC, et al.

J Mol Genet Med, Volume 15:S1, 2021

guanine of viral RNA. Additionally, patients begin to improve their sore throat, dry cough and dyspnea. This effect could be attributable to a direct antiinflammatory action, possibly through an action on inflammasomes and it is even postulated that it may be possible due to action on NETosis processes, through a mechanism that has not yet been clearly elucidated and that we are in the process of developing investigate in detail.

Figure 8. Blood samples with methahemoglobinemy and with ClO2.

Although we did not include the oxygen saturation measurement variable by pulse oximetry in the parameters of this investigation, we did the measurement in all patients, observing a considerable improvement in some cases in oxygen saturation; being a consistent observation, this improvement could be explained by the action of the dioxide through the oxidative mechanism of amino acids of the virus "anchored" to the unitβ1 of hemoglobin, improving oxygenation. We verified this mechanism as a possibility, in the research that we did in molecular biology through in silico simulation, in Japan, and it will be the subject of a future paper. In the same way, Pichert in his research suggests that chlorite interacts with the forms of hemoglobin and oxidized hemoglobin. Chlorite inactivates iron hemoglobin and methemoglobin. By these mechanisms, chlorite effectively decreases the yield of cytotoxic hemoglobin species that may appear later of excessive hemolysis of red blood cells in pathological situations.

Likewise, in the laboratory of the Liechtensteiner Verein für Wissenschaft und Gesundheit in Switzerland we were able to verify the immediate oxygenation of erythrocytes, by injecting chlorine dioxide at a concentration of 50 ppm to a sample of red blood cells with methemoglobinemia, using contrast microscopies. Phase and direct (Figure 8).

Figure 9. Variations in serum lactate before and 10 min after an IV clorine dioxide injection at 50 ppm. (A. Kalcker. SCHWEIZER VEREIN FUR NATURWISSENS CHAFTLICHE BIOPHYSIK).

An interesting characteristic when we observe the behavior of the reduction of thrombocytosis in the 21 days of treatment is that there could be an action of dioxide on the rheological behavior of the blood, an idea that is reinforced with the evident decrease of D-dimer in treated patients with the dioxide.

The lymphopenia observed in most patients could be due to a process that begins with disorganization of the cytoskeleton, condensation of chromatin and cytoplasm, loss of mitochondrial function, DNA fragmentation and terminal formation of ruptured membranes or apoptotic bodies, finally eliminated by macrophages. This phenomenon is mediated by the synthesis of the caspase-8 complex, which we consider could be activated by non-canonical means. The complex is favored by protein kinase-R, also accelerated by interferon generated in the presence of the virus. The signaling pathways that are activated by these receptors initiate an inflammatory response, with antiviral effects,

Figure 10. Lactate variations before and one hour after injection with saline sol.

or substance under investigation, in particular because chlorine dioxide has not yet complied with the corresponding process to be classified as a drug.

Regarding the results and the possible explanations for them, the most important thing in the experimental group is the negativization of the RT-PCR in one hundred percent of the cases at seven days, as well as the rapid way in which the results are reduced. general symptoms in patients, in particular fever, cough, dyspnea, asthenia and general pain, which is achieved in 70% of cases seven days after treatment, a very marked reduction in VAS values​​ in percentage similar to 7 days, in particular reduction of sore throat by 100%, reduction of low back pain by almost 80%, results that reveal a significant improvement in morbidity in patients.

In the initial stage of administration of chlorine dioxide, patients show improvement in their febrile symptoms, this could be attributed to its direct virucidal effect due to its action on the spike and on the virus RNA, through the mechanism that we explained above the oxidation of amino acids and Page 9 of 11

The virus also activates the transforming growth factor-b through neuraminidase, thus generating another cascade that ends with c-Jun-N-kinase and triggers transcription followed by the expression of pro apoptosis genes.

The virus could synthesize the virulence peptide PB1-F2, a small fragment responsible for destroying lymphocytes, a circumstance that could explain the lymphopenia observed in COVID-19. The action could also be mediated by action on inflammasomes, where the signaling (redox) by ATP can induce the de-subiquitination of NLRP3, possibly induced by chlorine dioxide.

Regarding the increase in lactate, it is clear that it can be caused by dysfunction of the enzyme pyruvate dehydrogenase (PDH), as has been demonstrated in patients with sepsis, by mitochondrial dysfunction, or by hyperlactacidemia. The observed improvement could be explained through a mechanism in which chlorine dioxide has a cellular oxygenating effect directly through the release of oxygen or by redox signaling mechanisms with enzyme activation/inhibition that activate the pyruvate pathway with efficient ATP production.

When we inject direct chlorine dioxide into the vein, the amount of lactate in the blood is reduced, as can be seen in the following graph which shows in a comparative way the level of lactate in the blood and after intravenous chlorine dioxide application at 50 ppm at ten minutes post injection (Figure 9).

In comparison, when administering normal saline solution via direct intravenous, and measuring the lactate levels, we observe that there is no variation in the lactate level (Figure 10).

Eduardo IC, et al.

The data obtained in this research validates the effectiveness of chlorine dioxide in COVID-19. This is the first phase IIa clinical trial in the world. In this study there were no inconclusive results. The phase IIa study design is appropriate for the research phase of a new molecule - such as chlorine dioxide - in its development as a potential drug.

There is much to explore about chlorine dioxide and its therapeutic possibilities, about which there is enough reasonable information to continue to study further.

The findings of this research open a complete world of possibilities for medical therapists in the world, as these initial results will motivate more formal research, will support the search for new therapeutic options, particularly in COVID-19, and will allow the generation of research spaces at the molecular level and genomics on the processes of cellular respiration, redox signaling and the implantation of oxidative therapies as a new possibility for the approach of multiple pathologies with altered common intracellular bioenergetic mechanisms. The results can be extended to the control of infectious diseases in agronomy, the development of new nanotechnologies in pharmaceuticals and humans in general by alleviating and controlling COVID-19 and other pandemics or epidemics that may occur in the short future. The results found here, rather than expanding previous studies, as it is a pilot study and the first of its kind in the world, enable the initiation of many studies on dioxide and the related issues noted.

Conclusion In conclusion, we can affirm without a doubt, based on test of comparison of proportions and its confidence interval, as well as the paired tests where we used the Wilcoxon – Mann – Whitney test (α: 95%), that the data in most of the variables (P<0.05) obtained indicate that chlorine dioxide is effective in the treatment of COVID-19, making RT-PCR negative in one hundred percent of cases at 7 days, significantly and rapidly modifying the symptoms of the disease, significantly reducing laboratory parameters to normality within 14 to 21 days. We recommend conducting randomized double-blind studies and delving into studies of toxicological safety and therapeutic efficacy of chlorine dioxide in pathologies of epidemiological impact in the near future.

Author Contributions and Recognitions • We want to express our gratitude for your collaboration and contributions to the doctor, Mitchell B. Liester, Doctor of Medicine, University of Colorado School of Medicine, Colorado Springs Branch for their conceptual contributions and selfless support in the search for new options for patients.

•

Our sincere thanks to Engineer Victor Yela for their permanent assistance with computer science topics, tabulation and research data; to the physicians of the eleven countries that are members of our research team, and to the thousands of physicians around the world who accompany us in this chlorine dioxide research process, to whom we thank for their support and testimonial, conceptual contributions, as well as their a voice of encouragement to remain in this investigation despite the immense difficulties of the way.

• To Andreas Kalcker for his lights, intelligence and injection of energy and motivation to advance being a beacon of wisdom born from the heart.

• To all the patients who participated in the research, in particular for their trust in us, for accompanying us in seeking new possibilities for a world better.

J Mol Genet Med, Volume 15:S1, 2021

Funding This work was supported with the researchers' own resources.

Conflict of Interests The authors declare no conflict of interest or no competing economic interests. Authors have completed the ICMJE conflict of interest declaration form, translated into Spanish by Medwave, and declare that they have not received funding for the article/research; have no financial relationships with organizations that could have an interest in the published article in the last three years; and have no other relationships or activities that could influence the published article. Forms can be requested by contacting the responsible author. This does not alter the authors' adherence to all policies on the exchange of data and materials.

References 1. https://www.naturalnews.com/2020-05-18-researchers-claim-100-percent-curerate-vs-COVID-19-ecuador-intravenous-chlorine-dioxide.html 2. https: //www.atsdr.cdc.gov/ToxProfiles/tp160-c1-b.pdf 3. Walls, Alexandra C, Young-Jun Park, M Alejandra Tortorici and Abigail Wall, et al. "Structure, Function, and Antigenicity of the SARS-Cov-2 Spike Glycoprotein." Cell 181 (2020): 281-292.

4. Grifoni, Alba, John Sidney, Yun Zhang and Richard H Scheuermann, et al. "Candidate targets for Immune Responses to 2019-Novel Coronavirus (nCoV): Sequence Homology-and Bioinformatic-Based Predictions." Cell-Host-Microbe-D-20-00119 (2020).

5. Alvarez, Maria E and RT O'Brien. "Mechanisms of Inactivation of Poliovirus by Chlorine Dioxide and Iodine." Appl Environ Microbiol 44 (1982): 1064-1071.

6. Calabrese, Edward J, Gary S Moore and Robert W Tuthill. "The Health Effects of Chlorine Dioxide as a Disinfectant in Potable Water: A literature survey." J Environ Health 41 (1978): 26-31.

7. Chen, Yu-Shiaw and James M Vaughn. "Inactivation of Human and Simian Rotaviruses by Chlorine Dioxide." Appl Environ Microb 56 (1990): 1363-1366.

8. Sun, Chunyun, Long Chen, Ji Yang and Chunxia Luo, et al. "SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development." Biorxiv (2020).

9. https: //clinicaltrials.gov/ct2/show/NCT04343742 10. https: //clinicaltrials.gov/ct2/show/NCT04409873 11. https: //clinicaltrials.gov/ct2/show/NCT04621149 12. Daniel, F Bernard, Lyman W Condie, Merrel Robinson and Judy A Stober, et al.

"Comparative Subchronic Toxicity Studies of Three Disinfectants." J Am Water Works Assoc 82 (1990): 61-69.

13. Department of Health and Human Services, DHSS, INH, United States.

14. DeZuane, John. Handbook of drinking water quality. John Wiley & Sons, 1997.

15. Food and Drug Administration. Food Additive Status, Specific Use Additives 173.300 Chlorine dioxide, 2020.

16. Guo, Yan-Rong, Qing-Dong Cao, Zhong-Si Hong and Yuan-Yang Tan, et al.

"The Origin, Transmission and Clinical Therapies on Coronavirus Disease 2019 (COVID-19) Outbreak–An Update on the Status." Military Med Res 7 (2020): 1-10.

17. Haag, HB. "The Effect on Rats of Chronic Administration of Sodium Chlorite and Chlorine Dioxide in the Drinking Water." Report to the Mathieson Alkali Works from the Medical College of Virginia (1949).

18. Heffernan, WP, C Guion and RJ Bull. "Oxidative Damage to the Erythrocyte Induced

by Sodium Chlorite, in vivo." J Environ Pathol Toxicol 2 (1979): 1487-1499.

19. https: //www.vyphidroasesores.com/

• To Nela who was our permanent support with patient data records.

20. Insignares-Carrione, Eduardo, Blanca Bolano Gómez and Andreas Ludwig Kalcker.

"Chlorine Dioxide in COVID-19: Hypothesis about the Pos-sible Mechanism of Molecular Action in SARS-CoV-2." (2020).

• To professors Luis Prieto, Jorge Gaupp and Pablo Campra, for his critical remarks to the work. With their observations they forced us to improve our effort.

22. Lan, Jun, Jiwan Ge, Jinfang Yu and Sisi Shan, et al. "Structure of the SARS-CoV-2

Page 10 of 11

21. Jin, Min, Jinyang Shan, Zhaoli Chen and Xuan Guo, et al. "Chlorine Dioxide Inactivation of Enterovirus 71 in Water and its Impact on Genomic Targets." Environ Sci Technol 47 (2013): 4590-4597.

Eduardo IC, et al.

Spike Receptor-Binding Domain Bound to the ACE2 Receptor." Nature 581 (2020): 215-220.

23. Junli, Huang, Wang Li, Ren Nenqi and Liu Xue Li, et al. "Disinfection Effect of Chlorine Dioxide on Viruses, Algae and Animal Planktons in Water." Water Res 31 (1997): 455-460.

24. Kalcker, Andreas. Forbidden health. Voedia, 2016.

J Mol Genet Med, Volume 15:S1, 2021 43. Schijven, Jack, Peter Teunis, Trudy Suylen and Henk Ketelaars. "QMRA of Adenovirus in Drinking Water at a Drinking Water Treatment Plant using UV and Chlorine Dioxide Disinfection." Water Res 158 (2019): 34-45.

44. Shi, L and C Xie. "Experimental Observation on Acute Toxicity and Irritative Effect of Stable Chlorine Dioxide." Zhongguo Xiaoduxue Zashi 16 (1999): 39-40.

25. Kály-Kullai, K, M Wittmann, Z Noszticzius and László Rosivall. "Can Chlorine Dioxide Prevent the Spreading of Coronavirus or other Viral Infections? Medical Hypotheses." Physiol Int 107 (2020): 1-11.

45. Simmons, Graham, Jacqueline D Reeves, Andrew J Rennekamp and Sean M Amberg, et al. "Characterization of Severe Acute Respiratory Syndrome-Associated Coronavirus (SARS-CoV) Spike Glycoprotein-Mediated Viral Entry." Proc Nat Acad Sci 101 (2004): 4240-4245.

26. Letko, Michael, Andrea Marzi and Vincent Munster. "Functional Assessment of Cell Entry and Receptor Usage for SARS-CoV-2 and Other Lineage B Beta corona viruses." Nature Microbiol 5 (2020): 562-569.

46. Song, Wenfei, Miao Gui, Xinquan Wang and Ye Xiang. "Cryo-EM Structure of the SARS Coronavirus Spike Glycoprotein in Complex with its Host Cell Receptor ACE2." PLoS Pathog 14 (2018): e1007236.

27. Li, Wenhui, Michael J Moore, Natalya Vasilieva and Jianhua Sui, et al.

"Angiotensin-Converting Enzyme 2 is a Functional Receptor for the SARS Coronavirus." Nature 426 (2003): 450-454.

47. https: //www.atsdr.cdc.gov/toxprofiles/tp160.pdf

28. Li, Jun Wen, Zhong Tao Xin, Xin Wei Wang and Jin Lai Zheng, et al. "Mechanisms of Inactivation of Hepatitis A Virus in Water by Chlorine Dioxide." Water Res 38 (2004): 1514-1519.

29. Lin, Ja-Liang and Paik-Seong Lim. "Acute Sodium Chlorite Poisoning associated with Renal Failure." Renal Failure 15 (1993): 645-648.

30. Zhang, Linlin, Daizong Lin, Xinyuanyuan Sun and Katharina Rox, et al. "X-ray Structure of Main Protease of the Novel Coronavirus SARS-Cov-2 Enables Design of Α-Ketoamide Inhibitors." BioRxiv (2020).

31. Lubbers, Judith R, Sudha Chauan and Joseph R Bianchine. "Controlled Clinical Evaluations of Chlorine Dioxide, Chlorite and Chlorate in Man." Environ Health Persp 46 (1982): 57-62.

32. Manterola, Carlos and Tamara Otzen. "Critical Analysis of Biomedical Literature." Int J Morphol 32 (2014): 599-607.

33. Moore, Gary S, Edward J Calabrese, Salvatore R DiNardi and Robert W Tuthill.

"Potential Health Effects of Chlorine Dioxide as a Disinfectant in Potable Water Supplies." Med Hypotheses 4 (1978): 481-496.

34. Musil J, Kontek Z, Chalupa J and Schmidt P (1964) Toxicological Aspects Of ClO2 Application for the Treatment of Water Containing Phenol. Chem-Technol Praze 8: 327-345.

35. https: //www.cdc.gov/niosh/idlh/10049044.html 36. National Institutes of Health. Novel Coronavirus Structure Reveals Targets for Vaccines and Treatments, 2020.

37. Noszticzius, Zoltán, Maria Wittmann, Kristóf Kály-Kullai and Zoltán Beregvári, et al. "Chlorine dioxide is a Size-Selective Antimicrobial Agent." PloS one 8 (2013): e79157.

38. Ogata, Norio. "Inactivation of influenza virus haemagglutinin by chlorine dioxide: Oxidation of the Conserved Tryptophan 153 Residue in the Receptor-Binding Site." J Gen Virol 93 (2012): 2558-2563.

39. Láng, Orsolya, Krisztina S Nagy, Julia Láng and Katalin Perczel-Kovách, et al. "Comparative Study of Hyper pure Chlorine Dioxide with Two other Irrigants Regarding the Viability of Periodontal Ligament Stem Cells." Clin Oral Invest 2 (2020): 1-12.

40. Pichert, Annelie and Jürgen Arnhold. "Interaction of the Chlorite-Based Drug WF10 and Chlorite with Hemoglobin, Methemoglobin and Ferryl Hemoglobin." Arch Biochem Biophys 585 (2015): 82-89.

41. Couri, Daniel, Mohamed S Abdel-Rahman and Richard J Bull. "Toxicological Effects of Chlorine Dioxide, Chlorite and Chlorate." Environ Health Persp 46 (1982): 13-17.

42. YanRH, ZhangYY. "Structural Basis for the Recognition of the SARS-CoV-2 by Full Length Human ACE2." bioRxiv.

48. https://cfpub.epa.gov/ncea/iris/iris_documents/documents/toxreviews/0648tr.pdf 49. https://www3.epa.gov/pesticides/chem_search/ppls/074986-00001-20040414.pdf 50. https://www3.epa.gov/pesticides/chem_search/cleared_reviews/csr_PC020503_26-Sep-08_a.pdf 51. https: //iaspub.epa.gov/apex/pesticides/f?

52. https://www.epa.gov/pesticide-registration/list-n-disinfectants-coronavirusCOVID-19 53. https://consumermedsafety.org/medication-safety-articles/item/179-miracletreatment-turns-into-potent-bleach 54. https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?fr=173.300 55. https://www.fda.gov/emergency-preparedness-and-response/coronavirus-disease2019-COVID-19/coronavirus-disease-2019-COVID-19-frequently-asked-questions.

56. Wang, Peigang, Jian Chen, Aihua Zheng and Yuchun Nie, et al. "Expression Cloning of Functional Receptor used by SARS Coronavirus." Biochem Biophys Res Commun 315 (2004): 439-444.

57. Wang, Xin-Wei, Jin-Song Li, Min Jin and Bei Zhen, et al. "Study on the Resistance of Severe Acute Respiratory Syndrome-Associated Coronavirus." J Virol Methods 126 (2005): 171-177.

58. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technicalguidance/naming-the-coronavirus-disease-(COVID-2019)-and-the-virus-thatcauses-it.

59. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/events-asthey-happen.

60. https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200522COVID-19-sitrep 61. https: //www.who.int/substance_abuse/facts/en/ 62. Zambrano-Estrada, Xochitl, Carlos A Dominguez-Sanchez, Marina BanuetMartinez and Fabiola Guerrero-de la Rosa, et al. "Evaluation of the Antiviral Effect of Chlorine Dioxide (ClO2) using a Vertebrate Model Inoculated with Avian Coronavirus." bioRxiv, 2020.

63. Xue, Bin, Min Jin, Dong Yang and Xuan Guo, et al. "Effects of Chlorine and Chlorine Dioxide on Human Rotavirus Infectivity and Genome Stability." Water Res 47 (2013): 3329-3338.

64. Young, RO. "Chlorine dioxide (ClO2) as a Non-Toxic Antimicrobial Agent for Virus, Bacteria and Yeast (Candida albicans)." Int J Vaccines Vaccin 2 (2016): 00052.

65. Zhu, Zhenbang, Yang Guo, Piao Yu and Xiaoying Wang, et al. "Chlorine Dioxide Inhibits the Replication of Porcine Reproductive and Respiratory Syndrome Virus

by Blocking Viral Attachment." Infect Genet Evol 67 (2019): 78-87.

How to cite this article: Insignares-Carrione E, Bolano Gomez B, Andrade Yohanny, and Callisperis Patricia et al. “Determination of the Effectiveness of Chlorine Dioxide in the Treatment of COVID-19.” J Mol Genet Med 15 (2021): S2 doi: 10.37421/ jmgm.2021.15.481

Page 11 of 11

Physiology International DOI: 10.1556/2060.2020.00015

Can chlorine dioxide prevent the spreading of coronavirus or other viral infections? Medical hypotheses 1

K. KALY-KULLAI , M. WITTMANN1, Z. NOSZTICZIUS1 and

ROSIVALL2* LASZL O

1

Department of Physics, Group of Chemical Physics, Budapest University of Technology and Economics, Budapest, Hungary 2

Institute of Translational Medicine and International Nephrology Research and Training Center, Semmelweis University, Budapest, Hungary

© 2020 The Author(s)

INTRODUCTION Motivation Viruses have caused many epidemics throughout human history. The novel coronavirus [10] is just the latest example. A new viral outbreak can be unpredictable, and development of specific defense tools and countermeasures against the new virus remains time-consuming even in today's era of modern medical science and technology. In the lack of effective and specific medication or vaccination, it would be desirable to have a nonspecific protocol or substance to render the virus inactive, a substance/protocol, which could be applied whenever a new viral outbreak occurs. This is especially important in cases when the emerging new virus is as infectious as SARS-CoV-2 [4].

Aim and structure of the present communication In this editorial, we propose to consider the possibility of developing and implementing antiviral protocols by applying high purity aqueous chlorine dioxide (ClO2) solutions. The aim of this proposal is to initiate research that could lead to the introduction of practical and effective antiviral protocols. To this end, we first discuss some important properties of the ClO2 molecule, which make it an advantageous antiviral agent, then some earlier results of ClO2 gas application

*

Corresponding author: Prof. emer. Laszlo Rosivall, MD, PHD, DSc, FERA, FAPS, Institute of Translational Medicine, International Nephrology Research and Training Center, Semmelweis University, Budapest, Nagyv arad t er 4., H-1089, Hungary. Tel/Fax: 36-1-2100-100, E-mail: rosivall.laszlo@med.semmelweis-univ.hu

2

Physiology International

against viruses will be reviewed. Finally, we hypothesize on methods to control the spread of viral infections using aqueous ClO2 solutions.

PREVIOUS EXPERIENCE AND BACKGROUND OF USING ClO2 AS AN ANTIVIRAL AGENT Inactivating viruses with ClO2 in aqueous phase To our present knowledge, an aqueous solution of ClO2 is able to inactivate all types of viruses.

Disinfectants (in water phase) are compared by their CT values, which is the concentration (measured in mg/L) multiplied by the contact time (measured in minute). In CT tables, ClO2 is indicated for viruses in general, without mentioning any exemptions. For example, according to [6], a CT value of 8.4 mg 3 min/L is needed to achieve a four-orders-of-magnitude (“4 log” or “99.99%”) inactivation of viruses in an aqueous medium at 25 8C.

Chemical mechanism of virus inactivation: reaction of ClO2 with amino acid residues In 1986, Noss et al. [19] proved that the inactivation of bacterial virus f2 by ClO2 was due to its reactions with the viral capsid proteins, and almost no inactivation of the infectious viral RNA occurred [8] when that was treated with ClO2 separately. They found [19], however, that three discrete chemical moieties in the viral protein, namely the cysteine, tyrosine, and tryptophan amino acid residues were able to react with ClO2 rapidly. In 1987, Tan et al. [28] tested the reactivity of ClO2 on 21 free amino acids. ClO2 reacted only with six amino acids dissolved in 0.1 M sodium phosphate buffer, pH 6.0. The reaction with cysteine, tryptophan, and tyrosine was too rapid to be followed by their technique. Three further amino acids (histidine, hydroxyproline, and proline) reacted with ClO2 much more slowly, at a measurable rate.

The reactivity of the three fast-reacting amino acids (cysteine [12], tyrosine [17], and tryptophan [27]) was studied in Margerum's laboratory between 2005 and 2008. They found that cysteine had the highest reactivity among these amino acids. From their experimental data they calculated second order-rate constants (at pH 7.0, 25 8C and 1 M ionic strength) and obtained the following sequence: cysteine 6.9 3 106 M 1 s 1 >> tyrosine 1.3 3 105 M 1 s 1 > tryptophan 3.4 3 104 M 1 s 1 >> guanosine 50 -monophosphate 4.5 3 102 M 1 s 1. (They studied guanosine 50 -monophosphate [18] as a model compound for guanine in nucleic acids.

Data presented here are taken from Table 3 of ref. [18]).

In 2007, Ogata [22] found that the antimicrobial activity of ClO2 is based on denaturation of certain proteins, which is primarily due to the oxidative modification of the tryptophan and tyrosine residues of the two model proteins (bovine serum albumin and glucose-6-phosphate dehydrogenase) used in his experiments. In 2012, it was again Ogata who showed [23] that the inactivation of influenza virus by ClO2 was caused by oxidation of a tryptophan residue (W153) in hemagglutinin (a spike protein of the virus), thereby abolishing its receptor-binding ability.

In this context it is interesting to remark that the spike protein of the new coronavirus SARS_CoV-2 contains 54 tyrosine, 12 tryptophan, and 40 cysteine residues [29]. If we assume that in an aqueous solution all of these residues are able to react with ClO2 just like the free amino acids, then the inactivation of the viruses can be extremely rapid even in a very dilute (e.g., in a 0.1 mg/L) ClO2 solution.

Physiology International

3

ClO2 is a water soluble gas Although chlorinedioxide in itself is a gas, it is highly soluble in water. When both air and water are present, ClO2 is distributed between the two phases in an equilibrium ratio determined by the temperature. This distribution coefficient of ClO2 was determined by Ishi [11] in 1958. The distribution coefficient, g 5 [ClO2]G/[ClO2]L gives the ratio of the concentrations expressed in the same units in the gas and the aqueous phases (e.g., g/L) and changes as a function of the temperature. For example, at 20 8C g 5 0.0316, indicating that in equilibrium 1 cm3 aqueous phase contains (0.0316) 1 5 31.6 times more ClO2 molecules than 1 cm3 gas phase.

In practice, the concentrations in the two phases are usually given in ppm. However, these dimensionless numbers are defined in a different way in the gas and liquid phases as ppm (V/V) and ppm (m/m), respectively. Therefore, for practical purposes, we need a distribution coefficient, which gives the ratio between these concentrations. Straightforward calculation yields that the distribution coefficient in terms of ppm is 357 times the distribution coefficient in terms of (g/L), so at 20 8C gppm 5 11.3. Thus, the following formula can be used to calculate the ClO2 concentration of the gas phase being in equilibrium with a ClO2 solution at 20 8C: ½ClO2 gas in ppmðV=VÞ ¼ 11:3 3 ½ClO2 aq in ppmðm=mÞ

Inactivating viruses with ClO2 in gas phase The virus-inactivating reactions (the reactions of ClO2 with the three amino acids) take place in an aqueous medium; consequently, ClO2 can inactivate microbes in their wet state only.

Therefore, ClO2 gas that is moisturized can be an ideal agent against viruses both in their wet and dry states. Viruses that are carried by water droplets could be easily inactivated even by ClO2 gas owing to the high solubility of ClO2 in water [11]. A dry ClO2 gas would be inappropriate as the water content of the aqueous droplet could evaporate, and in the absence of aqueous medium the reactions of ClO2 slow down extremely. Indeed, Morino et al. [16] reported that when applying a low concentration of ClO2 in the gas phase against FCV in dry state, atmospheric moisture – at least a 75–85% relative humidity – is indispensable to inactivate viruses. The advantage of using a moisturized ClO2 gas is that its water content is also able to wet viruses in dry environment. Most viruses are found on hard surfaces indoor, but a small fraction of viruses are “airborn”, attached to dust particles, which can also carry a single microbe or an aggregate of microbes. Therefore, it is a prerequisite of an effective disinfection that all microbes in all parts of the room should be wet and should be in contact with ClO2. If enough aqueous ClO2 solution is sprayed into the room, the droplets will saturate the atmosphere with water vapor everywhere, moreover, the atmosphere will also contain gaseous ClO2 everywhere.

The great advantage of this method is that H2O and ClO2 molecules of the gas phase can reach the microbes in every small corner of the room. Finely dispersed water droplets containing dissolved ClO2 can create an advantageous environment to maintain such conditions for a longer time.

This method using high ClO2 concentration allows fast disinfection of rooms when people are not present, e.g., intensive care units, buildings used as quarantine, or public transport vehicles. However, the application of ClO2 gas is limited when people are present, as it is harmful for humans and animals above certain concentrations. The US Occupational Safety and Health Administration (OSHA) limits the concentration of ClO2 gas allowed in workplace air to

4

Physiology International

0.1 ppm (V/V) time-weighted average (TWA) for an 8-h exposure, and to a temporarily higher 0.3 ppm Short-Term Exposure Limit (STEL) only for a 15-min period [30].

Previous research on preventing viral infections with gaseous ClO2 Ogata [21] realized first that ClO2 is able to inactivate viruses even under the 0.1 ppm (OSHA TWA) limit that is in concentrations which are not harmful to humans. In 2008, Ogata and Shibata [25] demonstrated that infection of mice with influenza A virus applied in an aerosol can be prevented by ClO2 gas present at 0.03 ppm concentration in the air, which is only 30% of the permissible TWA exposure level for humans at a workplace. They concluded that “ClO2 gas could, therefore, be useful as a preventive tool against influenza in places of human activity without necessitating evacuation.” They have even made attempts to decrease the incidence of flue infections among schoolchildren by applying low concentrations of ClO2 gas in a classroom [24].

In spite of these promising early results we are not aware of any wider-scale application of this method in the last decade. There are two problems which could hinder the widespread adoption of this method: 1. With the technique applied by the above-cited authors it is not an easy task to achieve and maintain a very low ClO2 concentration in a large space and for a long time, which is a prerequisite of achieving a satisfactory level of virus inactivation.

2. It is not understood why low ClO2 levels are not harmful to humans or animals and still effective against viruses.

Size selective effect of ClO2 Although cysteine, tyrosine, and tryptophan residues can also be found in human tissues, ClO2 is much less toxic for humans or animals than for microbes (bacteria, fungi, and viruses).

Noszticzius et al. [20] found that the main reason for this selectivity between humans and microbes is based not on their different biochemistries but on their different sizes. Based on experiments and calculations using a reaction-diffusion model Noszticzius et al. [20] found that the killing time of a living organism is proportional to the square of its characteristic size (e.g., its diameter), thus small ones will be killed extremely fast. Their calculations indicated that a bacterium 1 mm in diameter would be killed in a 300 mg/L ClO2 solution within 3 ms, and even in a much more dilute, 0.25 mg/L ClO2 solution it would be eliminated in only 3.6 s. During this time, ClO2 reaches all parts of the cell and kills it by destroying its cysteine-, tyrosine-, and tryptophan-containing proteins, which are essential for life processes.

The protective role of glutathione against ClO2 oxidation in a living cell According to Ison et al. [12] glutathione reacts with ClO2 at a rate, which is even higher than the rate of the very fast ClO2 – cysteine reaction. When ClO2 contacts a living cell containing glutathione, at first the ClO2 concentration remains very low even at the point of entry into the cell due to this rapid reaction. As a small molecule, glutathione can also diffuse rapidly to the point of entry from other parts of the cell consuming most of the ClO2 there, and preventing it from reaching the cysteine, tyrosine, and tryptophan residues of the proteins in the bulk of the cytoplasm. Consequently, the initial low ClO2 concentration cannot make too much harm.

Physiology International

5

However, continuous ClO2 entry can finally exhaust the cell's glutathione (and other antioxidant) capacity even if the cell produces such antioxidants continuously. At this point, ClO2 can enter into the previously protected zones of the cell and react with the reactive amino acid residues, causing denaturation of the affected proteins and ultimately cell death.

The effect of glutathione and other small antioxidant molecules present in living cells was not taken into account in the theoretical calculations of Noszticzius et al. [20]. Their experiments were done on a non-living and washed animal membrane, where membrane-fixed reactive proteins were present, but glutathione and other small molecules were absent. A living cell, however, continuously produces these antioxidants, thus their role cannot be neglected.

Indeed, looking at the experimentally measured disinfection dynamics of a 0.25 mg/L ClO2 solution against Escherichia coli bacteria [2] we can see a disinfection rate, which is surprisingly fast but still about one order of magnitude slower than the theoretical estimate. It is reasonable to assume that the delaying effect of these small reducing molecules is responsible for that deviation.

Protection of human tissues against the oxidative effect of ClO2 Human cells also contain glutathione in mM concentrations, as well as other antioxidants like vitamin C and E, which work together with glutathione to reduce ClO2 [7]. As a human cell is much larger than a bacterium, consequently its glutathione reserve and glutathione production potential are also greater, so even an isolated human cell can survive much longer in a ClO2 environment than a planktonic bacterium. Considering that human cells are not isolated but form tissues, their glutathione stock may be many orders of magnitude greater than that of a planktonic bacterium. Additionally, in multicellular organisms circulation transports antioxidants continuously to the cells of the tissue affected by a ClO2 attack, helping them to survive.

This strengthens the size-selectivity effect, and explains the surprising observation [15] that ClO2 solutions that are able to kill planktonic bacteria in a fraction of a second may be consumed, because they are safe for humans to drink in a small amount (e.g., drinking 1 L of 24 mg/L ClO2 solution in two portions on a single day caused no observable effects in humans [15]).

The effect of ClO2 on the lung While human tissues are not very sensitive to ClO2 in general, lungs should be considered differently. This is because the interalveolar septum separating the airspace of an alveolus from the blood stream of a capillary lumen is very thin. That diffusion barrier in the human lung is a mere 2 mm thick [1] in order to facilitate an efficient diffusional exchange of oxygen and carbon dioxide between the air and blood. The alveolus is covered by a thin layer of lining fluid called epithelial lung lining fluid (ELF) or hypophase. The ELF is only 0.2 mm thick in rat alveoli [1, 13]. It contains glutathione [3] and other antioxidants such as ascorbic and uric acids [5]. It is remarkable, that the ascorbic acid concentration is 2.5 times, and the glutathione concentration is more than 100 times higher in the ELF than in the plasma. The normal function of these nonenzymatic antioxidants in the ELF is to protect the epithelial cells from reactive oxygen species (ROS) like superoxide radicals or hydrogen peroxide, which are toxic products of the metabolism. They can also defend the lung against other toxic gases such as ozone (O3), nitrogen dioxide (NO2) or ClO2. However, high amounts of ClO2 can consume all reducing agents in the

6

Physiology International

ELF, at which point ClO2 starts to react with the epithelial cells causing a continuously growing damage to these cells. It is known that higher concentrations of ClO2 gas can be lethal. However, it is reasonable to assume that the effect of ClO2 on the lung depends not only on its concentration in the gas phase but also on the contact time. Thus, when considering the impact of ClO2 on the lung, it would be logical to regard the CT (concentration) 3 (contact time) product in a similar way as in the case of the microbes.

Estimating the inactivation time of viruses In the case of viruses, the inactivation mechanism differs from that of bacteria or other cells. It is feasible to assume that the inactivation time of a virus is probably much shorter than the inactivation time of a bacterium under the same conditions (ClO2 concentration, temperature, etc.). The following arguments support this assumption: 1. Viruses are about one order of magnitude smaller than bacteria e.g., the diameter of SARS_CoV-2 is about 120 nm [9]. The killing time of a virus as introduced in [20] would be 1–2 orders of magnitude shorter than that of a bacterium, i.e., the diffusion-controlled reaction with ClO2 would happen on a shorter time scale in the entire volume of the virus.

2. It is not necessary for ClO2 gas to penetrate the virus in order to inactivate it. It is enough if ClO2 reacts with one or some of the cysteine, tyrosine, and tryptophan amino acid residues of the spike, which are located on the surface of the virus. This means that the theoretical approach of ref. [20] overestimates the inactivation time of viruses. On the one hand, because diffusion is extremely fast over a 0.1 mm length scale, thus probably it is not limiting the rate of the reactions. On the other hand, ClO2 can reach a large part of the reactive amino acid residues of the spike without permeating through the protein envelope of the virus.

3. Viruses do not contain protective small molecular thiols like glutathione or other small molecular protective metabolic products, because viruses have no metabolism. In this respect viruses should be much more vulnerable than bacteria to an attack by ClO2.

These facts all suggest that once ClO2 contacts the surface of a virus, its inactivation is quick.

However, a virus ready to infect a cell is typically in aqueous phase, e.g., in a fluid droplet, or in the epithelial lining fluid covering the mucous membranes. The size of these aqueous phases is much larger than that of the virus. Therefore, in such cases, the rate-limiting step probably is the diffusion of ClO2 in the water and the reaction with other substances. The time required to inactivate the virus itself would be short compared to the time needed to transport enough ClO2 molecules to the virus.

SUGGESTIONS FOR PREVENTING THE SPREAD OF VIRAL INFECTIONS USING ClO2 Based on the previous arguments, some propositions will now be put forward on how aqueous ClO2 solutions could be applied for global and local (personal) disinfection purposes. Many of these propositions are based on hypotheses, and therefore can only be applied after careful research. It is a goal of the present work to initiate research to check these hypotheses and proposals experimentally, which could lead to new applications of high-purity ClO2 solutions against viral or other infections. These ideas might be further matured in time, but due to the threat of a global pandemic, we have chosen to move fast.

Physiology International

7

Global prevention Disinfection of air spaces, hard surfaces, and persons simultaneously with aqueous ClO2 solutions. What we are proposing here is basically the same idea what has been already proposed by Ogata et al. [21, 24, 25]: it is possible to create ClO2 atmospheres which can be safe for humans but at the same time harmful for microbes. There are differences, however, between their proposals and ours. Ogata's group regarded the ClO2 concentration (C) of the atmosphere as the sole important parameter of the treatment. They proposed to apply a ClO2 concentration below the 0.1 ppm (V/V) OSHA limit, for a time that is necessary to inactivate the microbes.

With such a method, however, the necessary contact time (T) can be very long. Here we suggest regarding the CT product as the parameter of disinfection. In this way it is possible to apply ClO2 concentrations above the OSHA limit but for a limited time only. The advantage of this method is that as higher C values are applied, the necessary contact times can be much shorter.

The idea will be illustrated by a numerical example below.

Another important difference is that Ogata's method focuses mostly on the role of the ClO2 gas, whereas we emphasize the importance of the simultaneous usage of ClO2 and H2O gases, as confirmed by the observations of Morino et al. [16]. For this purpose, we suggest a new way of creating a ClO2 atmosphere: to apply aqueous ClO2 solutions which can establish equilibrium ClO2 and H2O concentrations in the atmosphere, when these are sprayed into the air. Aqueous solutions are also easier to handle than to maintain stable and very low ClO2 levels in continuous gas streams.

It is advisable to apply high-purity ClO2 solutions for spraying to avoid any unwanted side effects to the persons or the surfaces treated. High-purity ClO2 solutions evaporate without any residue or trace.

An illustrative numerical example. Let us assume that we want to disinfect a closed space by spraying some aqueous stock ClO2 solution into it. The equilibrium ClO2 concentration cair in the gas and cw,e in the liquid phase can be calculated from the vapor-liquid equilibrium distribution measured by Ishi [11]: cair g¼ cw;e and from the component balance for ClO2: Vw $cw0 ¼ Vw $cw;e þ Vair $cair where Vair is the volume to be disinfected, Vw is the volume of the ClO2 stock solution, and cw0 is its ClO2 concentration.

With the help of the above two equations cw;e can be calculated as cw0 cw;e ¼ 1 þ g$VVairw Suppose we apply Vw 5 20 mL of cw0 5 40 ppm (m/m) aqueous stock ClO2 solution in a Vair 5 1 m3 closed space.

At 20 8C the value of the distribution coefficient is g 5 0.0316 (data of Ishi [11]) when all concentrations are given in the same units (e.g., in mg/L), and it is gppm 5 11.3 (see section ClO2

Physiology International

8

is a water soluble gas) when ppm (m/m) and ppm (V/V) are used in the aqueous and gaseous phases, respectively. Substituting our data the results are cw;e ¼ 0:025 ppmðm=mÞ; cair ¼ gppm cw;e ¼ 0:29 ppmðV=VÞ This result is just below the OSHA STEL (Short Term Exposition Limit) value which is 0.30 ppm (V/V) for 15 min. According to OSHA, STEL is the acceptable average exposure over a short period of time – usually 15 min – as long as the time-weighted average (TWA) is not exceeded. If a person is exposed to 0.30 ppm for 15 min, and right after that he or she stays in a ClO2-free atmosphere for 30 min, then the TWA value for this whole 45 min period is just the acceptable 0.10 ppm. All this means that an exposure of a person to a 0.30 ppm ClO2 atmosphere for 15 min on a single occasion, or even applying that treatment periodically with 30 min pauses within an 8 h period, should not cause any health problems.

Questions and remarks.

1. It is a major question, whether or not a 15-min stay in a 0.29 ppm (V/V) ClO2 atmosphere is enough to inactivate the viruses present? Regarding the wet atmosphere we can assume that the viruses are also wet, or even that they can be found in small water droplets containing 0.025 ppm (m/m) ClO2. We have no direct data for the inactivating time of viruses in such a solution, but we have an estimated 15 ± 5 s killing time value for an E. coli bacterium in a 0.25 ppm (m/m) ClO2 solution [2]. It is reasonable to assume that the killing time would be 10 times longer in a 10 times more dilute solution, i.e., 150 s 5 2.5 min. As viruses are probably inactivated faster than bacteria, and 15 min is six times longer than the estimated 2.5 min for E. coli, such a method can be successful, at least in theory.

2. To test such a method, we suggest constructing special disinfecting rooms with larger volume. Starting such experiments would be highly desirable, because this method could be an effective nonspecific defense against all types of viruses and could help to contain viral outbreaks.

Local prevention. Personal disinfection techniques against viral infections Disinfection of the mouth and the upper respiratory track with gargling. The current epidemic coronavirus is known to be present in the mouth and both in the upper and lower respiratory tract, but causes severe infections only in the lower respiratory tract, especially in the lung. The incubation period of the disease is several days, but the virus can often be detected in samples taken from the upper respiratory tract a few days before symptoms appear. As discussed in a previous chapter, chlorine dioxide will certainly inactivate the virus. With gargling, the upper respiratory tract is accessible except for the nasal cavity, but that is also accessible using e.g. nose drops or impregnated tampons. These parts can be disinfected by rinsing them regularly with high-purity chlorine dioxide solutions available commercially [31], thus the number of the viruses can be significantly reduced in the mouth and in the upper respiratory tract. We cannot be sure that such a treatment would be enough to prevent the development of the illness, as viruses living in other parts of the body can survive. However, inactivating part of the viruses with such a treatment surely helps the immune system to fight against the disease. In this respect, it is interesting to remark that

Physiology International

9

Japanese researchers have proven [26] that regular gargling with drinking water has reduced the incidence of upper respiratory tract infections to a statistically significant extent. The effect was explained by the fact that the drinking water used in the experiments contained 0.5 mg/L of chlorine, which was used to disinfect the water. We remark here, that in certain places chlorine dioxide is applied for the disinfection of drinking water instead of chlorine.

Disinfection of the lower respiratory track. The first problem is how ClO2 can be safely introduced into the lower respiratory tract. For this purpose any inhalation technique could be applied using aerosols of water droplets containing ClO2 [14].

The second and more important problem is how much ClO2 can be inhaled without damaging the lung? It would be helpful to know the dose of ClO2 that is not yet harmful for the lung. To our knowledge such direct data are not available in the literature, but can be calculated from other data. The starting point for such a calculation is the OSHA STEL value [30], according to which 0.30 ppm ClO2 in the workplace atmosphere is tolerable for a 15 min period without any damage.

The volume of air inhaled by a worker during 15 min is 15 times the so-called “minute volume ventilation” [32]. According to Table 3 of ref. [32], during light activities e.g., when sitting in a car the minute volume is around 12 L, thus the total inhaled air is about 180 L. In the case of 0.30 ppm concentration the total inhaled amount of ClO2 is 54 mL, which is (at 20 8C) 2.25 mmol ≈ 0.15 mg ClO2. Assuming a more vigorous activity it can be two times more, 0.30 mg.

This rough calculation indicates that approximately this is the amount of ClO2, which can be tolerated by the lung. The OSHA limit probably applied high safety factors, thus the real limit should be higher.

We suggest that animal experiments should be performed to obtain experimental values for the pulmonary toxicity of ClO2. Furthermore, it would be important to check in additional animal experiments, whether ClO2 applied in a nontoxic amount is able to treat infections of the lung caused by bacteria or viruses.

CONCLUSION In this editorial, we summarized the unique properties of chlorine dioxide, which make it an ideal and nonspecific antimicrobial agent at concentrations harmless to humans, and we reviewed previous research on preventing viral infections with gaseous ClO2. Based on this background, we suggested some novel hypothetical methods using chlorine dioxide to disinfect rooms, prevent human infection, and slow down viral spread. These are nonspecific methods, which could be used against any newfound virus as a first line of protection until effective specific countermeasures are developed.

Conflict of interest: Zolt an Noszticzius, Maria Wittmann and Krist of K aly-Kullai are co-inventors of the European patent 2069232 “Permeation method and apparatus for preparing fluids containing high purity chlorine dioxide”. Zolt an Noszticzius is a founder and owner of the Solumium Ltd (a company producing chlorine-dioxide), while Krist of K aly-Kullai is its payed employee.

10

Physiology International

REFERENCES 1. Bastacky J, Lee CY, Goerke J, Koushafar H, Yager D, Kenaga L, et al. Alveolar lining layer is thin and continuous: low-temperature scanning electron microscopy of rat lung. J Appl Physiol 1995; 79(5): 1615–28, https://doi.org/10.1152/jappl.1995.79.5.1615.

2. Benarde MA, Snow WB, Oliveri VP, Davidson B. Kinetics and mechanism of bacterial disinfection by chlorine dioxide. Appl Microbiol 1967; 15(2): 257–65.

3. Cantin AM, North SL, Hubbard RC, Crystal RG. Normal alveolar epithelial lining fluid contains high levels of glutathione. J Appl Physiol 1987; 63(1): 152–7, https://doi.org/10.1152/jappl.1987.63.1.152.

4. Cao Z, Zhang Q, Lu X, Pfeiffer D, Jia Z, Song H, et al. Estimating the effective reproduction number of the 2019-nCoV in China. Available online at http.medxriv.org; Jan. 29; 2020; https://doi.org/10.1101/2020.01.27.

20018952.

5. Cross CE, van der Vliet A, O’Neill CA, Louie S, Halliwell B. Oxidants, antioxidants, and respiratory tract lining fluids. Environ Health Perspect 1994; 102(Suppl. 10): 185–91, https://doi.org/10.1289/ehp.94102s10185.

6. EPA Guidance Manual for Compliance with the Filtration and Disinfection Requirements for Public Water Sources (AWWA; 1991). Table C-9: CT values for inactivation of viruses by chlorine dioxide. Available at http://www.opssys.com/InstantKB/article.aspx?id514495.

7. Forman HJ, Zhang H, Rinna A. Glutathione: overview of its protective roles, measurement, and biosynthesis.

Mol Aspects Med 2009; 30(1–2): 1–12, https://doi.org/10.1016/j.mam.2008.08.006.

8. Hauchman FS, Noss CI, Olivieri VP. Chlorine dioxide reactivity with nucleic acids. Water Res 1986; 20(3): 357–61, https://doi.org/10.1016/0043-1354(86)90083-7.

9. https://www.britannica.com/science/coronavirus-virus-group.

10. Hui DS, Azhar EI, Madani TA, Ntoumi F, Kock R, Dar O, et al. The continuing 2019-nCoV epidemic threat of novel coronaviruses to global health – The latest 2019 novel coronavirus outbreak in Wuhan, China. Int J Infect Dis 2020; 91: 264–6, https://doi.org/10.1016/j.ijid.2020.01.009.

11. Ishi G. Solubility of chlorine dioxide. Chem Eng Japan 1958; 22(3): 153–4, https://doi.org/10.1252/ kakoronbunshu1953.22.153.

12. Ison A, Odeh IN, Margerum DW. Kinetics and mechanisms of chlorine dioxide and chlorite oxidations of cysteine and glutathione. Inorg Chem 2006; 45: 8768–75, https://doi.org/10.1021/ic0609554.

13. Knudsen L, Ochs M. The micromechanics of lung alveoli: structure and function of surfactant and tissue components. Review Histochem Cell Biol 2018; 150: 661–76, https://doi.org/10.1007/s00418-018-1747-9.

14. Koren E. A suggestion. Personal information; 2020.

15. Lubbers JR, Chauan SR, Bianchine JR. Controlled clinical evaluations of chlorine dioxide, chlorite and chlorate in man. Environ Health Perspect 1982; 46: 57–62, https://doi.org/10.1289/ehp.824657.

16. Morino H, Fukuda T, Miura T, Lee C, Shibata T, Sanekata T. Inactivation of feline calicivirus, a Norovirus surrogate, by chlorine dioxide gas. Biocontrol Sci 2009; 14: 147–53, https://doi.org/10.4265/bio.14.147.

17. Napolitano MJ, Green BJ, Nicoson JS, Margerum DW. Chlorine dioxide oxidations of tyrosine, N-acetyltyrosine, and Dopa. Chem Res Toxicol 2005; 18: 501–8, https://doi.org/10.1021/tx049697i.

18. Napolitano MJ, Stewart DJ, Margerum DW. Chlorine dioxide oxidation of guanosine 50 -monophosphate.

Chem Res Toxicol 2006; 19: 1451–8, https://doi.org/10.1021/tx060124a.

19. Noss CI, Hauchman FS, Olivieri VP. Chlorine dioxide reactivity with proteins. Water Res. 1986; 20(3): 351–6, https://doi.org/10.1016/0043-1354(86)90083-7.

20. Noszticzius Z, Wittmann M, K aly-Kullai K, Beregv ari Z, Kiss I, Rosivall L, et al. Chlorine dioxide is a sizeselective antimicrobial agent. PloS One 2013; 8(11): e79157, https://doi.org/10.1371/journal.pone.0079157.

Physiology International

11

21. Ogata N. Chlorine dioxide gas for use in treating respiratory virus infection. European Patent Specification EP 1955719 B1. Priority to JP 2005342503; 2005.

22. Ogata N. Denaturation of protein by chlorine dioxide: oxidative modification of tryptophane and tyrosine residues. Biochemistry 2007; 46: 4898–911, https://doi.org/10.1021/bi061827u.

23. Ogata N. Inactivation of influenza virus haemagglutinin by chlorine dioxide: oxidation of the conserved tryptophan 153 residue in the receptor-binding site. J Gen Virol 2012; 93: 2558–63, https://doi.org/10.1099/ vir.0.044263-0.

24. Ogata N, Shibata T. Effect of chlorine dioxide gas of extremely low concentration on absenteeism of schoolchildren. Int J Med Med Sci 2009; 1(7): 288–9.

25. Ogata N, Shibata T. Protective effect of low-concentration chlorine dioxide gas against influenza A virus infection. J Gen Virol 2008; 89: 60–7, https://doi.org/10.1099/vir.0.83393-0.

26. Satomura K, Kitamura T, Kawamura T, Shimbo T, Watanabe M, Kamei M, et al. Great cold investigators: prevention of upper respiratory tract infections by gargling. A randomized trial. Am J PrevMed 2005; 29: 302–7, https://doi.org/10.1016/j.amepre.2005.06.013.

27. Stewart DJ, Napolitano MJ, Bakhmutova-Albert EV, Margerum DW. Kinetics and mechanisms of chlorine dioxide oxidation of tryptophan. Inorg Chem 2008; 47: 1639–47, https://doi.org/10.1021/ic701761p.

28. Tan H, Wheeler BW, Wei C. Reaction of chlorine dioxide with amino acids and peptides: Kinetics and mutagenicity studies. Mutat Res 1987; 188(4): 259–66, https://doi.org/10.1016/0165-1218(87)90002-4.

29. Tao Y, Queen K, Paden CR, Zhang J, Li Y, Uehara A, et al. Severe acute respiratory syndrome coronavirus 2 isolate 2019-nCoV/USA-IL1/2020, complete genome. NCBI GenBank; 2020. Available at https://www.ncbi.nlm. nih.gov/nucleotide/MN988713.1?report5genbank&log$5nuclalign&blast_rank51&RID5304U21XH016.

30. US Occupational Safety and Health Administration. Determination of chlorine dioxide in workplace atmospheres; 1991. Available at https://www.osha.gov/dts/sltc/methods/inorganic/id202/id202.html.

31. www.solumium.com.

32. Zuurbier M, Hoek G, van den Hazel P, Brunekreef B. Minute ventilation of cyclists, car and bus passengers: an experimental study. Environ Health 2009; 8: 48, https://doi.org/10.1186/1476-069X-8-48.

Open Access statement. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/),which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the CC License is provided, and changes – if any – are indicated. (SID_1)

Solutions For All Your Gaseous Chlorine Dioxide Needs

The Chlorine Dioxide People

Blakeslea trispora

28

E. coli O157:H7 G5303

1

Bordetella bronchiseptica

8

E. coli O157:H7 C7927

1

Brucella suis

30

Erwinia carotovora (soft rot)

21

Burkholderia mallei

36

Franscicella tularensis

30

Burkholderia pseudomallei

36

Fusarium sambucinum (dry rot)

21

Campylobacter jejuni

39

Fusarium solani var. coeruleum (dry rot)

21

Clostridium botulinum

32

Helicobacter pylori

8

Corynebacterium bovis

8

Helminthosporium solani (silver scurf)

21

Coxiella burneti (Q-fever)

35

Klebsiella pneumonia

3

E. coli ATCC 11229

3

Lactobacillus acidophilus NRRL B1910

1

E. coli ATCC 51739

1

Lactobacillus brevis

1

E. coli K12

1

Lactobacillus buchneri

1

E. coli O157:H7 13B88

1

Lactobacillus plantarum

5

E. coli O157:H7 204P

1

Legionella

38

E. coli O157:H7 ATCC 43895

1

Legionella pneumophila

42

E. coli O157:H7 EDL933

13

Leuconostoc citreum TPB85

1

P.O. Box 549

Lebanon, NJ 08833-0549

Ph: 908-236-4100

www.clordisys.com

Page 1 of 7

Leuconostoc mesenteroides

5

Listeria innocua ATCC 33090

1

Adenovirus Type 40

6

Listeria monocytogenes F4248

1

Calicivirus

42

Listeria monocytogenes F5069

19

Canine Parvovirus

8

Listeria monocytogenes LCDC-81-861

1

Coronavirus

3

Listeria monocytogenes LCDC-81-886

19

Feline Calici Virus

3

Listeria monocytogenes Scott A Methicillin-resistant Staphylococcus aureus (MRSA) Multiple Drug Resistant Salmonella typhimurium (MDRS) Mycobacterium bovis

1

Foot and Mouth disease

8

Hantavirus

8

Hepatitis A Virus

3

3

Hepatitis B Virus

8

8

Hepatitis C Virus

8

Mycobacterium fortuitum

42

Human coronavirus

8

Pediococcus acidilactici PH3

1

Human Immunodeficiency Virus

3

Pseudomonas aeruginosa

3

Human Rotavirus type 2 (HRV)

15

Pseudomonas aeruginosa

8

Influenza A

22

Salmonella

1

Minute Virus of Mouse (Parovirus)(MVM-i)

8

Salmonella spp.

2

Minute Virus of Mouse (Parovirus)(MVM-p)

8

Salmonella Agona

1

Mouse Hepatitis Virus (MHV-A59)

8

Salmonella Anatum Group E

1

Mouse Hepatitis Virus (MHV-JHM)

8

Salmonella Choleraesins ATCC 13076

1

Mouse Parvovirus type 1 (MPV-1)

8

Salmonella choleraesuis

8

Murine Parainfluenza Virus Type 1 (Sendai)

8

Salmonella Enterica (PT30) BAA-1045

1

Newcastle Disease Virus

8

Salmonella Enterica S. Enteritidis

13

Norwalk Virus

8

Salmonella Enterica S. Javiana

13

Poliovirus

20

Salmonella Enterica S. Montevideo

13

3

Salmonella Enteritidis E190-88

1

Salmonella Javiana

1

Salmonella newport

4

Salmonella Typhimurium C133117

1

Salmonella Anatum Group E

1

Shigella

38

Rotavirus Severe Acute Respiratory Syndrome (SARS) Coronavirus Sialodscryoadenitis Virus (Coronavirus)(SDAV) Simian rotavirus SA-11 Theiler’s Mouse Encephalomyelitis Virus (TMEV) Vaccinia Virus

Staphylococcus aureus

23

Staphylococcus aureus ATCC 25923

1

Staphylococcus faecalis ATCC 344

1

Alternaria alternata

26

Tuberculosis Vancomycin-resistant Enterococcus faecalis (VRE) Vibrio strain Da-2

3

Aspergillus aeneus

28

3

Aspergillus aurolatus

28

37

Aspergillus brunneo-uniseriatus

28

Vibrio strain Sr-3

37

Aspergillus caespitosus

28

Yersinia enterocolitica

40

Aspergillus cervinus

28

Yersinia pestis

30

Aspergillus clavatonanicus

28

Yersinia ruckerii ATCC 29473

31

Aspergillus clavatus

28

P.O. Box 549

Lebanon, NJ 08833-0549

3

Ph: 908-236-4100

www.clordisys.com

43 8 15 8 10

Page 2 of 7

Aspergillus egyptiacus

28

Phormidium boneri

3

Aspergillus elongatus

28

Pichia pastoris

3

Aspergillus fischeri

28

Poitrasia circinans

28

Aspergillus fumigatus

28

Rhizopus oryzae

28

Aspergillus giganteus

28

Roridin A

33

Aspergillus longivesica

28

Saccharomyces cerevisiae

3

Aspergillus niger

12

Stachybotrys chartarum

7

Aspergillus ochraceus

28

T-mentag (athlete’s foot fungus)

3

Aspergillus parvathecius

28

Verrucarin A

33

Aspergillus sydowii

28

Aspergillus unguis

28

Aspergillus ustus

28

Alicyclobacillus acidoterrestris

17

Aspergillus versicolor

28

Bacillus coagulans

12

Botrytis species

3

Bacillus anthracis

10

Candida spp.

5

Bacillus anthracis Ames

30

Candida albicans

28

Bacillus atrophaeus

14

Candida dubliniensis

28

Bacillus atrophaeus ATCC 49337

31

Candida maltosa

28

Bacillus megaterium

12

Candida parapsilosis

28

Bacillus polymyxa

12

Candida sake

28

Bacillus pumilus ATCC 27142

12

Candida sojae

28

Bacillus pumilus ATCC 27147

11

Candida spp.

5

Bacillus subtillis (globigii) ATCC 9372

11

Candida tropicalis

28

Bacillus subtillis ATCC 19659

31

Candida viswanathil

28

Bacillus subtillis 5230

12

7

Clostridium. sporogenes ATCC 19404

12

Cladosporium cladosporioides

7

Geobacillus stearothermophilus ATCC 12980

11

Debaryomyces etchellsii

28

Geobacillus stearothermophilus ATCC 7953

31

Eurotium spp.

5

Geobacillus stearothermophilus VHP

11

Fusarium solani

3

Bacillus thuringiensis

18

Lodderomyces elongisporus

28

Mucor circinelloides

28

Mucor flavus

28

Mucor indicus

28

Mucor mucedo

28

Mucor rademosus

28

Mucor ramosissimus

28

Mucor saturnus

28

Penicillium chrysogenum

7

Penicillium digitatum

3

Penicillium herquei

28

Penicillium spp.

5

Chaetomium globosum

P.O. Box 549

Lebanon, NJ 08833-0549

Mustard Gas Ricin Toxin

10

dihydronicotinamide adenine dinucleotide

24

microcystin-LR (MC-LR)

25

cylindrospermopsin (CYN)

25

Amoxicillin

29

Amplicillin

29

Cefadroxil

29

Cefazolin

29

Ph: 908-236-4100

www.clordisys.com

Page 3 of 7

Cephalexin

29

Cryptosporidium parvum Oocysts

9

Imipenem

29

Cyclospora cayetanensis oocysts

41

Penicillin G

29

Giardia

34

Penicillin V

29 Encephalitozoon intestinalis

27

Chironomid larvae

27

Cryptosporidium

34

P.O. Box 549

Lebanon, NJ 08833-0549

Ph: 908-236-4100

www.clordisys.com

Page 4 of 7

Solutions For All Your Gaseous Chlorine Dioxide Needs

The Chlorine Dioxide People

J PREV MED HYG 2007; 48: 91-95

ORIGINAL

ARTICLE

Investigation on virucidal activity of chlorine dioxide.

Experimental data on Feline calicivirus, HAV and Coxsackie B5 R. ZONI, R. ZANELLI, E. RIBOLDI, L. BIGLIARDI, G. SANSEBASTIANO Department of Public Health, Sec. Hygiene, University of Parma, Italy

Key words Chlorine dioxide • Virucidal activity • Disinfection treatment

Summary Introduction. The aim of this study was to evaluate the efficacy of ClO2 with regard to viruses which show a particular resistance to oxidizing agent such as HAV and Norwalk and Norwalk-like viruses, and which play an important role in the epidemiology of viral foodborne diseases.

In the food industry, disinfection of processing systems and equipment is a very important instrument to prevent secondary contamination and to guarantee food safety. Among disinfectants, chlorine dioxide (ClO2) presents a good efficacy at wide range of pH values, its action is rapid and generates few reaction byproducts if compared to hypoclorite. Experimental studies have highlighted that ClO2 shows a good bactericidal activity and it is also active towards viruses. Furthermore, the low concentrations and low contact times required to obtain microbial load reduction are favourable elements for the application of this compound in the industrial sanitizing practices.

Methods. As it is impossible to cultivate the Norwalk virus in vitro, we tested the resistance of Feline calicivirus (F9 strain) vs. ClO2, in comparison with HAV (strain HM-175) and CoxsackieB5. Chlorine dioxide was used at concentrations ranging from 0.2 to 0.8 mg/l in water solution, at pH 7 and at +20 °C. Viral suspensions were added

to disinfecting solution and, at pre-set times, were sampled to undergo to titration after blocking the disinfectant action with thiosulphate 0.05 M. On the basis of the data obtained, for each virus and in relation to different concentrations, mean reduction times were calculated for 99%, 99.9% and 99.99% using the regression analysis model.

Results. As regards Feline calicivirus, at a concentration of 0.8 mg/l of ClO2, we obtained the complete elimination of the viral titre in 2 min while 30 min were required at concentrations of 0.2 mg/l. Coxackie B5 showed a similar behaviour, being completely inactivated in 4 min with 0.4 mg/l of ClO2 and after 30 min at a concentration of 0.2 mg/l. Inactivation was quicker for HAV, which was eliminated after only 30 sec at a concentration of 0.8 mg/l and after 5 min at 0.4 mg/l.

Conclusion. Our data show that for complete inactivation of HAV and Feline calicivirus, concentrations ≥ 0.6 mg/l are required.

This observation is true for Coxsackie B5 too, but this virus has shown a good sensitivity at all concentration tested according to regression analysis results. For Feline calicivirus and HAV, at low concentrations of disinfectant, prolonged contact times were needed to obtain a 99.99% reduction of viral titres (about 16 and 20 minutes respectively).

Introduction

Hypochlorite and chlorine dioxide (ClO2) are highly oxidizing and are thus commonly used as disinfectants, especially when disinfecting water.

Studies carried out on potential genotoxic effects of chlorination have shown that the use of sodium hypochlorite leads to the formation of a larger number of mutagen substances than the use of chlorine dioxide [2].

As compared to other oxidizing disinfectants, chlorine dioxide offers certain advantages, such as its efficacy in a wide range of pH values and its rapid action.

If compared to hypochlorite, for instance, it has a higher selectivity and generates a smaller number of reaction products; furthermore, the bactericidal efficacy of ClO2 is greater than that of HClO since its greater oxidizing capacity has been demonstrated.

Inactivation assays carried out under controlled experimental conditions have shown a different sensitivity towards ClO2 on the part of various enteroviruses: at a concentration of 0.32 mg/l in conditions of neutral pH and at a temperature of 15°C, the inactivation time for

Although viruses, unlike bacteria, are incapable for replicating in food, they represent an important cause of foodborne infections.

Viruses with oral-faecal transmission play an important role in the etiological factors involved, and most cases of gastroenteritis are attributed to the Norwalk virus or to the Norwalk-like virus, viruses belonging to the family of caliciviruses, and to the hepatitis A virus.

Food is exposed to risks of primary contamination when the raw materials themselves are contaminated, or secondary contamination when they come into contact with surfaces/equipment which are contaminated [1].

Thus, sanitizing the processing systems and the equipment which comes into contact with the foodstuffs is extremely important in the food industry in order to reduce the risk of secondary contamination, and represents a fundamental instrument to reduce the risk of infection linked to the consumption of foodstuffs.

91

R. ZONI ET AL.

Tab. I. Reduction of bacterial load.

Microorganism Staphylococcus aureus Escherichia coli Escherichia coli Streptococcus Lactobacillus brevis Lactobacillus brevis Pseudomonas aeuruginosa

Concentration ClO2 mg/l

Contact Times

1.00 0.15 0.25 1.00 0.15 1.00 1.00

99.99% was 5 min for Echo 7, 3 min for Coxsakie B3 and 7 min for the polio1 virus [3, 4].

The low concentrations generally required, low contact times and reduced microbial load are favourable elements promoting the use of ClO2 in the food industry.

Table I illustrates the disinfecting activity of chlorine dioxide [5].

The aim of this study was to assess under experimental conditions the efficacy of chlorine dioxide when used with certain viruses which have proven to be particularly resistant to oxidizing agents, and which has not been throughly investigated in the literature.

Materials and methods The following were used for the inactivation assays: the feline Calicivirus F9 strain grown on CRFK (feline kidney) cultures, the Coxsakie B5 virus grown on RC37 (monkey kidney), and the Hepatitis A virus strain HM-175 grown on FRhK4 (monkey kidney embryonic) cultures.

Infected cell cultures were incubated at 37°C until the onset of the cytopathic effect, and subsequently frozen at -80°C to induce cell lysis; the contents of the flask were then submitted to ultra-filtration and re-suspended with sterile physiological solution, and finally aliquoted in sterile test tubes and stored in deepfreeze at -20°C. The titre of the viral suspension was determined for the hepatitis A virus using the plate method and expressed as PFU, while for the other viruses it was calculated as TCID50 (titre of the dose infecting 50% of the cell culture) according to the Reed-Muench method; we integrated the titres of 3 or 4 test for each virus and different concentrations.

Chlorine dioxide was used as disinfectant at the following concentrations: 0.2, 0.4, 0.6, 0.8 mg/l.

Viral inactivation assays were carried out at a constant temperature of 20°C with neutral pH kept constant by using a buffer solution at pH 7.

PERFORMING THE ASSAYS 1 ml of viral suspension was added to the disinfecting solution, then aliquots of samples were collected at pre-set times – T0,T0.5, T1, T2, T3, T4, T5, T15, T30, T45, T60, – and immediately placed in contact with 0.5 ml

60 300 60 15 5 5 60

sec sec sec sec min min sec

% inactivation

> > > >

99.999 99.900 99.999 99.999 99.900 99.999 99.999

thiosulphate 0.05 M in order to neutralize the disinfectant activity.

For hepatitis A virus, we proceeded by setting up scalar 10-folds dilutions for each contact time and by inoculating them in cell cultures previously grown in 24-wellsplates (0.1 ml/well); the plates were then incubated for 2 hours at 37°C (5% CO2). As soon as the contact time has expired, the inoculum was sucked up and a semi-solid medium was added (4% foetal calf serum, 43% MEM 2X, 43% carboxymethylcellulose). After a twelve-days incubation, the cell cultures were fixed with formalin (two hours-contact time), then washed with demineralised water and stained with Giemsa; the following day we washed away the exeeding stain and the cell lysis plaques were counted.

As regards the other viruses assayed, the 10-folds dilutions prepared for each contact time aliquot were placed in 96-wells plates (25 μl/well) previously prepared with 25 μl/well of medium and 50 μl of cell suspension has been added to each well. Finally, after an incubation at 37°C for 4 days, we detected the presence of the cytopathic effect induced by the virus.

Results FELINE CALICIVIRUS In order to assess the trend of the inactivation assays with Feline calicivirus, the logarithms of the titres were calculated and the trends at various concentrations at various times are shown in Figure 1.

At a concentration of 0.8 mg/l the viral titre was eliminated after two minutes, and after thirty minutes at a concentration of 0.2 mg/l.

Mean reduction times were then calculated for 99%, 99.9% and 99.99% inactivation through analysis of regression, giving the results shown in Table II. Table II shows that at a concentration of 0.8 mg/l a 99.99% reduction was obtained after 2.1 minutes, while 15.5 minutes were required for concentrations of 0.2 mg/l.

COXSACKIE B5 VIRUS In this case, logarithms of the titres, as TCID50, were calculated too, and results are shown below in Figure 2. It is possible to note that at a concentration of 0.6

92

INVESTIGATION ON VIRUCIDAL ACTIVITY OF CHLORINE DIOXIDE

Fig. 1. Trend of viral titre (log TCID50) at different ClO2 concentration and contact times Feline calicivirus.

Tab. II. Mean reduction times for various inactivation percentages.

Tab. III. Mean reduction times at various inactivation times.

ClO2 – Feline calicivirus – neutral pH [ClO2] (mg/l)

ClO2 – Coxsackie Virus B5 – neutral pH

Mean time (min) to obtain reduction of: 99% 99.9% 99.99% (CI 95%) (CI 95%) (CI 95%)

[ClO2] (mg/l)

Mean time (min) to obtain reduction of: 99% 99.9% 99.99% (CI 95%) (CI 95%) (CI 95%)

0.80

0.50 (0.16-0.74)

1.10 (1.05-1.26)

2.10 (2.02-2.22)

0.60

0.12 (0.08-0.17)

0.34 (0.27-0.45)

1.00 (0.87-1.15)

0.40

1.24 (0.98-1.52)

3.48 (3.10-3.73)

9.59 (9.16-9.81)

0.40

0.57 (0.41-0.79)

1.18 (0.94-1.47)

2.41 (2.20-2.73)

0.20

2.20 (1.80-2.09)

6.20 (5.90-6.90)

15.50 (15.20-15.90)

0.20

0.63 (0.45-0.89)

1.52 (1.18-1.95)

3.73 (3.18-4.38)

mg/l the titre was eliminated after 4 minutes, while at a concentration of 0.2 the titre was eliminated after 30 minutes.

By calculating the regression, it was possible to record the mean reduction times at 99%, 99.9% and 99.99%.

Results are shown in Table III. Results show that at concentrations of 0.6 mg/l a 99.99% reduction of the viral titre was obtained after 1 minute, while at a concentration of 0.2 mg/l it was obtained after 3.73 minutes.

At a concentration of 0.8 mg/l the titre was completely eliminated after only 30 seconds, while at concentrations of 0.4 complete elimination was obtained after 5 minutes.

Finally, mean reduction times for elimination at various percentages were calculated here, too, using the regression analysis as shown in Table IV.

HEPATITIS A VIRUS As regards the Hepatitis A virus, the viral titre was calculated according to the PFU method. Figure 3 shows the trends of viral titres at various concentrations

Chlorine dioxide is an excellent bactericidal agent, as has been reported in various studies, although its virucidal activity has not been thoroughly investigated as yet [6-12].

93

Considerations and Conclusions

R. ZONI ET AL.

Fig. 2. Trend of viral titre (log TCID50) at different ClO2 concentration and contact times Coxsackie B5 virus.

Fig. 3. Trend of viral titre (log PFU) at different ClO2 concentration and contact times Hepatitis A virus.

The inactivation kinetics of Poliovirus 1 under experimental conditions have shown that it has a good oxidizing effect [13].

Our results have shown that it is only with concentrations greater than 0.6 mg/l that inactivation is obtained quickly for HAV and for Feline calicivirus, and only Coxsackie B5 shows great sensitivity at all concentrations assayed.

If these data are compared to the inactivation kinetics we performed on Poliovirus 1, the greater resistance

of Calicivirus to Chlorine dioxide is confirmed [14].

This result is particularly important in that it confirms that the Norwalk virus, which the Feline calicivirus is a surrogate of, presents considerable resistance to hypochlorite and is also characterized by strong resistance to Chlorine dioxide. This needs to be taken into account in sanitizing procedures applied to processing systems as well as in direct disinfection treatments of foodstuffs which may be involved as carriers of this virus.

94

INVESTIGATION ON VIRUCIDAL ACTIVITY OF CHLORINE DIOXIDE

Tab. IV. Mean reduction times at various inactivation percentages.

ClO2 – Hepatitis A virus – neutral pH [ClO2] (mg/l)

Mean time (min) to obtain reduction of: 99% (CI 95%)

99.9% (CI 95%)

99.99% (CI 95%)

0.80

0.26 *

0.35 *

0.43 *

0.60

0.53 (0.41-0.58)

0.85 (0.76-0.94)

1.45 (1.38-1.53)

0.40

2.35 (1.63-3.40)

6.79 (5.52-8.16)

19.58 (18.70-20.50)

* It was impossible to calculate confidence limits values because of the very short viral inactivation time.

References [1] [2]

[3] [4]

[5]

[6] [7]

Koopmans M, Duizer E. Food viruses: an emerging problem.

Intern J Food Microbiol 2004;90:23-41.

Guzzella L, Monarca S, Zani C, Feretti D, Zerbini I, Buschini A, et al. In vitro potential genotoxic effect of surface drinking water treated with chlorine and alternative disinfectants.

Mutation research 2004;564:179-93.

Sansebastiano G, Cesari C, Bellelli E. Further investigation on water disinfection by chlorine dioxide. Igiene Moderna 1986;85:358-80.

Sansebastiano G. Epidemiological review on chlorine dioxide.

In: Chlorine Dioxide and Disinfection. Vol. 17. Milano: C.I.P.A. Publisher 1997, p. 63-71.

Ruzic C. Chlorine dioxide-based water treatment in the food industry. In: Chlorine Dioxide and Disinfection. Vol. 17.

Milano: C.I.P.A. Publisher 1997, p. 197-209.

Aieta EM, Berg JB. A review of chlorine dioxide in drinking water treatment. JAWWA 1986;78:62-72.

Alvarez ME. O’Brien RT. Mechanisms of inactivation of poliovirus by chlorine dioxide and iodine. Appl Environ Microbiol 1982;44:1064-71.

■ Received on June 6, 2006. Accepted on March 28, 2007.

■ Corrispondence: Prof G. Sansebastiano, Department of Public Health, Sec. Hygiene, University of Parma, Italy, via Volturno 39, 43100 Parma, Italy

95

[8]

Bernarde MA, Israel BM, Olivieri VP, Granstrom ML. Efficiency of chlorine dioxide as a bactericide. Appl Microbiol 1965;13:776-80.

[9]

Chen YS, Vaughn JM. Inactivation of human and simian rotaviruses by chlorine dioxide. Appl Environ Microbiol 1990;56:1363-6.

[10] Latshaw CL. Chlorine dioxide: effective broad-spectrum biocide for white-water systems. Tappi Journal 1994;78:163-6.

[11] Roller SD. Some aspects of the mode of action of chlorine dioxide on Bacteria. Baltimore: M.S. Thesis, Johns Hopkins University 1978.

[12] Scarpino PV. Virucidal effectiveness of disinfection processeschlorine dioxide. In: Proceedings of the American Water Works Association, Annual Conference. 1979, p. 1-35.

[13] Traenhart O, Kuwert E. Comparative studies on the action of chlorine and ozone on polioviruses in the reprocessing of drinking water in Essen. Zbl Bakt I Abt Orig 1975;160:305.

[14] Sansebastiano G, Mori G, Tanzi ML, Cesari C, Bellelli E. Chlorine dioxide: methods of analysis and kinetics of inactivation of poliovirus type 1. Igiene Moderna 1983;79:61-91.

Ungewöhnliche Merkmale des SARS-CoV-2-Genoms, die auf eine ausgeklügelte Labormodifikation anstelle einer natürlichen Evolution und Abgrenzung seines wahrscheinlichen Synthesewegs hindeuten.

Li-Meng Yan (MD, PhD)1, Shu Kang (PhD)1, Jie Guan (PhD)1, Shanchang Hu (PhD)1 1 Rule of Law Society & Rule of Law Foundation, New York, NY, USA. Korrespondenz: team.lmyan@gmail.com

Zusammenfassung Die durch das neue Coronavirus SARS-CoV-19 verursachte COVID-2-Pandemie hat mehr als 910.000 Todesfälle verursacht weltweit und ein beispielloser Rückgang der Weltwirtschaft. Trotz seiner enormen Auswirkungen ist der Ursprung von SARS-CoV-2 rätselhaft und kontrovers geblieben. Die Theorie des natürlichen Ursprungs, obwohl weithin akzeptiert, fehlt wesentliche Unterstützung. Die alternative Theorie, dass das Virus von einem Forschungslabor stammen könnte, wird jedoch in von Expertengutachteten in wissenschaftlichen Fachzeitschriften streng zensiert. Jedoch zeigt SARS-CoV2 biologische Eigenschaften, die mit einem natürlichen Phänomen, dem Zoonose, unvereinbar sind. In diesem Bericht beschreiben wir genomische, strukturelle, medizinische und literarische Beweise, die wann zusammengenommen, der Theorie des natürlichen Ursprungs stark widersprechen. Es gibt Hinweise darauf, dass SARS-CoV-2 ein Laborprodukt sein muss, das mit dem Fledermaus-Coronavirus ZC45 und / oder ZXC21 als Vorlage erstellt wurde und / oder als Grundlage. Basierend auf den Beweisen postulieren wir weiterhin einen Syntheseweg für SARS-CoV-2. Dies zeigt, dass die Laborerstellung dieses Coronavirus bequem ist und in ungefähr sechs Monaten erreicht werden kann.

Unsere Arbeit betont die Notwendigkeit einer unabhängigen Untersuchung der einschlägigen Forschungslabors. Es befürwortet auch eine kritische Prüfung bestimmter kürzlich veröffentlichter Daten, die obwohl problematisch, verwendet wurden, um einen natürlichen Ursprung von SARS-CoV2 zu unterstützen und zu behaupten. Wegen der öffentlichen Gesundheitsperspektive sind diese Maßnahmen notwendig, um die Herkunft von SARS-CoV-2 zu kennen und wie das Virus die menschlichen Bevölkerung erreicht hat und ist von grundlegender Bedeutung für die Kontrolle der COVID-19 Pandemie sowie bei der Prävention ähnlicher Pandemien in der Zukunft.

Weiter im englischen Original…

Unusual Features of the SARS-CoV-2 Genome Suggesting Sophisticated Laboratory Modification Rather Than Natural Evolution and Delineation of Its Probable Synthetic Route Li-Meng Yan (MD, PhD)1, Shu Kang (PhD)1, Jie Guan (PhD)1, Shanchang Hu (PhD)1

1

Rule of Law Society & Rule of Law Foundation, New York, NY, USA.

Correspondence: team.lmyan@gmail.com

Abstract The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to over 910,000 deaths worldwide and unprecedented decimation of the global economy. Despite its tremendous impact, the origin of SARS-CoV-2 has remained mysterious and controversial. The natural origin theory, although widely accepted, lacks substantial support. The alternative theory that the virus may have come from a research laboratory is, however, strictly censored on peer-reviewed scientific journals. Nonetheless, SARS-CoV-2 shows biological characteristics that are inconsistent with a naturally occurring, zoonotic virus. In this report, we describe the genomic, structural, medical, and literature evidence, which, when considered together, strongly contradicts the natural origin theory. The evidence shows that SARS-CoV2 should be a laboratory product created by using bat coronaviruses ZC45 and/or ZXC21 as a template and/or backbone. Building upon the evidence, we further postulate a synthetic route for SARS-CoV-2, demonstrating that the laboratory-creation of this coronavirus is convenient and can be accomplished in approximately six months. Our work emphasizes the need for an independent investigation into the relevant research laboratories. It also argues for a critical look into certain recently published data, which, albeit problematic, was used to support and claim a natural origin of SARS-CoV-2. From a public health perspective, these actions are necessary as knowledge of the origin of SARS-CoV-2 and of how the virus entered the human population are of pivotal importance in the fundamental control of the COVID-19 pandemic as well as in preventing similar, future pandemics.

1

Introduction COVID-19 has caused a world-wide pandemic, the scale and severity of which are unprecedented.

Despite the tremendous efforts taken by the global community, management and control of this pandemic remains difficult and challenging.

As a coronavirus, SARS-CoV-2 differs significantly from other respiratory and/or zoonotic viruses: it attacks multiple organs; it is capable of undergoing a long period of asymptomatic infection; it is highly transmissible and significantly lethal in high-risk populations; it is well-adapted to humans since the very start of its emergence1; it is highly efficient in binding the human ACE2 receptor (hACE2), the affinity of which is greater than that associated with the ACE2 of any other potential host2,3.

The origin of SARS-CoV-2 is still the subject of much debate. A widely cited Nature Medicine publication has claimed that SARS-CoV-2 most likely came from nature4. However, the article and its central conclusion are now being challenged by scientists from all over the world5-15. In addition, authors of this Nature Medicine article show signs of conflict of interests16,17, raising further concerns on the credibility of this publication.

The existing scientific publications supporting a natural origin theory rely heavily on a single piece of evidence – a previously discovered bat coronavirus named RaTG13, which shares a 96% nucleotide sequence identity with SARS-CoV-218. However, the existence of RaTG13 in nature and the truthfulness of its reported sequence are being widely questioned6-9,19-21. It is noteworthy that scientific journals have clearly censored any dissenting opinions that suggest a non-natural origin of SARS-CoV-28,22. Because of this censorship, articles questioning either the natural origin of SARS-CoV-2 or the actual existence of RaTG13, although of high quality scientifically, can only exist as preprints5-9,19-21 or other non-peerreviewed articles published on various online platforms10-13,23. Nonetheless, analyses of these reports have repeatedly pointed to severe problems and a probable fraud associated with the reporting of RaTG136,8,9,1921 . Therefore, the theory that fabricated scientific data has been published to mislead the world’s efforts in tracing the origin of SARS-CoV-2 has become substantially convincing and is interlocked with the notion that SARS-CoV-2 is of a non-natural origin.

Consistent with this notion, genomic, structural, and literature evidence also suggest a non-natural origin of SARS-CoV-2. In addition, abundant literature indicates that gain-of-function research has long advanced to the stage where viral genomes can be precisely engineered and manipulated to enable the creation of novel coronaviruses possessing unique properties. In this report, we present such evidence and the associated analyses. Part 1 of the report describes the genomic and structural features of SARS-CoV2, the presence of which could be consistent with the theory that the virus is a product of laboratory modification beyond what could be afforded by simple serial viral passage. Part 2 of the report describes a highly probable pathway for the laboratory creation of SARS-CoV-2, key steps of which are supported by evidence present in the viral genome. Importantly, part 2 should be viewed as a demonstration of how SARS-CoV-2 could be conveniently created in a laboratory in a short period of time using available materials and well-documented techniques. This report is produced by a team of experienced scientists using our combined expertise in virology, molecular biology, structural biology, computational biology, vaccine development, and medicine.

2

1. Has SARS-CoV-2 been subjected to in vitro manipulation?

We present three lines of evidence to support our contention that laboratory manipulation is part of the history of SARS-CoV-2: i.

The genomic sequence of SARS-CoV-2 is suspiciously similar to that of a bat coronavirus discovered by military laboratories in the Third Military Medical University (Chongqing, China) and the Research Institute for Medicine of Nanjing Command (Nanjing, China).

ii.

The receptor-binding motif (RBM) within the Spike protein of SARS-CoV-2, which determines the host specificity of the virus, resembles that of SARS-CoV from the 2003 epidemic in a suspicious manner. Genomic evidence suggests that the RBM has been genetically manipulated.

iii.

SARS-CoV-2 contains a unique furin-cleavage site in its Spike protein, which is known to greatly enhance viral infectivity and cell tropism. Yet, this cleavage site is completely absent in this particular class of coronaviruses found in nature. In addition, rare codons associated with this additional sequence suggest the strong possibility that this furin-cleavage site is not the product of natural evolution and could have been inserted into the SARS-CoV-2 genome artificially by techniques other than simple serial passage or multi-strain recombination events inside co-infected tissue cultures or animals.

1.1 Genomic sequence analysis reveals that ZC45, or a closely related bat coronavirus, should be the backbone used for the creation of SARS-CoV-2 The structure of the ~30,000 nucleotides-long SARS-CoV-2 genome is shown in Figure 1. Searching the NCBI sequence database reveals that, among all known coronaviruses, there were two related bat coronaviruses, ZC45 and ZXC21, that share the highest sequence identity with SARS-CoV-2 (each bat coronavirus is ~89% identical to SARS-CoV-2 on the nucleotide level). Similarity between the genome of SARS-CoV-2 and those of representative β coronaviruses is depicted in Figure 1. ZXC21, which is 97% identical to and shares a very similar profile with ZC45, is not shown. Note that the RaTG13 virus is excluded from this analysis given the strong evidence suggesting that its sequence may have been fabricated and the virus does not exist in nature2,6-9. (A follow-up report, which summarizes the up-to-date evidence proving the spurious nature of RaTG13, will be submitted soon)

3

Figure 1. Genomic sequence analysis reveals that bat coronavirus ZC45 is the closest match to SARS-CoV-2.

Top: genomic organization of SARS-CoV-2 (2019-nCoV WIV04). Bottom: similarity plot based on the full-length genome of 2019-nCoV WIV04. Full-length genomes of SARS-CoV BJ01, bat SARSr-CoV WIV1, bat SARSr-CoV HKU3-1, bat coronavirus ZC45 were used as reference sequences.

When SARS-CoV-2 and ZC45/ZXC21 are compared on the amino acid level, a high sequence identity is observed for most of the proteins. The Nucleocapsid protein is 94% identical. The Membrane protein is 98.6% identical. The S2 portion (2nd half) of the Spike protein is 95% identical. Importantly, the Orf8 protein is 94.2% identical and the E protein is 100% identical.

Orf8 is an accessory protein, the function of which is largely unknown in most coronaviruses, although recent data suggests that Orf8 of SARS-CoV-2 mediates the evasion of host adaptive immunity by downregulating MHC-I24. Normally, Orf8 is poorly conserved in coronaviruses25. Sequence blast indicates that, while the Orf8 proteins of ZC45/ZXC21 share a 94.2% identity with SARS-CoV-2 Orf8, no other coronaviruses share more than 58% identity with SARS-CoV-2 on this particular protein. The very high homology here on the normally poorly conserved Orf8 protein is highly unusual.

Figure 2. Sequence alignment of the E proteins from different β coronaviruses demonstrates the E protein’s permissiveness and tendency toward amino acid mutations. A. Mutations have been observed in different strains of SARS-CoV. GenBank accession numbers: SARS_GD01: AY278489.2, SARS_ExoN1: ACB69908.1, SARS_TW_GD1: AY451881.1, SARS_Sino1_11: AY485277.1. B. Alignment of E proteins from related bat coronaviruses indicates its tolerance of mutations at multiple positions. GenBank accession numbers: Bat_AP040581.1: APO40581.1, RsSHC014: KC881005.1, SC2018: MK211374.1, Bat_NP_828854.1: NP_828854.1, BtRs-BetaCoV/HuB2013: AIA62312.1, BM48-31/BGR/2008: YP_003858586.1. C. While the early copies of SARS-CoV-2 share 100% identity on the E protein with ZC45 and ZXC21, sequencing data of SARS-CoV2 from April 2020 indicates that mutation has occurred at multiple positions. Accession numbers of viruses: Feb_11: MN997409, ZC45: MG772933.1, ZXC21: MG772934, Apr_13: MT326139, Apr_15_A: MT263389, Apr_15_B: MT293206, Apr_17: MT350246.

Alignments were done using the MultAlin webserver (http://multalin.toulouse.inra.fr/multalin/).

4

The coronavirus E protein is a structural protein, which is embedded in and lines the interior of the membrane envelope of the virion26. The E protein is tolerant of mutations as evidenced in both SARS (Figure 2A) and related bat coronaviruses (Figure 2B). This tolerance to amino acid mutations of the E protein is further evidenced in the current SARS-CoV-2 pandemic. After only a short two-month spread of the virus since its outbreak in humans, the E proteins in SARS-CoV-2 have already undergone mutational changes. Sequence data obtained during the month of April reveals that mutations have occurred at four different locations in different strains (Figure 2C). Consistent with this finding, sequence blast analysis indicates that, with the exception of SARS-CoV-2, no known coronaviruses share 100% amino acid sequence identity on the E protein with ZC45/ZXC21 (suspicious coronaviruses published after the start of the current pandemic are excluded18,27-31). Although 100% identity on the E protein has been observed between SARS-CoV and certain SARS-related bat coronaviruses, none of those pairs simultaneously share over 83% identity on the Orf8 protein32. Therefore, the 94.2% identity on the Orf8 protein, 100% identity on the E protein, and the overall genomic/amino acid-level resemblance between SARS-CoV-2 and ZC45/ZXC21 are highly unusual. Such evidence, when considered together, is consistent with a hypothesis that the SARS-CoV-2 genome has an origin based on the use of ZC45/ZXC21 as a backbone and/or template for genetic gain-of-function modifications.

Importantly, ZC45 and ZXC21 are bat coronaviruses that were discovered (between July 2015 and February 2017), isolated, and characterized by military research laboratories in the Third Military Medical University (Chongqing, China) and the Research Institute for Medicine of Nanjing Command (Nanjing, China). The data and associated work were published in 201833,34. Clearly, this backbone/template, which is essential for the creation of SARS-CoV-2, exists in these and other related research laboratories.

What strengthens our contention further is the published RaTG13 virus18, the genomic sequence of which is reportedly 96% identical to that of SARS-CoV-2. While suggesting a natural origin of SARSCoV-2, the RaTG13 virus also diverted the attention of both the scientific field and the general public away from ZC45/ZXC214,18. In fact, a Chinese BSL-3 lab (the Shanghai Public Health Clinical Centre), which published a Nature article reporting a conflicting close phylogenetic relationship between SARSCoV-2 and ZC45/ZXC21 rather than with RaTG1335, was quickly shut down for “rectification”36. It is believed that the researchers of that laboratory were being punished for having disclosed the SARS-CoV2—ZC45/ZXC21 connection. On the other hand, substantial evidence has accumulated, pointing to severe problems associated with the reported sequence of RaTG13 as well as questioning the actual existence of this bat virus in nature6,7,19-21. A very recent publication also indicated that the receptor-binding domain (RBD) of the RaTG13’s Spike protein could not bind ACE2 of two different types of horseshoe bats (they closely relate to the horseshoe bat R. affinis, RaTG13’s alleged natural host)2, implicating the inability of RaTG13 to infect horseshoe bats. This finding further substantiates the suspicion that the reported sequence of RaTG13 could have been fabricated as the Spike protein encoded by this sequence does not seem to carry the claimed function. The fact that a virus has been fabricated to shift the attention away from ZC45/ZXC21 speaks for an actual role of ZC45/ZXC21 in the creation of SARS-CoV-2.

1.2 The receptor-binding motif of SARS-CoV-2 Spike cannot be born from nature and should have been created through genetic engineering The Spike proteins decorate the exterior of the coronavirus particles. They play an important role in infection as they mediate the interaction with host cell receptors and thereby help determine the host range and tissue tropism of the virus. The Spike protein is split into two halves (Figure 3). The front or Nterminal half is named S1, which is fully responsible for binding the host receptor. In both SARS-CoV 5

and SARS-CoV-2 infections, the host cell receptor is hACE2. Within S1, a segment of around 70 amino acids makes direct contacts with hACE2 and is correspondingly named the receptor-binding motif (RBM) (Figure 3C). In SARS-CoV and SARS-CoV-2, the RBM fully determines the interaction with hACE2.

The C-terminal half of the Spike protein is named S2. The main function of S2 includes maintaining trimer formation and, upon successive protease cleavages at the S1/S2 junction and a downstream S2’ position, mediating membrane fusion to enable cellular entry of the virus.

Figure 3. Structure of the SARS Spike protein and how it binds to the hACE2 receptor. Pictures were generated based on PDB ID: 6acj37. A) Three spike proteins, each consisting of a S1 half and a S2 half, form a trimer. B) The S2 halves (shades of blue) are responsible for trimer formation, while the S1 portion (shades of red) is responsible for binding hACE2 (dark gray). C) Details of the binding between S1 and hACE2. The RBM of S1, which is important and sufficient for binding, is colored in orange. Residues within the RBM that are important for either hACE2 interaction or protein folding are shown as sticks (residue numbers follow the SARS Spike sequence).

6

Figure 4. Sequence alignment of the spike proteins from relevant coronaviruses. Viruses being compared include SARS-CoV-2 (Wuhan-Hu-1: NC_045512, 2019-nCoV_USA-AZ1: MN997409), bat coronaviruses (Bat_CoV_ZC45: MG772933, Bat_CoV_ZXC21: MG772934), and SARS coronaviruses (SARS_GZ02: AY390556, SARS: NC_004718.3). Region marked by two orange lines is the receptor-binding motif (RBM), which is important for interaction with the hACE2 receptor. Essential residues are additionally highlighted by red sticks on top. Region marked by two green lines is a furin-cleavage site that exists only in SARS-CoV-2 but not in any other lineage B β coronavirus.

7

Similar to what is observed for other viral proteins, S2 of SARS-CoV-2 shares a high sequence identity (95%) with S2 of ZC45/ZXC21. In stark contrast, between SARS-CoV-2 and ZC45/ZXC21, the S1 protein, which dictates which host (human or bat) the virus can infect, is much less conserved with the amino acid sequence identity being only 69%.

Figure 4 shows the sequence alignment of the Spike proteins from six β coronaviruses. Two are viruses isolated from the current pandemic (Wuhan-Hu-1, 2019-nCoV_USA-AZ1); two are the suspected template viruses (Bat_CoV_ZC45, Bat_CoV_ZXC21); two are SARS coronaviruses (SARS_GZ02, SARS). The RBM is highlighted in between two orange lines. Clearly, despite the high sequence identity for the overall genomes, the RBM of SARS-CoV-2 differs significantly from those of ZC45 and ZXC21.

Intriguingly, the RBM of SARS-CoV-2 resembles, on a great deal, the RBM of SARS Spike. Although this is not an exact “copy and paste”, careful examination of the Spike-hACE2 structures37,38 reveals that all residues essential for either hACE2 binding or protein folding (orange sticks in Figure 3C and what is highlighted by red short lines in Figure 4) are “kept”. Most of these essential residues are precisely preserved, including those involved in disulfide bond formation (C467, C474) and electrostatic interactions (R444, E452, R453, D454), which are pivotal for the structural integrity of the RBM (Figure 3C and 4). The few changes within the group of essential residues are almost exclusively hydrophobic “substitutions” (I428àL, L443àF, F460àY, L472àF, Y484àQ), which should not affect either protein folding or the hACE2-interaction. At the same time, majority of the amino acid residues that are non-essential have “mutated” (Figure 4, RBM residues not labeled with short red lines). Judging from this sequence analysis alone, we were convinced early on that not only would the SARS-CoV-2 Spike protein bind hACE2 but also the binding would resemble, precisely, that between the original SARS Spike protein and hACE223. Recent structural work has confirmed our prediction39.

As elaborated below, the way that SARS-CoV-2 RBM resembles SARS-CoV RBM and the overall sequence conservation pattern between SARS-CoV-2 and ZC45/ZXC21 are highly unusual. Collectively, this suggests that portions of the SARS-CoV-2 genome have not been derived from natural quasi-species viral particle evolution.

If SARS-CoV-2 does indeed come from natural evolution, its RBM could have only been acquired in one of the two possible routes: 1) an ancient recombination event followed by convergent evolution or 2) a natural recombination event that occurred fairly recently.

In the first scenario, the ancestor of SARS-CoV-2, a ZC45/ZXC21-like bat coronavirus would have recombined and “swapped” its RBM with a coronavirus carrying a relatively “complete” RBM (in reference to SARS). This recombination would result in a novel ZC45/ZXC21-like coronavirus with all the gaps in its RBM “filled” (Figure 4). Subsequently, the virus would have to adapt extensively in its new host, where the ACE2 protein is highly homologous to hACE2. Random mutations across the genome would have to have occurred to eventually shape the RBM to its current form – resembling SARS-CoV RBM in a highly intelligent manner. However, this convergent evolution process would also result in the accumulation of a large amount of mutations in other parts of the genome, rendering the overall sequence identity relatively low. The high sequence identity between SARS-CoV-2 and ZC45/ZXC21 on various proteins (94-100% identity) do not support this scenario and, therefore, clearly indicates that SARS-CoV2 carrying such an RBM cannot come from a ZC45/ZXC21-like bat coronavirus through this convergent evolutionary route.

In the second scenario, the ZC45/ZXC21-like coronavirus would have to have recently recombined and swapped its RBM with another coronavirus that had successfully adapted to bind an animal ACE2 8

highly homologous to hACE2. The likelihood of such an event depends, in part, on the general requirements of natural recombination: 1) that the two different viruses share significant sequence similarity; 2) that they must co-infect and be present in the same cell of the same animal; 3) that the recombinant virus would not be cleared by the host or make the host extinct; 4) that the recombinant virus eventually would have to become stable and transmissible within the host species.

In regard to this recent recombination scenario, the animal reservoir could not be bats because the ACE2 proteins in bats are not homologous enough to hACE2 and therefore the adaption would not be able to yield an RBM sequence as seen in SARS-CoV-2. This animal reservoir also could not be humans as the ZC45/ZXC21-like coronavirus would not be able to infect humans. In addition, there has been no evidence of any SARS-CoV-2 or SARS-CoV-2-like virus circulating in the human population prior to late 2019. Intriguingly, according to a recent bioinformatics study, SARS-CoV-2 was well-adapted for humans since the start of the outbreak1.

Only one other possibility of natural evolution remains, which is that the ZC45/ZXC21-like virus and a coronavirus containing a SARS-like RBM could have recombined in an intermediate host where the ACE2 protein is homologous to hACE2. Several laboratories have reported that some of the Sunda pangolins smuggled into China from Malaysia carried coronaviruses, the receptor-binding domain (RBD) of which is almost identical to that of SARS-CoV-227-29,31. They then went on to suggest that pangolins are the likely intermediate host for SARS-CoV-227-29,31. However, recent independent reports have found significant flaws in this data40-42. Furthermore, contrary to these reports27-29,31, no coronaviruses have been detected in Sunda pangolin samples collected for over a decade in Malaysia and Sabah between 2009 and 201943. A recent study also showed that the RBD, which is shared between SARS-CoV-2 and the reported pangolin coronaviruses, binds to hACE2 ten times stronger than to the pangolin ACE22, further dismissing pangolins as the possible intermediate host. Finally, an in silico study, while echoing the notion that pangolins are not likely an intermediate host, also indicated that none of the animal ACE2 proteins examined in their study exhibited more favorable binding potential to the SARS-CoV-2 Spike protein than hACE2 did3. This last study virtually exempted all animals from their suspected roles as an intermediate host3, which is consistent with the observation that SARS-CoV-2 was well-adapted for humans from the start of the outbreak1. This is significant because these findings collectively suggest that no intermediate host seems to exist for SARS-CoV-2, which at the very least diminishes the possibility of a recombinant event occurring in an intermediate host.

Even if we ignore the above evidence that no proper host exists for the recombination to take place and instead assume that such a host does exist, it is still highly unlikely that such a recombination event could occur in nature.

As we have described above, if natural recombination event is responsible for the appearance of SARSCoV-2, then the ZC45/ZXC21-like virus and a coronavirus containing a SARS-like RBM would have to recombine in the same cell by swapping the S1/RBM, which is a rare form of recombination. Furthermore, since SARS has occurred only once in human history, it would be at least equally rare for nature to produce a virus that resembles SARS in such an intelligent manner – having an RBM that differs from the SARS RBM only at a few non-essential sites (Figure 4). The possibility that this unique SARS-like coronavirus would reside in the same cell with the ZC45/ZXC21-like ancestor virus and the two viruses would recombine in the “RBM-swapping” fashion is extremely low. Importantly, this, and the other recombination event described below in section 1.3 (even more impossible to occur in nature), would both have to happen to produce a Spike as seen in SARS-CoV-2.

9

While the above evidence and analyses together appear to disapprove a natural origin of SARS-CoV2’s RBM, abundant literature shows that gain-of-function research, where the Spike protein of a coronavirus was specifically engineered, has repeatedly led to the successful generation of humaninfecting coronaviruses from coronaviruses of non-human origin44-47.

Record also shows that research laboratories, for example, the Wuhan Institute of Virology (WIV), have successfully carried out such studies working with US researchers45 and also working alone47. In addition, the WIV has engaged in decades-long coronavirus surveillance studies and therefore owns the world’s largest collection of coronaviruses. Evidently, the technical barrier is non-existent for the WIV and other related laboratories to carry out and succeed in such Spike/RBM engineering and gain-offunction research.

Figure 5. Two restriction sites are present at either end of the RBM of SARS-CoV-2, providing convenience for replacing the RBM within the spike gene. A. Nucleotide sequence of the RBM of SARS-CoV-2 (Wuhan-Hu-1). An EcoRI site is found at the 5’-end of the RBM and a BstEII site at the 3’-end. B. Although these two restriction sites do not exist in the original spike gene of ZC45, they can be conveniently introduced given that the sequence discrepancy is small (2 nucleotides) in either case. C. Amino acid sequence alignment with the RBM region highlighted (color and underscore). The RBM highlighted in orange (top) is what is defined by the EcoRI and BstEII sites in the SARS-CoV-2 (Wuhan-Hu-1) spike. The RBM highlighted in magenta (middle) is the region swapped by Dr. Fang Li and colleagues into a SARS Spike backbone39. The RBM highlighted in blue (bottom) is from the Spike protein (RBM: 424-494) of SARS-BJ01 (AY278488.2), which was swapped by the Shi lab into the Spike proteins of different bat coronaviruses replacing the corresponding segments47.

10

Strikingly, consistent with the RBM engineering theory, we have identified two unique restriction sites, EcoRI and BstEII, at either end of the RBM of the SARS-CoV-2 genome, respectively (Figure 5A). These two sites, which are popular choices of everyday molecular cloning, do not exist in the rest of this spike gene. This particular setting makes it extremely convenient to swap the RBM within spike, providing a quick way to test different RBMs and the corresponding Spike proteins.

Such EcoRI and BstEII sites do not exist in the spike genes of other β coronaviruses, which strongly indicates that they were unnatural and were specifically introduced into this spike gene of SARS-CoV-2 for the convenience of manipulating the critical RBM. Although ZC45 spike also does not have these two sites (Figure 5B), they can be introduced very easily as described in part 2 of this report.

It is noteworthy that introduction of the EcoRI site here would change the corresponding amino acids from -WNT- to -WNS- (Figure 5AB). As far as we know, all SARS and SARS-like bat coronaviruses exclusively carry a T (threonine) residue at this location. SARS-CoV-2 is the only exception in that this T has mutated to an S (serine), save the suspicious RaTG13 and pangolin coronaviruses published after the outbreak48.

Once the restriction sites were successfully introduced, the RBM segment could be swapped conveniently using routine restriction enzyme digestion and ligation. Although alternative cloning techniques may leave no trace of genetic manipulation (Gibson assembly as one example), this oldfashioned approach could be chosen because it offers a great level of convenience in swapping this critical RBM. Given that RBM fully dictates hACE2-binding and that the SARS RBM-hACE2 binding was fully characterized by high-resolution structures (Figure 3)37,38, this RBM-only swap would not be any riskier than the full Spike swap. In fact, the feasibility of this RBM-swap strategy has been proven39,47. In 2008, Dr. Zhengli Shi’s group swapped a SARS RBM into the Spike proteins of several SARS-like bat coronaviruses after introducing a restriction site into a codon-optimized spike gene (Figure 5C)47. They then validated the binding of the resulted chimeric Spike proteins with hACE2. Furthermore, in a recent publication, the RBM of SARS-CoV-2 was swapped into the receptor-binding domain (RBD) of SARSCoV, resulting in a chimeric RBD fully functional in binding hACE2 (Figure 5C)39. Strikingly, in both cases, the manipulated RBM segments resemble almost exactly the RBM defined by the positions of the EcoRI and BstEII sites (Figure 5C). Although cloning details are lacking in both publications39,47, it is conceivable that the actual restriction sites may vary depending on the spike gene receiving the RBM insertion as well as the convenience in introducing unique restriction site(s) in regions of interest. It is noteworthy that the corresponding author of this recent publication39, Dr. Fang Li, has been an active collaborator of Dr. Zhengli Shi since 201049-53. Dr. Li was the first person in the world to have structurally elucidated the binding between SARS-CoV RBD and hACE238 and has been the leading expert in the structural understanding of Spike-ACE2 interactions38,39,53-56. The striking finding of EcoRI and BstEII restriction sites at either end of the SARS-CoV-2 RBM, respectively, and the fact that the same RBM region has been swapped both by Dr. Shi and by her long-term collaborator, respectively, using restriction enzyme digestion methods are unlikely a coincidence. Rather, it is the smoking gun proving that the RBM/Spike of SARS-CoV-2 is a product of genetic manipulation.

Although it may be convenient to copy the exact sequence of SARS RBM, it would be too clear a sign of artificial design and manipulation. The more deceiving approach would be to change a few nonessential residues, while preserving the ones critical for binding. This design could be well-guided by the high-resolution structures (Figure 3)37,38. This way, when the overall sequence of the RBM would appear 11

to be more distinct from that of the SARS RBM, the hACE2-binding ability would be well-preserved. We believe that all of the crucial residues (residues labeled with red sticks in Figure 4, which are the same residues shown in sticks in Figure 3C) should have been “kept”. As described earlier, while some should be direct preservation, some should have been switched to residues with similar properties, which would not disrupt hACE2-binding and may even strengthen the association further. Importantly, changes might have been made intentionally at non-essential sites, making it less like a “copy and paste” of the SARS RBM. 1.3 An unusual furin-cleavage site is present in the Spike protein of SARS-CoV-2 and is associated with the augmented virulence of the virus Another unique motif in the Spike protein of SARS-CoV-2 is a polybasic furin-cleavage site located at the S1/S2 junction (Figure 4, segment in between two green lines). Such a site can be recognized and cleaved by the furin protease. Within the lineage B of β coronaviruses and with the exception of SARSCoV-2, no viruses contain a furin-cleavage site at the S1/S2 junction (Figure 6)57. In contrast, furincleavage site at this location has been observed in other groups of coronaviruses57,58. Certain selective pressure seems to be in place that prevents the lineage B of β coronaviruses from acquiring or maintaining such a site in nature.

Figure 6. Furin-cleavage site found at the S1/S2 junction of Spike is unique to SARS-CoV-2 and absent in other lineage B β coronaviruses. Figure reproduced from Hoffmann, et al57.

12

As previously described, during the cell entry process, the Spike protein is first cleaved at the S1/S2 junction. This step, and a subsequent cleavage downstream that exposes the fusion peptide, are both mediated by host proteases. The presence or absence of these proteases in different cell types greatly affects the cell tropism and presumably the pathogenicity of the viral infection. Unlike other proteases, furin protease is widely expressed in many types of cells and is present at multiple cellular and extracellular locations. Importantly, the introduction of a furin-cleavage site at the S1/S2 junction could significantly enhance the infectivity of a virus as well as greatly expand its cell tropism — a phenomenon well-documented in both influenza viruses and other coronaviruses59-65.

If we leave aside the fact that no furin-cleavage site is found in any lineage B β coronavirus in nature and instead assume that this site in SARS-CoV-2 is a result of natural evolution, then only one evolutionary pathway is possible, which is that the furin-cleavage site has to be derived from a homologous recombination event. Specifically, an ancestor β coronavirus containing no furin-cleavage site would have to recombine with a closely related coronavirus that does contain a furin-cleavage site.

However, two facts disfavor this possibility. First, although some coronaviruses from other groups or lineages do contain polybasic furin-cleavage sites, none of them contains the exact polybasic sequence present in SARS-CoV-2 (-PRRAR/SVA-). Second, between SARS-CoV-2 and any coronavirus containing a legitimate furin-cleavage site, the sequence identity on Spike is no more than 40%66. Such a low level of sequence identity rules out the possibility of a successful homologous recombination ever occurring between the ancestors of these viruses. Therefore, the furin-cleavage site within the SARS-CoV-2 Spike protein is unlikely to be of natural origin and instead should be a result of laboratory modification.

Consistent with this claim, a close examination of the nucleotide sequence of the furin-cleavage site in SARS-CoV-2 spike has revealed that the two consecutive Arg residues within the inserted sequence (PRRA-) are both coded by the rare codon CGG (least used codon for Arg in SARS-CoV-2) (Figure 7)8.

In fact, this CGGCGG arrangement is the only instance found in the SARS-CoV-2 genome where this rare codon is used in tandem. This observation strongly suggests that this furin-cleavage site should be a result of genetic engineering. Adding to the suspicion, a FauI restriction site is formulated by the codon choices here, suggesting the possibility that the restriction fragment length polymorphism, a technique that a WIV lab is proficient at67, could have been involved. There, the fragmentation pattern resulted from FauI digestion could be used to monitor the preservation of the furin-cleavage site in Spike as this furincleavage site is prone to deletions in vitro68,69. Specifically, RT-PCR on the spike gene of the recovered viruses from cell cultures or laboratory animals could be carried out, the product of which would be subjected to FauI digestion. Viruses retaining or losing the furin-cleavage site would then yield distinct patterns, allowing convenient tracking of the virus(es) of interest.

Figure 7. Two consecutive Arg residues in the -PRRA- insertion at the S1/S2 junction of SARS-CoV-2 Spike are both coded by a rare codon, CGG. A FauI restriction site, 5’-(N)6GCGGG-3’, is embedded in the coding sequence of the “inserted” PRRA segment, which may be used as a marker to monitor the preservation of the introduced furin-cleavage site.

In addition, although no known coronaviruses contain the exact sequence of -PRRAR/SVA- that is present in the SARS-CoV-2 Spike protein, a similar -RRAR/AR- sequence has been observed at the S1/S2 junction of the Spike protein in a rodent coronavirus, AcCoV-JC34, which was published by Dr. Zhengli 13

Shi in 201770. It is evident that the legitimacy of -RRAR- as a functional furin-cleavage site has been known to the WIV experts since 2017.

The evidence collectively suggests that the furin-cleavage site in the SARS-CoV-2 Spike protein may not have come from nature and could be the result of genetic manipulation. The purpose of this manipulation could have been to assess any potential enhancement of the infectivity and pathogenicity of the laboratory-made coronavirus59-64. Indeed, recent studies have confirmed that the furin-cleavage site does confer significant pathogenic advantages to SARS-CoV-257,68.

1.4 Summary Evidence presented in this part reveals that certain aspects of the SARS-CoV-2 genome are extremely difficult to reconcile to being a result of natural evolution. The alternative theory we suggest is that the virus may have been created by using ZC45/ZXC21 bat coronavirus(es) as the backbone and/or template.

The Spike protein, especially the RBM within it, should have been artificially manipulated, upon which the virus has acquired the ability to bind hACE2 and infect humans. This is supported by the finding of a unique restriction enzyme digestion site at either end of the RBM. An unusual furin-cleavage site may have been introduced and inserted at the S1/S2 junction of the Spike protein, which contributes to the increased virulence and pathogenicity of the virus. These transformations have then staged the SARSCoV-2 virus to eventually become a highly-transmissible, onset-hidden, lethal, sequelae-unclear, and massively disruptive pathogen.

Evidently, the possibility that SARS-CoV-2 could have been created through gain-of-function manipulations at the WIV is significant and should be investigated thoroughly and independently.

2. Delineation of a synthetic route of SARS-CoV-2 In the second part of this report, we describe a synthetic route of creating SARS-CoV-2 in a laboratory setting. It is postulated based on substantial literature support as well as genetic evidence present in the SARS-CoV-2 genome. Although steps presented herein should not be viewed as exactly those taken, we believe that key processes should not be much different. Importantly, our work here should serve as a demonstration of how SARS-CoV-2 can be designed and created conveniently in research laboratories by following proven concepts and using well-established techniques.

Importantly, research labs, both in Hong Kong and in mainland China, are leading the world in coronavirus research, both in terms of resources and on the research outputs. The latter is evidenced not only by the large number of publications that they have produced over the past two decades but also by their milestone achievements in the field: they were the first to identify civets as the intermediate host for SARS-CoV and isolated the first strain of the virus71; they were the first to uncover that SARS-CoV originated from bats72,73; they revealed for the first time the antibody-dependent enhancement (ADE) of SARS-CoV infections74; they have contributed significantly in understanding MERS in all domains (zoonosis, virology, and clinical studies)75-79; they made several breakthroughs in SARS-CoV-2 research18,35,80. Last but not least, they have the world’s largest collection of coronaviruses (genomic sequences and live viruses). The knowledge, expertise, and resources are all readily available within the Hong Kong and mainland research laboratories (they collaborate extensively) to carry out and accomplish the work described below.

14

Figure 8. Diagram of a possible synthetic route of the laboratory-creation of SARS-CoV-2.

15

2.1 Possible scheme in designing the laboratory-creation of the novel coronavirus In this sub-section, we outline the possible overall strategy and major considerations that may have been formulated at the designing stage of the project.

To engineer and create a human-targeting coronavirus, they would have to pick a bat coronavirus as the template/backbone. This can be conveniently done because many research labs have been actively collecting bat coronaviruses over the past two decades32,33,70,72,81-85. However, this template virus ideally should not be one from Dr. Zhengli Shi’s collections, considering that she is widely known to have been engaged in gain-of-function studies on coronaviruses. Therefore, ZC45 and/or ZXC21, novel bat coronaviruses discovered and owned by military laboratories33, would be suitable as the template/backbone. It is also possible that these military laboratories had discovered other closely related viruses from the same location and kept some unpublished. Therefore, the actual template could be ZC45, or ZXC21, or a close relative of them. The postulated pathway described below would be the same regardless of which one of the three was the actual template.

Once they have chosen a template virus, they would first need to engineer, through molecular cloning, the Spike protein so that it can bind hACE2. The concept and cloning techniques involved in this manipulation have been well-documented in the literature44-46,84,86. With almost no risk of failing, the template bat virus could then be converted to a coronavirus that can bind hACE2 and infect humans44-46.

Second, they would use molecular cloning to introduce a furin-cleavage site at the S1/S2 junction of Spike. This manipulation, based on known knowledge60,61,65, would likely produce a strain of coronavirus that is a more infectious and pathogenic.

Third, they would produce an ORF1b gene construct. The ORF1b gene encodes the polyprotein Orf1b, which is processed post-translationally to produce individual viral proteins: RNA-dependent RNA polymerase (RdRp), helicase, guanidine-N7 methyltransferase, uridylate-specific endoribonuclease, and 2’-O-methyltransferase. All of these proteins are parts of the replication machinery of the virus. Among them, the RdRp protein is the most crucial one and is highly conserved among coronaviruses. Importantly, Dr. Zhengli Shi’s laboratory uses a PCR protocol, which amplifies a particular fragment of the RdRp gene, as their primary method to detect the presence of coronaviruses in raw samples (bat fecal swap, feces, etc).

As a result of this practice, the Shi group has documented the sequence information of this short segment of RdRp for all coronaviruses that they have successfully detected and/or collected.

Here, the genetic manipulation is less demanding or complicated because Orf1b is conserved and likely Orf1b from any β coronavirus would be competent enough to do the work. However, we believe that they would want to introduce a particular Orf1b into the virus for one of the two possible reasons: 1. Since many phylogenetic analyses categorize coronaviruses based on the sequence similarity of the RdRp gene only18,31,35,83,87, having a different RdRp in the genome therefore could ensure that SARS-CoV-2 and ZC45/ZXC21 are separated into different groups/sub-lineages in phylogenetic studies. Choosing an RdRp gene, however, is convenient because the short RdRp segment sequence has been recorded for all coronaviruses ever collected/detected. Their final choice was the RdRp sequence from bat coronavirus RaBtCoV/4991, which was discovered in 2013. For RaBtCoV/4991, the only information ever published was the sequence of its short RdRp segment83, while neither its full genomic sequence nor virus isolation were ever reported. After amplifying the RdRp segment (or the whole ORF1b gene) of RaBatCoV/4991, they would have then used it for subsequent assembly and creation of the genome of SARS-CoV-2. Small changes in the RdRp 16

sequence could either be introduced at the beginning (through DNA synthesis) or be generated via passages later on. On a separate track, when they were engaged in the fabrication of the RaTG13 sequence, they could have started with the short RdRp segment of RaBtCoV/4991 without introducing any changes to its sequence, resulting in a 100% nucleotide sequence identity between the two viruses on this short RdRp segment83. This RaTG13 virus could then be claimed to have been discovered back in 2013.

2. The RdRp protein from RaBatCoV/4991 is unique in that it is superior than RdRp from any other β coronavirus for developing antiviral drugs. RdRp has no homologs in human cells, which makes this essential viral enzyme a highly desirable target for antiviral development. As an example, Remedesivir, which is currently undergoing clinical trials, targets RdRp. When creating a novel and human-targeting virus, they would be interested in developing the antidote as well. Even though drug discovery like this may not be easily achieved, it is reasonable for them to intentionally incorporate a RdRp that is more amenable for antiviral drug development.

Fourth, they would use reverse genetics to assemble the gene fragments of spike, ORF1b, and the rest of the template ZC45 into a cDNA version of the viral genome. They would then carry out in vitro transcription to obtain the viral RNA genome. Transfection of the RNA genome into cells would allow the recovery of live and infectious viruses with the desired artificial genome.

Fifth, they would carry out characterization and optimization of the virus strain(s) to improve the fitness, infectivity, and overall adaptation using serial passage in vivo. One or several viral strains that meet certain criteria would then be obtained as the final product(s).

2.2 A postulated synthetic route for the creation of SARS-CoV-2 In this sub-section, we describe in more details how each step could be carried out in a laboratory setting using available materials and routine molecular, cellular, and virologic techniques. A diagram of this process is shown in Figure 8. We estimate that the whole process could be completed in approximately 6 months.

Step 1: Engineering the RBM of the Spike for hACE2-binding (1.5 months) The Spike protein of a bat coronavirus is either incapable of or inefficient in binding hACE2 due to the missing of important residues within its RBM. This can be exemplified by the RBM of the template virus ZC45 (Figure 4). The first and most critical step in the creation of SARS-CoV-2 is to engineer the Spike so that it acquires the ability to bind hACE2. As evidenced in the literature, such manipulations have been carried out repeatedly in research laboratories since 200844, which successfully yielded engineered coronaviruses with the ability to infect human cells44-46,88,89. Although there are many possible ways that one can engineer the Spike protein, we believe that what was actually undertaken was that they replaced the original RBM with a designed and possibly optimized RBM using SARS’ RBM as a guide. As described in part 1, this theory is supported by our observation that two unique restriction sites, EcoRI and BstEII, exist at either end of the RBM in the SARS-CoV-2 genome (Figure 5A) and by the fact that such RBM-swap has been successfully carried out by Dr. Zhengli Shi and by her long-term collaborator and structure biology expert, Dr. Fang Li39,47.

Although ZC45 spike does not contain these two restriction sites (Figure 5B), they can be introduced very easily. The original spike gene would be either amplified with RT-PCR or obtained through DNA synthesis (some changes could be safely introduced to certain variable regions of the sequence) followed

by PCR. The gene would then be cloned into a plasmid using restriction sites other than EcoRI and BstEII. 17

Once in the plasmid, the spike gene can be modified easily. First, an EcoRI site can be introduced by converting the highlighted “gaacac” sequence (Figure 5B) to the desired “gaattc” (Figure 5A). The difference between them are two consecutive nucleotides. Using the commercially available QuikChange Site-Directed Mutagenesis kit, such a di-nucleotide mutation can be generated in no more than one week.

Subsequently, the BstEII site could be similarly introduced at the other end of the RBM. Specifically, the “gaatacc” sequence (Figure 5B) would be converted to the desired “ggttacc” (Figure 5A), which would similarly require a week of time.

Once these restriction sites, which are unique within the spike gene of SARS-CoV-2, were successfully introduced, different RBM segments could be swapped in conveniently and the resulting Spike protein subsequently evaluated using established assays.

As described in part 1, the design of an RBM segment could be well-guided by the high-resolution structures (Figure 3)37,38, yielding a sequence that resembles the SARS RBM in an intelligent manner.

When carrying out the structure-guided design of the RBM, they would have followed the routine and generated a few (for example a dozen) such RBMs with the hope that some specific variant(s) may be superior than others in binding hACE2. Once the design was finished, they could have each of the designed RBM genes commercially synthesized (quick and very affordable) with an EcoRI site at the 5’-end and a BstEII site at the 3’-end. These novel RBM genes could then be cloned into the spike gene, respectively.

The gene synthesis and subsequent cloning, which could be done in a batch mode for the small library of designed RBMs, would take approximately one month.

These engineered Spike proteins might then be tested for hACE2-binding using the established pseudotype virus infection assays45,49,50. The engineered Spike with good to exceptional binding affinities would be selected. (Although not necessary, directed evolution could be involved here (error-prone PCR on the RBM gene), coupled with either an in vitro binding assay39,90 or a pseudotype virus infection assay45,49,50, to obtain an RBM that binds hACE2 with exceptional affinity.)

Given the abundance of literature on Spike engineering44-46,84,86 and the available high-resolution structures of the Spike-hACE2 complex37,38, the success of this step would be very much guaranteed. By the end of this step, as desired, a novel spike gene would be obtained, which encodes a novel Spike protein capable of binding hACE2 with high affinity.

Step 2: Engineering a furin-cleavage site at the S1/S2 junction (0.5 month) The product from Step 1, a plasmid containing the engineered spike, would be further modified to include a furin-cleavage site (segment indicated by green lines in Figure 4) at the S1/S2 junction. This short stretch of gene sequence can be conveniently inserted using several routine cloning techniques, including QuikChange Site-Directed PCR60, overlap PCR followed by restriction enzyme digestion and ligation91, or Gibson assembly. None of these techniques would leave any trace in the sequence.

Whichever cloning method was the choice, the inserted gene piece would be included in the primers, which would be designed, synthesized, and used in the cloning. This step, leading to a further modified Spike with the furin-cleavage site added at the S1/S2 junction, could be completed in no more than two weeks.

Step 3: Obtain an ORF1b gene that contains the sequence of the short RdRp segment from RaBtCoV/4991 (1 month, yet can be carried out concurrently with Steps 1 and 2)

18

Unlike the engineering of Spike, no complicated design is needed here, except that the RdRp gene segment from RaBtCoV/4991 would need to be included. Gibson assembly could have been used here. In this technique, several fragments, each adjacent pair sharing 20-40 bp overlap, are combined together in one simple reaction to assemble a long DNA product. Two or three fragments, each covering a significant section of the ORF1b gene, would be selected based on known bat coronavirus sequences. One of these fragments would be the RdRp segment of RaBtCoV/499183. Each fragment would be PCR amplified with proper overlap regions introduced in the primers. Finally, all purified fragments would be pooled in equimolar concentrations and added to the Gibson reaction mixture, which, after a short incubation, would yield the desired ORF1b gene in whole.

Step 4: Produce the designed viral genome using reverse genetics and recover live viruses (0.5 month) Reverse genetics have been frequently used in assembling whole viral genomes, including coronavirus genomes67,92-96. The most recent example is the reconstruction of the SARS-CoV-2 genome using the transformation-assisted recombination in yeast97. Using this method, the Swiss group assembled the entire viral genome and produced live viruses in just one week97. This efficient technique, which would not leave any trace of artificial manipulation in the created viral genome, has been available since 201798,99. In addition to the engineered spike gene (from steps 1 and 2) and the ORF1b gene (from step 3), other fragments covering the rest of the genome would be obtained either through RT-PCR amplification from the template virus or through DNA synthesis by following a sequence slightly altered from that of the template virus. We believe that the latter approach was more likely as it would allow sequence changes introduced into the variable regions of less conserved proteins, the process of which could be easily guided by multiple sequence alignments. The amino acid sequences of more conserved functions, such as that of the E protein, might have been left unchanged. All DNA fragments would then be pooled together and transformed into yeast, where the cDNA version of the SARS-CoV-2 genome would be assembled via transformation-assisted recombination. Of course, an alternative method of reverse genetics, one of which the WIV has successfully used in the past67, could also be employed67,92-96,100. Although some earlier reverse genetics approaches may leave restriction sites at where different fragments would be joined, these traces would be hard to detect as the exact site of ligation can be anywhere in the ~30kb genome. Either way, a cDNA version of the viral genome would be obtained from the reverse genetics experiment.

Subsequently, in vitro transcription using the cDNA as the template would yield the viral RNA genome, which upon transfection into Vero E6 cells would allow the production of live viruses bearing all of the designed properties.

Step 5: Optimize the virus for fitness and improve its hACE2-binding affinity in vivo (2.5-3 months) Virus recovered from step 4 needs to be further adapted undergoing the classic experiment – serial passage in laboratory animals101. This final step would validate the virus’ fitness and ensure its receptororiented adaptation toward its intended host, which, according to the analyses above, should be human.

Importantly, the RBM and the furin-cleavage site, which were introduced into the Spike protein separately, would now be optimized together as one functional unit. Among various available animal models (e.g. mice, hamsters, ferrets, and monkeys) for coronaviruses, hACE2 transgenic mice (hACE2-mice) should be the most proper and convenient choice here. This animal model has been established during the study of SARS-CoV and has been available in the Jackson Laboratory for many years102-104.

The procedure of serial passage is straightforward. Briefly, the selected viral strain from step 4, a precursor of SARS-CoV-2, would be intranasally inoculated into a group of anaesthetized hACE2-mice.

Around 2-3 days post infection, the virus in lungs would usually amplify to a peak titer. The mice would 19

then be sacrificed and the lungs homogenized. Usually, the mouse-lung supernatant, which carries the highest viral load, would be used to extract the candidate virus for the next round of passage. After approximately 10~15 rounds of passage, the hACE2-binding affinity, the infection efficiency, and the lethality of the viral strain would be sufficiently enhanced and the viral genome stabilized101. Finally, after a series of characterization experiments (e.g. viral kinetics assay, antibodies response assay, symptom observation and pathology examination), the final product, SARS-CoV-2, would be obtained, concluding the whole creation process. From this point on, this viral pathogen could be amplified (most probably using Vero E6 cells) and produced routinely.

It is noteworthy that, based on the work done on SARS-CoV, the hACE2-mice, although suitable for SARS-CoV-2 adaptation, is not a good model to reflect the virus’ transmissibility and associated clinical symptoms in humans. We believe that those scientists might not have used a proper animal model (such as the golden Syrian hamster) for testing the transmissibility of SARS-CoV-2 before the outbreak of COVID-19. If they had done this experiment with a proper animal model, the highly contagious nature of SARS-CoV-2 would be extremely evident and consequently SARS-CoV-2 would not have been described as “not causing human-to-human transmission” at the start of the outbreak.

We also speculate that the extensive laboratory-adaptation, which is oriented toward enhanced transmissibility and lethality, may have driven the virus too far. As a result, SARS-CoV-2 might have lost the capacity to attenuate on both transmissibility and lethality during its current adaptation in the human population. This hypothesis is consistent with the lack of apparent attenuation of SARS-CoV-2 so far despite its great prevalence and with the observation that a recently emerged, predominant variant only shows improved transmissibility105-108.

Serial passage is a quick and intensive process, where the adaptation of the virus is accelerated.

Although intended to mimic natural evolution, serial passage is much more limited in both time and scale.

As a result, less random mutations would be expected in serial passage than in natural evolution. This is particularly true for conserved viral proteins, such as the E protein. Critical in viral replication, the E protein is a determinant of virulence and engineering of it may render SARS-CoV-2 attenuated109-111 Therefore, at the initial assembly stage, these scientists might have decided to keep the amino acid sequence of the E protein unchanged from that of ZC45/ZXC21. Due to the conserved nature of the E protein and the limitations of serial passage, no amino acid mutation actually occurred, resulting in a 100% sequence identity on the E protein between SARS-CoV-2 and ZC45/ZXC21. The same could have happened to the marks of molecular cloning (restriction sites flanking the RBM). Serial passage, which should have partially naturalized the SARS-CoV-2 genome, might not have removed all signs of artificial manipulation.

3. Final remarks Many questions remain unanswered about the origin of SARS-CoV-2. Prominent virologists have implicated in a Nature Medicine letter that laboratory escape, while not being entirely ruled out, was unlikely and that no sign of genetic manipulation is present in the SARS-CoV-2 genome4. However, here we show that genetic evidence within the spike gene of SARS-CoV-2 genome (restriction sites flanking the RBM; tandem rare codons used at the inserted furin-cleavage site) does exist and suggests that the SARS-CoV-2 genome should be a product of genetic manipulation. Furthermore, the proven concepts, well-established techniques, and knowledge and expertise are all in place for the convenient creation of this novel coronavirus in a short period of time.

20

Motives aside, the following facts about SARS-CoV-2 are well-supported: 1. If it was a laboratory product, the most critical element in its creation, the backbone/template virus (ZC45/ZXC21), is owned by military research laboratories.

2. The genome sequence of SARS-CoV-2 has likely undergone genetic engineering, through which the virus has gained the ability to target humans with enhanced virulence and infectivity.

3. The characteristics and pathogenic effects of SARS-CoV-2 are unprecedented. The virus is highly transmissible, onset-hidden, multi-organ targeting, sequelae-unclear, lethal, and associated with various symptoms and complications.

4. SARS-CoV-2 caused a world-wide pandemic, taking hundreds of thousands of lives and shutting down the global economy. It has a destructive power like no other.

Judging from the evidence that we and others have gathered, we believe that finding the origin of SARS-CoV-2 should involve an independent audit of the WIV P4 laboratories and the laboratories of their close collaborators. Such an investigation should have taken place long ago and should not be delayed any further.

We also note that in the publication of the chimeric virus SHC015-MA15 in 2015, the attribution of funding of Zhengli Shi by the NIAID was initially left out. It was reinstated in the publication in 2016 in a corrigendum, perhaps after the meeting in January 2016 to reinstate NIH funding for gain-of-function research on viruses. This is an unusual scientific behavior, which needs an explanation for.

What is not thoroughly described in this report is the various evidence indicating that several coronaviruses recently published (RaTG1318, RmYN0230, and several pangolin coronaviruses27-29,31) are highly suspicious and likely fraudulent. These fabrications would serve no purpose other than to deceive the scientific community and the general public so that the true identity of SARS-CoV-2 is hidden.

Although exclusion of details of such evidence does not alter the conclusion of the current report, we do believe that these details would provide additional support for our contention that SARS-CoV-2 is a laboratory-enhanced virus and a product of gain-of-function research. A follow-up report focusing on such additional evidence is now being prepared and will be submitted shortly.

Acknowledgements We would like to thank Daoyu Zhang for sharing with us the findings of mutations in the E proteins in different sub-groups of β coronaviruses. We also thank all the anonymous scientists and other individuals, who have contributed in uncovering various facts associated with the origin of SARS-CoV-2.

References: 1.

2.

3.

4.

Zhan, S.H., Deverman, B.E. & Chan, Y.A. SARS-CoV-2 is well adapted for humans. What does this mean for re-emergence? bioRxiv, https://doi.org/10.1101/2020.05.01.073262 (2020).

Mou, H. et al. Mutations from bat ACE2 orthologs markedly enhance ACE2-Fc neutralization of SARSCoV-2. bioRxiv, https://doi.org/10.1101/2020.06.29.178459 (2020).

Piplani, S., Singh, P.K., Winkler, D.A. & Petrovsky, N. In silico comparison of spike protein-ACE2 binding affinities across species; significance for the possible origin of the SARS-CoV-2 virus. arXiv, arXiv:2005.06199 (2020).

Andersen, K.G., Rambaut, A., Lipkin, W.I., Holmes, E.C. & Garry, R.F. The proximal origin of SARSCoV-2. Nat Med 26, 450-452 (2020).

21

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

Maiti, A.K. On The Origin of SARS-CoV-2 Virus. Preprint (authorea.com), DOI: 10.22541/au.159355977.76503625 (2020).

Lin, X. & Chen, S. Major Concerns on the Identification of Bat Coronavirus Strain RaTG13 and Quality of Related Nature Paper. Preprints, 2020060044 (2020).

Bengston, D. All journal articles evaluating the origin or epidemiology of SARS-CoV-2 that utilize the RaTG13 bat strain genomics are potentially flawed and should be retracted. OSFPreprints, DOI: 10.31219/osf.io/wy89d (2020).

Segreto, R. & Deigin, Y. Is considering a genetic-manipulation origin for SARS-CoV-2 a conspiracy theory that must be censored? Preprint (Researchgate) DOI: 10.13140/RG.2.2.31358.13129/1 (2020).

Rahalkar, M.C. & Bahulikar, R.A. Understanding the Origin of ‘BatCoVRaTG13’, a Virus Closest to SARS-CoV-2. Preprints, 2020050322 (2020).

Robinson, C. Was the COVID-19 virus genetically engineered? (https://gmwatch.org/en/news/latestnews/19383, 2020).

Robinson, C. Another expert challenges assertions that SARS-CoV-2 was not genetically engineered. (https://gmwatch.org/en/news/latest-news/19383, 2020).

Sørensen, B., Dalgleish, A. & Susrud, A. The Evidence which Suggests that This Is No Naturally Evolved Virus. Preprint, https://www.minervanett.no/files/2020/07/13/TheEvidenceNoNaturalEvol.pdf (2020).

Zhang, B. SARS-CoV-2 Could Come from a Lab - A Critique of “The Proximal Origin of SARS-CoV-2” Published in Nature Medicine. (https://www.linkedin.com/pulse/sars-cov-2-could-come-from-labcritique-proximal-origin-billy-zhang?articleId=6651628681431175168#comments6651628681431175168&trk=public_profile_article_view, 2020).

Sirotkin, K. & Sirotkin, D. Might SARS‐CoV‐2 Have Arisen via Serial Passage through an Animal Host or Cell Culture? BioEssays, https://doi.org/10.1002/bies.202000091 (2020).

Seyran, M. et al. Questions concerning the proximal origin of SARS-CoV-2. J Med Virol (2020).

China Honors Ian Lipkin. (https://www.publichealth.columbia.edu/public-health-now/news/china-honorsian-lipkin, 2020).

Holmes, E. Academic CV. (https://www.sydney.edu.au/AcademicProfiles/profile/resource?urlid=edward.holmes&type=cv, 2020).

Zhou, P. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature (2020).

Rahalkar, M. & Bahulikar, R. The Abnormal Nature of the Fecal Swab Sample used for NGS Analysis of RaTG13 Genome Sequence Imposes a Question on the Correctness of the RaTG13 Sequence.

Preprints.org, 2020080205 (2020).

Singla, M., Ahmad, S., Gupta, C. & Sethi, T. De-novo Assembly of RaTG13 Genome Reveals Inconsistencies Further Obscuring SARS-CoV-2 Origins. Preprints, 2020080595 (doi: 10.20944/preprints202008.0595.v1) (2020).

Zhang, D. Anomalies in BatCoV/RaTG13 sequencing and provenance. Preprint (zenodo.org), https://zenodo.org/record/3987503#.Xz9GzC-z3GI (2020).

Robinson, C. Journals censor lab origin theory for SARS-CoV-2. (https://www.gmwatch.org/en/news/latest-news/19475-journals-censor-lab-origin-theory-for-sars-cov-2, 2020).

Scientific evidence and logic behind the claim that the Wuhan coronavirus is man-made. https://nerdhaspower.weebly.com (2020).

Zhang, Y. et al. The ORF8 Protein of SARS-CoV-2 Mediates Immune Evasion through Potently Downregulating MHC-I. bioRxiv, https://doi.org/10.1101/2020.05.24.111823 (2020).

Muth, D. et al. Attenuation of replication by a 29 nucleotide deletion in SARS-coronavirus acquired during the early stages of human-to-human transmission. Sci Rep 8, 15177 (2018).

Schoeman, D. & Fielding, B.C. Coronavirus envelope protein: current knowledge. Virol J 16, 69 (2019).

Lam, T.T. et al. Identifying SARS-CoV-2-related coronaviruses in Malayan pangolins. Nature (2020).

Liu, P. et al. Are pangolins the intermediate host of the 2019 novel coronavirus (SARS-CoV-2)? PLoS Pathog 16, e1008421 (2020).

Xiao, K. et al. Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins. Nature (2020).

22

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

Zhou, H. et al. A Novel Bat Coronavirus Closely Related to SARS-CoV-2 Contains Natural Insertions at the S1/S2 Cleavage Site of the Spike Protein. Curr Biol 30, 2196-2203 e3 (2020).

Zhang, T., Wu, Q. & Zhang, Z. Probable Pangolin Origin of SARS-CoV-2 Associated with the COVID19 Outbreak. Curr Biol 30, 1578 (2020).

Yang, X.L. et al. Isolation and Characterization of a Novel Bat Coronavirus Closely Related to the Direct Progenitor of Severe Acute Respiratory Syndrome Coronavirus. J Virol 90, 3253-6 (2015).

Hu, D. et al. Genomic characterization and infectivity of a novel SARS-like coronavirus in Chinese bats.

Emerg Microbes Infect 7, 154 (2018).

Wang, Y. Preliminary investigation of viruses carried by bats on the southeast coastal area (东南沿海地 区蝙蝠携带病毒的初步调查研究). Master Thesis (2017).

Wu, F. et al. A new coronavirus associated with human respiratory disease in China. Nature 579, 265-269 (2020).

Lab That First Shared Novel Coronavirus Genome Still Shut Down by Chinese Government. Global Biodefense, https://globalbiodefense.com/headlines/chinese-lab-that-first-shared-novel-coronavirusgenome-shut-down/ (2020).

Song, W., Gui, M., Wang, X. & Xiang, Y. Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2. PLoS Pathog 14, e1007236 (2018).

Li, F., Li, W., Farzan, M. & Harrison, S.C. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309, 1864-8 (2005).

Shang, J. et al. Structural basis of receptor recognition by SARS-CoV-2. Nature (2020).

Hassanin, A. The SARS-CoV-2-like virus found in captive pangolins from Guangdong should be better sequenced. bioRxiv, https://doi.org/10.1101/2020.05.07.077016 (2020).

Zhang, D. The Pan-SL-CoV/GD sequences may be from contamination. Preprint (zenodo.org), DOI: 10.5281/zenodo.3885333 (2020).

Chan, Y.A. & Zhan, S.H. Single source of pangolin CoVs with a near identical Spike RBD to SARSCoV-2. bioRxiv, https://doi.org/10.1101/2020.07.07.184374 (2020).

Lee, J. et al. No evidence of coronaviruses or other potentially zoonotic viruses in Sunda pangolins (Manis javanica) entering the wildlife trade via Malaysia. bioRxiv, https://doi.org/10.1101/2020.06.19.158717 (2020).

Becker, M.M. et al. Synthetic recombinant bat SARS-like coronavirus is infectious in cultured cells and in mice. Proc Natl Acad Sci U S A 105, 19944-9 (2008).

Menachery, V.D. et al. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nat Med 21, 1508-13 (2015).

Menachery, V.D. et al. SARS-like WIV1-CoV poised for human emergence. Proc Natl Acad Sci U S A 113, 3048-53 (2016).

Ren, W. et al. Difference in receptor usage between severe acute respiratory syndrome (SARS) coronavirus and SARS-like coronavirus of bat origin. J Virol 82, 1899-907 (2008).

Li, X. et al. Emergence of SARS-CoV-2 through Recombination and Strong Purifying Selection. bioRxiv (2020).

Hou, Y. et al. Angiotensin-converting enzyme 2 (ACE2) proteins of different bat species confer variable susceptibility to SARS-CoV entry. Arch Virol 155, 1563-9 (2010).

Yang, Y. et al. Two Mutations Were Critical for Bat-to-Human Transmission of Middle East Respiratory Syndrome Coronavirus. J Virol 89, 9119-23 (2015).

Luo, C.M. et al. Discovery of Novel Bat Coronaviruses in South China That Use the Same Receptor as Middle East Respiratory Syndrome Coronavirus. J Virol 92(2018).

Cui, J., Li, F. & Shi, Z.L. Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol 17, 181192 (2019).

Wan, Y. et al. Molecular Mechanism for Antibody-Dependent Enhancement of Coronavirus Entry. J Virol 94(2020).

Li, F. Receptor recognition mechanisms of coronaviruses: a decade of structural studies. J Virol 89, 195464 (2015).

Li, F. Structure, Function, and Evolution of Coronavirus Spike Proteins. Annu Rev Virol 3, 237-261 (2016).

23

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

Shang, J. et al. Cell entry mechanisms of SARS-CoV-2. Proc Natl Acad Sci U S A 117, 11727-11734 (2020).

Hoffmann, M., Kleine-Weber, H. & Pohlmann, S. A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells. Mol Cell 78, 779-784 e5 (2020).

Coutard, B. et al. The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antiviral Res 176, 104742 (2020).

Claas, E.C. et al. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus.

Lancet 351, 472-7 (1998).

Watanabe, R. et al. Entry from the cell surface of severe acute respiratory syndrome coronavirus with cleaved S protein as revealed by pseudotype virus bearing cleaved S protein. J Virol 82, 11985-91 (2008).

Belouzard, S., Chu, V.C. & Whittaker, G.R. Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites. Proc Natl Acad Sci U S A 106, 5871-6 (2009).

Kido, H. et al. Role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure. Biochim Biophys Acta 1824, 186-94 (2012).

Sun, X., Tse, L.V., Ferguson, A.D. & Whittaker, G.R. Modifications to the hemagglutinin cleavage site control the virulence of a neurotropic H1N1 influenza virus. J Virol 84, 8683-90 (2010).

Cheng, J. et al. The S2 Subunit of QX-type Infectious Bronchitis Coronavirus Spike Protein Is an Essential Determinant of Neurotropism. Viruses 11(2019).

Ito, T. et al. Generation of a highly pathogenic avian influenza A virus from an avirulent field isolate by passaging in chickens. J Virol 75, 4439-43 (2001).

Canrong Wu, Y.Y., Yang Liu, Peng Zhang, Yali Wang, Hua Li, Qiqi Wang, Yang Xu, Mingxue Li, Mengzhu Zheng, Lixia Chen. Furin, a potential therapeutic target for COVID-19. Preprint (chinaXiv), http://www.chinaxiv.org/abs/202002.00062 (2020).

Zeng, L.P. et al. Bat Severe Acute Respiratory Syndrome-Like Coronavirus WIV1 Encodes an Extra Accessory Protein, ORFX, Involved in Modulation of the Host Immune Response. J Virol 90, 6573-6582 (2016).

Lau, S.Y. et al. Attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction. Emerg Microbes Infect 9, 837-842 (2020).

Liu, Z. et al. Identification of common deletions in the spike protein of SARS-CoV-2. J Virol (2020).

Ge, X.Y. et al. Detection of alpha- and betacoronaviruses in rodents from Yunnan, China. Virol J 14, 98 (2017).

Guan, Y. et al. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 302, 276-8 (2003).

Ge, X.Y. et al. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor.

Nature 503, 535-8 (2013).

Lau, S.K. et al. Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc Natl Acad Sci U S A 102, 14040-5 (2005).

Kam, Y.W. et al. Antibodies against trimeric S glycoprotein protect hamsters against SARS-CoV challenge despite their capacity to mediate FcgammaRII-dependent entry into B cells in vitro. Vaccine 25, 729-40 (2007).

Chan, J.F. et al. Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARS-like disease. Clin Microbiol Rev 28, 465-522 (2015).

Zhou, J., Chu, H., Chan, J.F. & Yuen, K.Y. Middle East respiratory syndrome coronavirus infection: virus-host cell interactions and implications on pathogenesis. Virol J 12, 218 (2015).

Yeung, M.L. et al. MERS coronavirus induces apoptosis in kidney and lung by upregulating Smad7 and FGF2. Nat Microbiol 1, 16004 (2016).

Chu, D.K.W. et al. MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity. Proc Natl Acad Sci U S A 115, 3144-3149 (2018).

Ommeh, S. et al. Genetic Evidence of Middle East Respiratory Syndrome Coronavirus (MERS-Cov) and Widespread Seroprevalence among Camels in Kenya. Virol Sin 33, 484-492 (2018).

Sia, S.F. et al. Pathogenesis and transmission of SARS-CoV-2 in golden hamsters. Nature (2020).

Ren, W. et al. Full-length genome sequences of two SARS-like coronaviruses in horseshoe bats and genetic variation analysis. J Gen Virol 87, 3355-9 (2006).

24

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

99.

100.

101.

102.

103.

104.

105.

106.

107.

Yuan, J. et al. Intraspecies diversity of SARS-like coronaviruses in Rhinolophus sinicus and its implications for the origin of SARS coronaviruses in humans. J Gen Virol 91, 1058-62 (2010).

Ge, X.Y. et al. Coexistence of multiple coronaviruses in several bat colonies in an abandoned mineshaft.

Virol Sin 31, 31-40 (2016).

Hu, B. et al. Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus. PLoS Pathog 13, e1006698 (2017).

Luo, Y. et al. Longitudinal Surveillance of Betacoronaviruses in Fruit Bats in Yunnan Province, China During 2009-2016. Virol Sin 33, 87-95 (2018).

Kuo, L., Godeke, G.J., Raamsman, M.J., Masters, P.S. & Rottier, P.J. Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier. J Virol 74, 1393406 (2000).

Drexler, J.F. et al. Genomic characterization of severe acute respiratory syndrome-related coronavirus in European bats and classification of coronaviruses based on partial RNA-dependent RNA polymerase gene sequences. J Virol 84, 11336-49 (2010).

Agnihothram, S. et al. A mouse model for Betacoronavirus subgroup 2c using a bat coronavirus strain HKU5 variant. mBio 5, e00047-14 (2014).

Johnson, B.A., Graham, R.L. & Menachery, V.D. Viral metagenomics, protein structure, and reverse genetics: Key strategies for investigating coronaviruses. Virology 517, 30-37 (2018).

Wu, K., Peng, G., Wilken, M., Geraghty, R.J. & Li, F. Mechanisms of host receptor adaptation by severe acute respiratory syndrome coronavirus. J Biol Chem 287, 8904-11 (2012).

Follis, K.E., York, J. & Nunberg, J.H. Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry. Virology 350, 358-69 (2006).

Yount, B., Denison, M.R., Weiss, S.R. & Baric, R.S. Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59. J Virol 76, 11065-78 (2002).

Yount, B. et al. Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci U S A 100, 12995-3000 (2003).

Almazan, F. et al. Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis. J Virol 80, 10900-6 (2006).

Scobey, T. et al. Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus. Proc Natl Acad Sci U S A 110, 16157-62 (2013).

Almazan, F., Marquez-Jurado, S., Nogales, A. & Enjuanes, L. Engineering infectious cDNAs of coronavirus as bacterial artificial chromosomes. Methods Mol Biol 1282, 135-52 (2015).

Thao, T.T.N. et al. Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform. Nature (2020).

Oldfield, L.M. et al. Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods. Proc Natl Acad Sci U S A 114, E8885-E8894 (2017).

Vashee, S. et al. Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination. mSphere 2(2017).

Xie, X. et al. An Infectious cDNA Clone of SARS-CoV-2. Cell Host Microbe 27, 841-848 e3 (2020).

Roberts, A. et al. A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice.

PLoS Pathog 3, e5 (2007).

Roberts, A. et al. Animal models and vaccines for SARS-CoV infection. Virus Res 133, 20-32 (2008).

Takayama, K. In Vitro and Animal Models for SARS-CoV-2 research. Trends Pharmacol Sci 41, 513517 (2020).

Wang, Q. hACE2 Transgenic Mouse Model For Coronavirus (COVID-19) Research. The Jackson Laboratory Research Highlight, https://www.jax.org/news-and-insights/2020/february/introducingmouse-model-for-corona-virus# (2020).

Zhang, L. et al. The D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity. bioRxiv, https://doi.org/10.1101/2020.06.12.148726 (2020).

Yurkovetskiy, L. et al. Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant. bioRxiv, https://doi.org/10.1101/2020.07.04.187757 (2020).

Korber, B. et al. Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus. Cell 182, 812-827 e19 (2020).

25

108.

109.

110.

111.

Plante, J.A. et al. Spike mutation D614G alters SARS-CoV-2 fitness and neutralization susceptibility. bioRxiv, https://doi.org/10.1101/2020.09.01.278689 (2020).

Poon, L.L. et al. Recurrent mutations associated with isolation and passage of SARS coronavirus in cells from non-human primates. J Med Virol 76, 435-40 (2005).

Pervushin, K. et al. Structure and inhibition of the SARS coronavirus envelope protein ion channel. PLoS Pathog 5, e1000511 (2009).

Nieto-Torres, J.L. et al. Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis. PLoS Pathog 10, e1004077 (2014).

26

Chlorine Dioxide Is a Size-Selective Antimicrobial Agent Zoltán Noszticzius1, Maria Wittmann1*, Kristóf Kály-Kullai1, Zoltán Beregvári2, István Kiss3, László Rosivall4, János Szegedi2 1 Department of Physics, Budapest University of Technology and Economics, Budapest, Hungary, 2 Jósa András Hospital, Nyíregyháza, Hungary, 3 St. Imre Hospital, Budapest, Hungary, 4 Semmelweis University, Budapest, Hungary

Abstract Background / Aims: ClO2, the so-called “ideal biocide”, could also be applied as an antiseptic if it was understood why the solution killing microbes rapidly does not cause any harm to humans or to animals. Our aim was to find the source of that selectivity by studying its reaction-diffusion mechanism both theoretically and experimentally.

Methods: ClO2 permeation measurements through protein membranes were performed and the time delay of ClO2 transport due to reaction and diffusion was determined. To calculate ClO2 penetration depths and estimate bacterial killing times, approximate solutions of the reaction-diffusion equation were derived. In these calculations evaporation rates of ClO2 were also measured and taken into account.

Results: The rate law of the reaction-diffusion model predicts that the killing time is proportional to the square of the characteristic size (e.g. diameter) of a body, thus, small ones will be killed extremely fast. For example, the killing time for a bacterium is on the order of milliseconds in a 300 ppm ClO2 solution. Thus, a few minutes of contact time (limited by the volatility of ClO2) is quite enough to kill all bacteria, but short enough to keep ClO2 penetration into the living tissues of a greater organism safely below 0.1 mm, minimizing cytotoxic effects when applying it as an antiseptic. Additional properties of ClO2, advantageous for an antiseptic, are also discussed. Most importantly, that bacteria are not able to develop resistance against ClO2 as it reacts with biological thiols which play a vital role in all living organisms.

Conclusion: Selectivity of ClO2 between humans and bacteria is based not on their different biochemistry, but on their different size. We hope initiating clinical applications of this promising local antiseptic.

Citation: Noszticzius Z, Wittmann M, Kály-Kullai K, Beregvári Z, Kiss I, et al. (2013) Chlorine Dioxide Is a Size-Selective Antimicrobial Agent . PLoS ONE 8(11): e79157. doi:10.1371/journal.pone.0079157 Editor: Patrick M Schlievert, University of Iowa Carver College of Medicine, United States of America Received June 17, 2013; Accepted September 25, 2013; Published November 5, 2013 Copyright: © 2013 Noszticzius et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by OTKA Grant 77908. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: ZN, MW and KKK declare competing financial interest as they are co-inventors of the European patent 2069232 “Permeation method and apparatus for preparing fluids containing high purity chlorine dioxide”, see also reference 13. In addition ZN is also a founder of the Solumium Ltd. The other four authors have no competing financial interest. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: wittmann@eik.bme.hu

Introduction

When treating local infections of wounds, ulcers or an infected mucous membrane, the application of antiseptics instead of antibiotics is a reasonable alternative especially because bacteria are less able to develop resistance against them [5]. Presently the majority of the antiseptics used for wounds [6] are organic compounds. The most frequently applied ones [6] are chlorhexidine (chlorhexidine digluconate), octenidine (octanidine dihydrochloride), polyhexanide (polyhexametylene biguanide) and triclosan (5-chlorine-2-(2,4dichlorphenoxy)-phenol). Notable exceptions are PVP-iodine (poly(vinylpirrolidone)-iodine complex) [6] where the active ingredient is iodine, and silver [7], both being inorganic compounds.

The emergence and dissemination of new antibiotic-resistant bacterial strains caused by an overuse of antibiotics [1] is a global public-health concern.

Methicillin Resistant Staphylococcus aureus (MRSA) [1,2] and Carbapenem- or Extreme Drug-Resistant Acinetobacter baumannii [3,4] are only two well known examples for such bacteria attracting world wide attention. Moreover, while the number of antibiotic resistant infections is on the rise, the number of new antibiotics is declining [1,2]. As a result of such a dangerous situation, searches for new antimicrobial agents, as well as strategies including a switch from antibiotic to antiseptic therapies, whenever that is feasible, have been initiated.

PLOS ONE | www.plosone.org

1

November 2013 | Volume 8 | Issue 11 | e79157

ClO₂ Is a Size-Selective Antimicrobial Agent

with ClO2, can be calculated. (Details of the reaction-diffusion model and the derivation of formulae estimating the killing time are given in the Information S1.) It was found that the characteristic time necessary to kill a microbe is only a few milliseconds. As ClO2 is a rather volatile compound its contact time (its staying on the treated surface) is limited to a few minutes. While this stay is safely long enough (being at least 3 orders of magnitude longer than the killing time) to inactivate all bacteria on the surface of the organism, it is too short for ClO2 to penetrate deeper than few tenths of a millimetre; thus, it cannot cause any real harm to an organism which is much larger than a bacterium.

In the Discussion part, it is shown that ClO2 can meet the safety and effectiveness requirements for a local antiseptic.

Next, the chemical mechanism of the antiseptic action of ClO2 is discussed and compared with that of hypochlorous and hypoiodous acids (HOCl and HOI) which are „natural” antiseptics. These hypohalous acids are used by neutrophil granulocytes, the most abundant type of white blood cells in mammals, to kill bacteria after phagocytosis. Both hypohalous acids and also ClO2 attack sulfhydryl groups [18,19] which play an essential role in the life processes of all living systems, e.g. in ATP synthesis. That explains why bacteria were not able to develop resistance against HOCl during eons of evolution and why the emergence of ClO2 resistant bacterial strains cannot be expected either. Besides this similarity, however, there are also important dissimilarities among these reagents, e.g. ClO2 is more selective than HOCl. Last of all, circulation in multicellular organisms can provide some additional protection to these organisms against ClO2.

There are some other, less used, inorganic antiseptics such as aqueous sodium hypochlorite (NaOCl), or hydrogen peroxide (H2O2) solutions, or ozone (O3) gas which have some applications in dentistry [8]. These compounds, however, are mainly used as disinfectants because they can be toxic even in low concentrations, a property seriously limiting their antiseptic applications. NaOCl, for example, one of the most commonly used components of irrigating solutions in endodontic practice, can cause poisoning and extensive tissue destruction if it is injected (inadvertently) into periapical tissues in the course of endodontic therapy [9]. H2O2 is also a double edged sword against bacteria as it also hurts living tissue [10]. Moreover, many bacteria are able to resist H2O2 as their catalase enzyme is able to decompose H2O2 rapidly [11]. Thus, beside toxicity, resistance can be also a problem even with the use of inorganic disinfectants [5]. It would be therefore reasonable to choose an antiseptic which would be free of such problems.

We believe that in this respect chlorine dioxide (ClO2) may be the right choice, moreover ClO2 has other characteristic features favourable for antiseptic applications.

In the last twenty or more years chlorine dioxide emerged as a new and popular inorganic disinfectant. It is often referred to as „the ideal biocide” [12] because of its advantageous properties. In spite of that, as far as we know, ClO2 solutions are not frequently used as antiseptic. This is because the available ClO2 solutions were more or less contaminated with other chemicals applied in its synthesis and that contamination formed a major obstacle in medical applications like treating infected wounds, for example. Since 2006, however, with the help of an invention [13], it is relatively easy to produce high purity aqueous ClO2 solutions. These solutions are already commercially available [14] and have been successfully used in dentistry [15] since 2008. Thus, it seems reasonable to ask the question whether the “ideal biocide” in its pure form can also be an “ideal local antiseptic” at the same time.

Such an ideal local antiseptic should satisfy many criteria.

First of all, it should be safe: it should act only locally to avoid the danger of systemic poisoning and should not inflict cytotoxic effects even in the disinfected area. In this respect, it is one of the main aims of the present work to find a reasonable answer for the following intriguing question: how is it possible that contacting or even drinking ClO2 solution is practically harmless for animals [16] and human beings [17], while the same aqueous solution can be a very effective and a rapid killer for bacteria, fungi, and viruses? What is the basis of this unexpected selectivity?

The answer suggested in the Results section is the following: the selectivity between humans or animals and microbes is based not on their different biochemistry, but on their different size. Denominating ClO2 in the title as a „size selective” antimicrobial agent aims to emphasize this new type of selectivity. To reach that conclusion, ClO2 transport was studied experimentally via protein membranes. The results of these experiments were evaluated applying a reaction-diffusion model for the ClO2 transport in a reactive medium to obtain the diffusion coefficient of ClO2, and the concentration of reactive groups in a protein medium. Based on these parameters the killing time, the time needed to flood a bacterium completely

PLOS ONE | www.plosone.org

Methods Materials Reagent grade chemicals were purchased from SigmaAldrich and pork skin gelatine from Fluka (48719). High purity chlorine dioxide solutions were produced according to our invention [13]. Dried pig bladders were purchased in the Great Market Hall of Budapest at the shop “Solvent” (www.solvent.hu). These bladders are usually applied for kulen sausage production.

Physico-chemical methods Measurement of ClO2 permeation through protein membranes. The rate of ClO2 transport was measured with the apparatus shown in Figure 1 through two kinds of protein membranes: gelatin and pig bladder membranes, respectively.

Choosing a membrane geometry for the experiments is advantageous because then the problem is „one dimensional”, the concentration is a function of only one spatial coordinate x, which is perpendicular to the membrane, and the concentration distribution can be given as c=c(x,t).

As Figure 1 shows, the membrane is in a horizontal position and the transport of ClO2 takes place across the membrane bounded by two horizontal planes we denote by x=0 and x=d in our calculations, where d is the thickness of the membrane.

Constant ClO2 concentrations are maintained at both boundaries of the membrane, i.e. we have constant boundary

2

November 2013 | Volume 8 | Issue 11 | e79157

ClO₂ Is a Size-Selective Antimicrobial Agent

of 1 M sulphuric acid, 1 ml of 1 M KI, and 0.5 ml of 0.01 M Na2S2O3 and as an indicator, two drops of 5 % starch solution is also added. When ClO2 enters the upper solution, it oxidizes iodide to iodine, which, in turn, is reduced back to iodide again

by Na2S2O3 as long as thiosulphate is in excess. However, when all thiosulphate is consumed, the intense blue-black colour of the starch-triiodide complex appears suddenly. The time t when the whole solution becomes homogeneously black (the time of the „black burst”) was recorded and another 0.5 ml of Na2S2O3 solution was added with the help of the syringe shown in the Figure. Addition of the thiosulphate eliminated the blue-black colour immediately but, after a certain period, when enough new ClO2 was transported across the membrane, it reappeared again. Then the cycle was repeated starting with the injection of a new 0.5 ml portion of the Na2S2O3 solution.

The results of the measurements were depicted in a V=V(t) diagram where t is the time of the n-th dark burst and V = n×0.5 ml that is the total volume of the thiosulphate solution added before the n-th breakthrough.

The experiments were performed at laboratory temperature 24 ± 2 °C.

Preparation of the gelatin membrane To prepare a mechanically strong membrane, it was reinforced by filter paper and the gelatin was cross-linked with glutaraldehyde. As the cellulose in the filter paper does not react with ClO2 from the point of our experiments, it is an inert material.

10 ml of 10 % aqueous gelatin solution was mixed rapidly with 0.5 ml of 25 % glutaraldehyde solution at room temperature, and a filter paper disk (diameter: 10 cm) was soaked with the mixture. Then the disk was placed between two glass plates covered with polyethylene foils. Spacers were applied to produce a 0.5 mm thick membrane. After a 2 hour setting time the filter paper reinforced gelatin membrane was removed from the form and it was placed into distilled water overnight before the measurements.

Figure 1. Apparatus to measure ClO2 transport through gelatine or pig bladder membranes. The two glass parts of the apparatus are held together by a pair of extension clamps (not shown in the Figure) which are fixed to a support stand by clamp holders. The active cross-section of the membranes is 28 cm2. See text for the working principle. doi: 10.1371/journal.pone.0079157.g001

conditions: c(0,t)=c0 and c(d,t)=0, respectively. There is no ClO2 in the membrane at the start of the experiment, so the initial condition: c(0<x≤d,0)=0 (see Figure S1 in Information S1).

While the lower face of the protein membrane is not in direct contact with the liquid phase, such direct contact would not make any difference regarding the ClO2 transport. This is because the chemical potential of ClO2 in the liquid and the vapour phase is the same due to the equilibrium between the liquid and the vapour phase established by continuous stirring.

Above the protein membrane there is a silicone rubber membrane in order to block the transport of any other chemicals except ClO2. Silicone rubber is highly permeable for chlorine dioxide, but it is practically impermeable for other reagents [13]. This way the ClO2 transport across the test membrane can be measured selectively.

Both protein membranes had a thickness of 0.5 mm and a diameter of 10 cm. The diameter of the active area in the apparatus was 6 cm resulting in an active area of 28 cm2. The volume of the aqueous ClO2 solution was 40 ml and its ClO2 concentration was around 1000 ppm. (The exact value is given at each experiment.)

After crossing the membranes, ClO2 enters the upper aqueous solution which is made by mixing 10 ml of water, 2 ml

PLOS ONE | www.plosone.org

Preparation of the pig bladder membrane For the experiments, membrane disks with 10 cm diameters were cut from commercially available pig bladders and they were kept in distilled water for one day at +4 °C to stabilize their water content. The pig bladder membranes are slightly asymmetric: the surface of one side is smoother than the other.

To obtain reproducible results, the membrane was always fixed in the apparatus with its smoother side facing downwards.

Results Our results cover the following themes: First, we present and evaluate membrane transport experiments aiming to determine i) the diffusion coefficient of ClO2 D in a reactive protein medium, and ii) the concentration of reactive groups s0 in that medium.

To evaluate the membrane transport experiments we applied a reaction-diffusion model for the transport of ClO2 in a medium containing reactive proteins. The details of that theory and the

3

November 2013 | Volume 8 | Issue 11 | e79157

ClO₂ Is a Size-Selective Antimicrobial Agent

can be observed. In the first regime, the amount of the permeated ClO2 is very small, then, after a rapid transition period the cumulated amount of ClO2 increases linearly with time. Real dynamics can be approximated with the following simplified model: zero permeation is assumed at the beginning during a waiting period but right after that a constant diffusion current appears, thus, the permeated amount increases linearly with time. To characterize such a dynamic behaviour the concept of „time lag” can be introduced: it is the time where the asymptote of the linear regime crosses the time axis [21].

Regarding the asymptotes of the corresponding curves, the time lag in the first and in the second experiment is TL1 = 627 s and TL2 = 175 s, respectively. A logical explanation for this difference is that some ClO2 is consumed inside the gelatin in the rapid reaction with methionine and tyrosine. So ClO2 can break through only after it eliminates all these highly reactive amino acid residues. In the case of the second experiment, the breakthrough occurs earlier as most of these residues already reacted with ClO2 during the first experiment.

If we assume that in the second experiment the reaction plays a minor role only, then in that case, the time lag is entirely due to diffusion. Roughly speaking the diffusional time lag is the time necessary to establish a steady state concentration profile inside the membrane that is to “fill up” the membrane with ClO2. Based on dimensional analysis considerations (the dimension of the diffusion coefficient is (length)2/(time) ) we can expect that the time lag should be proportional with the square of the thickness and inversely proportional with the diffusional coefficient. Really, the exact result [21] is that the diffusional time lag TDM for a membrane of thickness d can be calculated as:

Figure 2. Permeation of ClO2 through a gelatin membrane as a function of time t. Each point in the diagram represents a „black burst” (see Methods). V is the cumulative volume of the 0.01 M Na2S2O3 titrant added before the burst and N is the amount of ClO2 permeated until time t. TL1 = 627 s and TL2 = 175 s are time lags of the first and the second experiments, respectively. The concentration of ClO2 source in the magnetically stirred aqueous solution was 1360 ppm (mg/kg) or 20.1 mM. doi: 10.1371/journal.pone.0079157.g002

mathematical derivation of formulas applied in this section are given in the Information S1. Then, based on the experimentally determined D and s0 we calculate TKILL, the time needed to kill bacteria by ClO2, and p, the penetration depth of ClO2 into human tissue during a wound healing treatment.

ClO2 permeation was measured via gelatin and pig bladder membranes. The apparatus is shown in Figure 1 of the Methods section.

1 d2 6 D

T DM = ⋅

Thus, with the assumption TL2 = TDM = 175 s, D, the diffusion coefficient of ClO2 in the gelatin membrane can be calculated knowing that d = 0.5 mm. The result: D = 2.4×10-6 cm2s-1.

D can be determined in another way as well, from the steady state regime. The steady state ClO2 current is the slope of the curve in the linear regime. For the 2nd experiment J2 = 30 nmol/s. Then Fick’s law of diffusion

Permeation of ClO2 through an artificial gelatin membrane Gelatin was our first choice for a model material because we wanted to study the ClO2 transport in a protein medium with a known amino acid composition. Pork skin gelatin (Fluka 48719) contains only two amino acids that can react with ClO2: methionine (0.88 %) and tyrosine (0.6 %) [20].

Figure 2 shows the results of two consecutive experiments performed with the same gelatin membrane (see the two curves denoted as 1st exp. and 2nd exp.). After the first experiment, the membrane was removed from the apparatus and was kept in distilled water for 1 hour before the second experiment.

J = A⋅ D⋅

Δc d

(2)

can be applied to calculate D. Here A= 28.3 cm2 is the active cross-section of the membrane and Δc is the concentration difference between the two sides of the membrane. Regarding our boundary conditions Δc=c0 = 20.1×10-3 M. This way D = 2.6×10-6 cm2s-1 is obtained.

The two D values, the one calculated from the time lag and the other calculated from the steady state, agree reasonably well indicating that indeed the 175 s time lag is caused mostly by diffusion and any delay due to chemical reactions is negligible in the second experiment.

On the other hand, in the first experiment, the time lag TRM is caused mostly by the reaction between ClO2 and the reactive amino acid residues (in short “substrates”) in the membrane. It is important to realize that TRM is not due to a slowness of the

Calculating the ClO2 diffusion coefficient D and the effective concentration of ClO2 consuming substrates s0 in gelatin Figure 2 shows N, the cumulated amount of ClO2 permeated through the membrane as a function of time.

(N was calculated from the titrant volumes V that are given in Table S1 in Information S1. together with the times t of addition.) It is a common feature of both curves shown in Figure 2 that two characteristically different dynamical regimes

PLOS ONE | www.plosone.org

(1)

4

November 2013 | Volume 8 | Issue 11 | e79157

ClO₂ Is a Size-Selective Antimicrobial Agent

All the three measurements (indicated as 1st day, 2nd day and 3rd day) were performed with the same pig bladder membrane but on three successive days. The membrane was kept in distilled water at +4 °C overnight between the experiments which were always started with fresh solutions.

To check the reproducibility of our measurements, we repeated the measurements with another pig bladder membrane (not shown in the Figure). While the new membrane was from a different pig bladder and its blood vessel pattern was also different, the relative deviation between the results of the two series of experiments was surprisingly small: only about 10 %. (The blood vessel structure of the membrane becomes visible as a dark network before a „black burst” because the permeability of the membrane is somewhat higher through those vessels.)

Another interesting observation was that the pig bladder membrane maintained its integrity and its mechanical strength even after the third experiment. This is because ClO2 reacts selectively with certain amino acid residues of the proteins but does not destroy the peptide bonds thus the primary protein structure can survive.

Figure 3. Permeation of ClO2 through a pig bladder membrane as a function of time t. V and N have the same meaning like in Figure 2. TL1 = 2770 s, TL2 = 586 s and TL3 = 226 s are time lags of the experiments performed on the 1st, 2nd, and 3rd day, respectively. The concentration of the ClO2 source was 946 ppm (14.0 mM) in these experiments. doi: 10.1371/journal.pone.0079157.g003

Calculating the ClO2 diffusion coefficient and the effective concentration of ClO2 consuming substrates in pig bladder

reaction kinetics (as the rate constants of the relevant ClO2 – amino acid reactions are relatively high [22–24]), but it is due to the actual ClO2 consumption by the reactions within the membrane delaying the breakthrough. If we assume that the rate of the chemical reaction is limited by the diffusional transport of ClO2 across a zone already without reactive amino acids toward a zone of unreacted ones, then a sharp reaction front will develop on the boundary of the two zones (see Figure S1 in Information S1). The front starting from one side of the membrane and driven by diffusion, propagates slowly through the membrane and TRM is the time when it arrives to the other side of the membrane. According to a detailed derivation in the Information S1, TRM can be given by the so-called parabolic rate law (see equation (S12) in Information S1):

1 s0 d 2 ⋅ 2 c0 D

T RM = ⋅

Evaluation of the results was made in a similar way as in the case of the gelatin membrane. It was assumed that the time lag measured in the third experiment TL3 =226 s is a purely diffusional time lag that is TL3 ≈ TDM. The diffusion coefficient of ClO2 in a pig bladder membrane calculated from the above assumption is D = 1.84×10-6 cm2s-1. That value is in good agreement with the D = 1.80×10-6 cm2s-1 value calculated from the steady state current J3 = 14.1 nmol/s of the 3rd experiment.

As we can see, the diffusion coefficient of ClO2 in a pig bladder tissue is only 30 % smaller than in the unstructured gelatin. This supports our assumption that the cellular structure of the pig bladder tissue does not matter too much from the point of the diffusional transport of ClO2 as it can penetrate through the external and internal lipid membranes of the individual cells of the tissue.

However, there is a more significant deviation between the pig bladder and the gelatin regarding s0, the effective substrate concentration. Assuming that the time lag in the first experiment TL1 = 2770 s is due to the chemical reaction, TL1 ≈ TRM, then from (3) we get s0 = 56 mM, indicating that the concentration of the reactive components in the pig bladder tissue is about two times higher than that is in the gelatin. This is a reasonable result as the animal tissue is denser, and it contains not only methionine and tyrosine like gelatin, but also cysteine and tryptophan residues.

We would like to add that in a series of measurements performed with the same membrane the steady state ClO2 current in the first experiment is always smaller than in the subsequent ones, although the ClO2 source is not changed.

This effect is more pronounced in the case of an animal membrane (compare the slope of the 1st day experiment with that of the other days). The phenomenon can be understood if we assume that some components, which are able to react

(3)

where s0 is the initial effective substrate concentration, i.e. the ClO2 consuming capacity of the membrane in unit volume, and c0 is ClO2 concentration at the boundary of the membrane.

Substituting the more reliable diffusion coefficient measured in the steady-state of the second experiment D = 2.6×10-6 cm2s-1 and applying the assumption that TRM = TL1 = 627 s, the effective substrate concentration of the gelatin membrane s0 can be calculated. The result: s0 = 26.2 mM.

Permeation of ClO2 through a pig bladder membrane In this experiment, we studied the ClO2 permeability of a pig bladder membrane which is a relatively thin (in our case it was 0.5 mm thick) but sturdy animal tissue. The same apparatus was applied as in the case of the gelatin membrane and the experimental points were depicted in Figure 3 also with the same method. (Titrant volumes and the time of addition are given in Table S2 in Information S1.)

PLOS ONE | www.plosone.org

5

November 2013 | Volume 8 | Issue 11 | e79157

ClO₂ Is a Size-Selective Antimicrobial Agent

with ClO2 but only slowly, can remain in the pig bladder even after the first ClO2 breakthrough. As it is shown in equation (S40) in the Information S1, the slow ClO2 consumption of these components can explain a smaller quasi-steady state current. The fact that these components disappear from the membrane after keeping it in water overnight suggests that they are reaction products which can be leached out from the membrane or are unstable intermediates which decompose.

μm we obtain that the killing time for a cylindrical bacterium with a diameter of 1 μm is

Estimating the killing time for bacteria with cylindrical and spherical geometries

As we can see, the killing time for a bacterium is only a few ms due to its small size. Even if s0, the effective substrate concentration of a bacterium would be an order of magnitude higher than we assumed, the killing time would be still less than 0.1 s. Other approximations applied in our calculations can only overestimate the real killing time. For example, the diffusion coefficient of ClO2 in the pig bladder was measured at 24 ± 2 °C. If ClO2 is used to disinfect a living human tissue, the temperature is higher, which means a larger diffusion coefficient and an even shorter killing time. Another approximation is the concept of fixed substrates. Inside a bacterium mobile substrates like glutathione [25], free amino acids and various antioxidants also occur. These small molecules can diffuse by and large freely within the bacterium.

Nevertheless TKILL would still work as a good upper estimate because the mobility of the substrate can only shorten the time needed for ClO2 to reach these substrates and react with them.

Furthermore, when the killing time TKILL is regarded as the time when the sharp front reaches the center of the sphere or the symmetry axis of the cylindrical bacterium, it will surely be overestimated, as it is not necessary to oxidize all the available substrate content of a bacterium to kill it. For example, it is enough to oxidize less than 40 % of the methionine content of E. coli to achieve a 100 % kill [26].

T KILL,C = 4.4 ms, while the killing time for a spherical bacterium with a diameter of 1 μm is

T KILL,S = 2.9 ms.

We assume that a bacterium is killed when its whole volume is flooded by ClO2. To calculate the killing time, if we know the shape and the size of the bacterium, we would need two more parameters, the diffusion coefficient of ClO2 D and the effective concentration of ClO2 consuming substrates s0 in the bacterial medium. In the absence of bacterial data it will be assumed that the parameters D and s0 in the single cell of a bacterium are close to that what we have measured above in the animal cell aggregates of the pig bladder. A further simplifying assumption is that only spherical and cylindrical bacteria are considered. Numerical results are calculated for a diameter of 1 μm, which is a characteristic length-scale for bacteria.

Mathematical formulas for the killing time and the penetration depth are derived in the Information S1. In this section only the results of those derivations will be given together with some qualitative explanations on their meaning.

It will be assumed that the rate of the “ClO2 – bacterium reaction” is also limited by the diffusion of ClO2 to the fast reacting amino acid residues fixed in protein molecules like in the case of the much larger membranes and this way a sharp reaction front propagates from the cell wall toward the centre of the bacterium.

Intuitively, the killing time TKILL should be analogous to the time lag TRM in a membrane caused by a chemical reaction, because these are the times needed to flood the whole volume.

We can expect, however, that the geometric factor should be different depending on the shape of the bacterium. For a cylindrical bacterium with a diameter of d the killing time is

T KILL,C =

1 s0 d 2 ⋅ ⋅ 16 c0 D

Contact time and penetration depth of ClO2 into human skin or wound When an organism is not submerged in the aqueous ClO2 solution but the solution is applied on its surface only, as in the case of disinfecting wounds, the volatility of ClO2 also has to be taken into account. The effective contact time is much shorter using a ClO2 solution than with less or non-volatile disinfectants. According to our measurements, when a wound is covered with 3 wet and 3 dry layers of gauze more than 80 % of ClO2 evaporates from the bandage within one minute due to the high volatility of ClO2 and to the high specific surface of the gauze. Thus, to give an upper limit for the penetration depth into the human tissue, we will assume that the initial ClO2 concentration (c0 = 4.45 mM, Solumium Oral ®) is maintained for 60 s, that is TCON = 60 s, where TCON denotes the contact time.

As a zero-th estimate, we assume again that the human tissue has the same D and s0 values like that of the pig bladder tissue.

Applying the parabolic rate law (see equation (S13) in Information S1 where t = TCON) the penetration depth p can be estimated:

(4)

see equation (S18) in the Information S1, and for a spherical bacterium also with a diameter of d it is

T KILL,S =

1 s0 d 2 ⋅ ⋅ 24 c0 D

(5)

according to equation (S24) in Information S1. We can see that (4) and (5) are analogous to (3) but the geometric factors for a cylinder and for a sphere are much smaller than for the planar membrane indicating that in these geometries the surface from where diffusion current is starting is relatively larger compared to the volume that has to be flooded.

Substituting the pig bladder parameters D = 1.8×10-6 cm2s-1 and s0 = 56 mM into formulas (4,5) together with the ClO2 concentration applied in the wound healing experiments (see later) c0 = 4.45 mM (Solumium Oral ®, 300 ppm) and using d = 1

PLOS ONE | www.plosone.org

p=

6

2c0D⋅T CON s0

(6)

November 2013 | Volume 8 | Issue 11 | e79157

ClO₂ Is a Size-Selective Antimicrobial Agent

p(TCON = 60 s) = 41.5 μm. We remark that (6) can be derived from (3) directly if we realize that for the present problem d = p and TRM = TCON. Nevertheless, the actual penetration depth into a living tissue - either its surface is a wound or an intact human skin – should be even much smaller than the above estimate. This is due to the capillary circulation which is present in living tissue but is absent from dead tissue like the pig bladder membrane used for the measurements. The serum in the blood vessels and also the extracellular fluid contain many components capable of reacting rapidly with ClO2. The fluid transport of these reactive components in the blood capillaries of the dermis [27] can maintain a finite reactant concentration in that region. Then the diffusive transport of these reactants outward from the dermis into the epidermis [27] can halt an inward propagating reaction front establishing a steady state.

Moreover, in the case of intact human skin, ClO2 should permeate through the stratum corneum [28] first, which is the 10–40 μm thick outermost layer of epidermis consisting of several layers of dead cells. This keratinous layer forms a barrier to protect the underlying tissue from infection, dehydration and chemicals. The diffusion coefficient of ClO2 in that layer should be much lower compared to the underlying tissue.

As we can see, the penetration depth into human skin is only few tens of a micrometer even if we neglect circulation. Such shallow penetration cannot really harm human tissues. On the other hand, this short contact time is still several orders of magnitude larger than the killing time, TCON >> TKILL, which is the necessary criterion of a successful disinfection.

ClO2 but their reaction products can be different. The importance of that difference and the protective role of SH groups and of the circulatory system, existing in a multicellular organism only, is also discussed.

Safety and effectiveness requirements for a local antiseptic A local antiseptic should meet the following requirements to be considered as safe: i) it should act only locally to avoid systemic poisoning, and ii) it should not prevent or delay the process of healing, i.e. it should not be cytotoxic. and as effective: iii) it should be effectual in relatively low concentrations, and even in biofilms (biofilms are medically important, accounting for over 80 percent of microbial infections in the body [29]) as well, and iv) microbes should not be able to develop resistance against it (a problem related to the biochemical mechanism of action).

As it was shown in the Results section ClO2 as a size selective antiseptic, meets requirements i) and ii). Thus only criteria iii) and iv) are discussed here.

Comparing the biocidal activity of ClO2 to that of other antiseptics (criterion iii) In free aqueous solutions, the strongest chemical disinfectant is ozone. In biofilms, however, the performance of ozone is rather poor. In addition, ozone is toxic and decomposes in aqueous solutions rapidly. (Its half life is only 15 min at 25 °C at pH 7.) All of these disadvantageous properties of ozone prevent its use as an antiseptic in most applications.

The second strongest disinfectant after ozone is chlorine dioxide. Tanner [30] made a comparative testing of eleven disinfectants on three test organisms (including two bacteria: Staphylococcus aureus and Pseudomonas aeruginosa and one yeast: Saccharomyces cerevisiae). He found that the disinfectant containing ClO2 had the highest biocidal activity on a mg/l basis against the test organisms. Beside antibacterial and antifungal properties, ClO2 also shows strong antiviral activity, about ten times higher than that of sodium hypochlorite [31]. And it inactivates practically all microbes including algae and animal planktons [32] and protozoans [33].

Moreover ClO2 can remove biofilms swiftly [12] because it is highly soluble in water and unlike ozone it does not react with the extracellular polysaccharides of the biofilm. This way ClO2 can penetrate into biofilms rapidly to reach and kill the microbes living within the film.

Therapeutic window The above formulas and calculations indicate that disinfection of living tissues with aqueous ClO2 solutions has a very wide therapeutic window: while surprisingly low concentrations and short contact times are able to kill bacteria, much higher concentrations and residence times are still safe to use.

There is one notable exception: inhaling high concentration ClO2 gases for an extended time can be dangerous for human health because the alveolar membrane is extremely thin (a mere 1-2 microns and in some places even below 1 micron).

The effect of ClO2 in these membranes is somewhat counterbalanced, however, by the intense blood circulation there.

Discussion In this section first we discuss whether ClO2 should be regarded as an “exotic” antiseptic only or it has the promise to become a commonly used antiseptic to treat local infections.

To this end safety and effectiveness requirements for a local antiseptic are collected to check how ClO2 can meet these requirements compared to other antiseptics.

Next a biochemical action mechanism, explaining the antiseptic effect of ClO2 is discussed, which is partly analogous to that of hypochlorous and hypoiodous acids. These “natural” antiseptics also react, among others, with sulfhydryl groups like

PLOS ONE | www.plosone.org

Impossibility of bacterial resistance against ClO2 (criterion iv) ClO2 is a strong, but a rather selective oxidizer. Unlike other oxidants it does not react (or reacts extremely slowly) with most organic compounds of a living tissue. ClO2 reacts rather fast, however, with cysteine [22] and methionine [34] (two sulphur containing amino acids), with tyrosine [23] and tryptophan [24]

7

November 2013 | Volume 8 | Issue 11 | e79157

ClO₂ Is a Size-Selective Antimicrobial Agent

iii) HOCl is a more drastic reagent and causes irreversible damage. For example ClO2 oxidizes glutathione (GSH) mainly to glutathione disulfide (GSSG) [22] which can be reduced back to GSH easily in a natural way in the body. On the other hand, HOCl can attack disulfide bonds and oxidizes GSH mostly to glutathione sulfonamide (GSA) [44] causing an irreversible loss of the cellular GSH.

(two aromatic amino acids) and with two inorganic ions: Fe2+ and Mn2+. It is generally assumed that the antimicrobial effect of ClO2 is due mostly to its reactions with the previously mentioned four amino acids and their residues in proteins and peptides. In the peptide group it is important to mention glutathione – a small tripeptide containing cysteine – which is a major antioxidant in cells, with an intracellular concentration of 0.1-10 mM [35].

Margerum’s group [22–24] reported the following second order rate constants at pH 7 and 25 °C: cysteine 1×107 M-1s-1 >> tyrosine 1.8×105 M-1s-1 > tryptophan 3.4×104 M-1s-1. As can be seen, cysteine is the far most reactive amino acid because of its thiol group. As the above mentioned four amino acids and especially cysteine and biological thiols play a crucial role in all living systems, including microbes, it is impossible for any microbe to develop a resistance against chlorine dioxide.

As an important analogy we can mention that bacteria have never been able to become resistant against hypochlorous acid (HOCl) either, which is an important natural antiseptic used by neutrophils for millions of years. Neutrophils, a type of white blood cells, are phagocytes which kill the engulfed microbes by applying various hydrolytic enzymes and hypohalogeneous acids, chiefly HOCl [36,37]. On that basis Robson and coworkers applied HOCl as a kind of „natural” wound care agent [38,39]. Thus, it is reasonable to compare the action mechanisms and other properties of ClO2 and HOCl as antiseptic agents.

Sulfhydryl groups and circulation can protect multicellular organisms from ClO2 inflicted irreversible damage As it was mentioned, the ClO2 –SH group reaction has the highest rate constant among the ClO2 – amino acid reactions.

(Cysteine or GSH [22] reacts about 50 times faster than the runner up tyrosine.) Consequently, as long as some SH groups are present (mostly in the form of GSH), these groups react with ClO2 rapidly protecting other amino acid residues from oxidative damage. Moreover the oxidation of SH groups to disulfide bonds can be reversed. An interesting example was presented by Müller and Kramer [45,46]. They found that the cytotoxic effect of povidone-iodine after a 30 min contact with murine fibroblast was only temporal: after a 24 hour culture without the antiseptic an unexpected revitalization of the fibroblasts was observed [45]. According to Winterbourn and co-workers [47], HOI (the reactive hydrolysis product of iodine) also oxidises GSH to GSSG but not to GSA. That parallelism between the reversible HOI-GSH and the ClO2-GSH reactions raises the question whether an analogous revitalization might be also possible in the case of ClO2. This question is all the more justified since in some animal experiments [16] rats were drinking water containing 200 ppm ClO2 for 90 days but without developing any gastrointestinal problems. In those experiments all ClO2 must have reacted with the animal tissues as it cannot evaporate from the stomach of the rats. To interpret that result it is reasonable to assume that SH groups transported by the circulation system of the rodent protected the epithelial cells in its gastrointestinal tract from an irreversible oxidation by ClO2.

Above a certain limit, however, when a too high percentage of the protective SH groups is already oxidized, ClO2 would inflict irreversible changes to the higher order protein structures by oxidizing the tyrosine and tryptophan residues [48]. That would certainly happen with the bacteria on the surface of an infected tissue as their GSH supply [26] can be rapidly exhausted by ClO2. Mammalian cells below the surface, however, might survive being supported by the circulation which transports protective sulfhydryl and other reductive compounds to the cells, continuously repairing or even revitalizing them.

Thus beside their size there is another important difference between single cell and more complex multicellular organisms: it is the circulation which can help the cells of a multicellular organism to survive while that type of help is not available for a bacterium.

Comparison of ClO2 and HOCl as possible antiseptic agents HOCl, like ClO2, reacts rapidly with the sulphur containing amino acid residues of methionine and cysteine, the second order rate constant (at pH 7.4 and 22 °C) being 3.8×107 M-1s-1 and 3.0×107 M-1s-1, respectively, and also reacts with tryptophan (1.1×104 M-1s-1) and tyrosine (44 M-1s-1) [40].

However, unlike ClO2, HOCl reacts rapidly with many other amino acid residues and even with peptide bonds [40], and many other compounds such as carbohydrates, lipids, nucleobases, and amines [41].

As we can see the important similarity is the fast reaction of both HOCl and ClO2 with the SH group of cysteine. This is important because it is assumed that abolition of ATP synthesis and killing bacteria by HOCl is due to its reaction with sulfhydryl groups [18]. It is a logical assumption that ClO2 can also stop the ATP synthesis as it reacts with the very same SH groups like HOCl. At the same time, however, there are important dissimilarities between HOCl and ClO2: i) HOCl is much less specific and reacts rapidly with numerous other substrates. Thus killing bacteria with HOCl requires more reagent than with ClO2. ii) While ClO2 evaporates rapidly from its aqueous solution and can reach and kill bacteria even through a gas phase, e.g. through an air bubble blocking a dental root canal [42], evaporation of HOCl is not significant. Thus HOCl stays at the disinfected area for a long time even after killing all bacteria which can cause inflammation there [43].

PLOS ONE | www.plosone.org

Conclusion Chlorine dioxide is a size selective antimicrobial agent which can kill micron sized organisms rapidly but cannot make real

8

November 2013 | Volume 8 | Issue 11 | e79157

ClO₂ Is a Size-Selective Antimicrobial Agent

harm to much larger organisms like animals or humans as it is not able to penetrate deeply into their living tissues. Moreover the circulation of multicellular organisms can provide an additional protection to these organisms against ClO2.

It is an aim of the present work to initiate clinical studies hoping that ClO2 could be applied to treat various local infections, especially where bacterial resistance is a problem.

We have already obtained an official permission [49] to start such studies.

(PDF)

Acknowledgements The authors thank Ms. Marianna Megyesi for performing the physico-chemical experiments and Dr. György Berencsi, Dr. Mihály Kádár, Dr. Márta Milassin, Prof. Ferenc Rozgonyi and Dr. Gusztáv Várnai for helpful discussions. We also thank Ms. Mary-Beth Sinamon for proofreading our MS as a native English speaker.

Supporting Information

Author Contributions

Information S1. This file contains the description of a reaction-diffusion (RD) model for the transport of ClO2 in a medium containing reactive proteins, and its quasi steady state solution when the ClO2 – substrate reaction is fast and when it is slow. Figure S1. Schematic ClO2 and substrate concentration profiles in a hydrogel slab. Table S1. Data depicted in Figure 2 (the cumulative volume V of the 0.01 M Na2S2O3 titrant added until time t). Table S2. Data depicted in Figure 3 (the cumulative volume V of the 0.01 M Na2S2O3 titrant added until time t).

Conceived and designed the experiments: ZN MW KKK ZB IK LR JSz. Performed the experiments: ZN MW KKK. Analyzed the data: ZN MW KKK ZB IK LR JSz. Contributed reagents/ materials/analysis tools: ZN MW KKK. Wrote the manuscript: ZN MW KKK. Evaluated the physico-chemical experiments from the point of medical relevance and future research protocols: ZB IK LR JSz.

References 1. Amyes SGB (2010) Antibacterial Chemotherapy. Theory, Problems and Practice. Oxford: Oxford University Press.

2. Spellberg B (2009) Rising Plague. The Global Threat from Deadly Bacteria and Our Dwindling Arsenal to Fight Them. Amherst, NY: Prometheus Books.

3. Evans BA, Hamouda A, Amyes SGB (2013) The rise of carbapenemresistant Acinetobacter baumannii. Curr Pharm Des 19(2): 223-238. doi:10.2174/138161213804070285. PubMed: 22894617.

4. Luo G, Lin L, Ibrahim AS, Baquir B, Pantapalangkoor P et al. (2012) Active and Passive Immunization Protects against Lethal, Extreme Drug Resistant-Acinetobacter baumannii Infection. PLOS ONE 7(1): e29446. doi:10.1371/journal.pone.0029446. PubMed: 22253723.

5. McDonnell G, Russell AD (1999) Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev 12: 147–179. PubMed: 9880479.

6. Koburger T, Hübner NO, Braun M, Siebert J, Kramer A (2010) Standardized comparison of antiseptic efficacy of triclosan, PVP-iodine, octenidine dihydrochloride, polyhexanide and chlorhexidine digluconate. J Antimicrob Chemother 65: 1712–1719. doi:10.1093/jac/ dkq212. PubMed: 20551215.

7. Lansdown ABG (2010) Silver in Healthcare: Its Antimicrobial Efficacy and Safety in Use. Issues in Toxicology. Cambridge: RSC Publications.

8. Das S (2011) Application of ozone therapy in dentistry. Rev Ind J Dent Adv 3: 538–542.

9. Mehra P, Clancy C, Wu J (2000) Formation of facial hematoma during endodontic therapy. J Am Dent Assoc 131: 67–71. PubMed: 10649874.

10. Cadenas E (1989) Biochemistry of oxygen toxicity. Annu Rev Biochem 58: 79–110. doi:10.1146/annurev.bi.58.070189.000455. PubMed: 2673022.

11. Harris AG, Hinds FE, Beckhouse AG, Kolesnikow T, Hazell SL (2002) Resistance to hydrogen peroxide in Helicobacter pylori: role of catalase. Microbiology 148: 3813–3825. PubMed: 12480885.

12. Simpson GD, Miller RF, Laxton GD, Clements WR (1993) A focus on chlorine dioxide: the "ideal" biocide. Corrosion 93. New Orleans, La, March 8-12. paper No. 472.. Available: http://www.clo2.gr/en/pdf/ secure/chlorinedioxideidealbiocide.pdf. Accessed 2013 April 15.

13. Noszticzius Z, Balogh S, Gyökérné Wittmann M, Kály-Kullai K, Megyesi M et al. (2006) Permeation method and apparatus for preparing fluids containing high purity chlorine dioxide.

Available: http:// patentscope.wipo.int/search/en/WO2008035130 Accessed 2013 April 15.

14. Solumium website. Available: www.solumium.com. Accessed 2013 April 15.

15. Csikány Cs, Várnai G, Noszticzius Z (2009) SOLUMIUM DENTAL: a hipertiszta klórdioxid oldat és alkalmazása a fogorvosi gyakorlatban II.

PLOS ONE | www.plosone.org

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

9

(SOLUMIUM DENTAL: Application of high purity chlorine dioxide solution in the dental practice II., in Hungarian). Dental Hírek 2009/5: 36-38 Daniel FB, Condie LW, Robinson M, Stober JA, York RG et al. (1990) Comparative 90-day subchronic toxicity studies on three drinking water disinfectants, chlorine, monochloramine and chlorine dioxide in the Sprague-Dawley rats. J Am Water Works Assoc 82: 61–69.

Lubbers JR, Chauan SR, Bianchine JR (1982) Controlled clinical evaluations of chlorine dioxide, chlorite and chlorate in man. Environ Health Perspect 46: 57–62. doi:10.1289/ehp.824657. PubMed: 6961033.

Barrette WC Jr, Hannum DM, Wheeler WD, Hurst JK (1989) General mechanism for the bacterial toxicity of hypochlorous acid: abolition of ATP production. Biochemistry 28: 9172–9178. doi:10.1021/ bi00449a032. PubMed: 2557918.

Smit MJ, Anderson R (1992) Biochemical mechanisms of hydrogen peroxide- and hypochlorous acid-mediated inhibition of human mononuclear leukocyte functions in vitro: protection and reversal by anti-oxidants. Agents Actions 36: 58-65. doi:10.1007/BF01991229.

PubMed: 1329447.

Eastoe JE (1955) The amino acid composition of mammalian collagen and gelatin. Biochem J 61: 589–600. PubMed: 13276342.

Crank J (1975) Mathematics of Diffusion. Oxford: Clarendon. 51 pp.

Ison A, Odeh IN, Margerum DW (2006) Kinetics and mechanisms of chlorine dioxide and chlorite oxidations of cysteine and glutathione.

Inorg Chem 45: 8768–8775. doi:10.1021/ic0609554. PubMed: 17029389.

Napolitano MJ, Green BJ, Nicoson JS, Margerum DW (2005) Chlorine dioxide oxidations of tyrosine, N-acetyltyrosine, and Dopa. Chem Res Toxicol 18: 501–508. doi:10.1021/tx049697i. PubMed: 15777090.

Stewart DJ, Napolitano MJ, Bakhmutova-Albert EV, Margerum DW (2008) Kinetics and mechanisms of chlorine dioxide oxidation of tryptophan. Inorg Chem 47: 1639–1647. doi:10.1021/ic701761p.

PubMed: 18254588.

Chesney JA, Eaton JW, Mahoney JR (1996) Bacterial glutathione: a sacrificial defense against chlorine compounds. J Bacteriol 178: 2131– 2135. PubMed: 8606194.

Rosen H, Klebanoff SJ, Wang Y, Brot N, Heinecke JW et al. (2009) Methionine oxidation contributes to bacterial killing by the myeloperoxidase system of neutrophils. Proc Natl Acad Sci U S A 106: 18686–18691. 18688 p. Fig. 3B doi:10.1073/pnas.0909464106.

PubMed: 19833874.

Wikipedia: Skin. Available: http://en.wikipedia.org/wiki/Skin. Accessed 2013 April 15.

November 2013 | Volume 8 | Issue 11 | e79157

ClO₂ Is a Size-Selective Antimicrobial Agent

28. Wikipedia: Stratum corneum. Available: http://en.wikipedia.org/wiki/ Stratum_corneum. Accessed 2013 April 15.

29. NIH Guide: Research on microbial biofilms. Available: http:// grants.nih.gov/grants/guide/pa-files/PA-03-047.html. Accessed 2013 April 15 30. Tanner R (1989) Comparative testing and evaluation of hard-surface disinfectants. J Ind Microbiol 4: 145–154. doi:10.1007/BF01569799.

31. Sanekata T, Fukuda T, Miura T, Morino H, Lee C et al. (2010) Evaluation of the antiviral activity of chlorine dioxide and sodium hypochlorite against feline calicivirus, human influenza virus, measles virus, canine distemper virus, human herpesvirus, human adenovirus, canine adenovirus and canine parvovirus. Biocontrol Sci 15/2: 45–49. doi:10.4265/bio.15.45. PubMed: 20616431.

32. Junli H, Li W, Nenqi R, Li LX, Fun SR et al. (1997) Disinfection effect of chlorine dioxide on viruses, algae and animal planktons in water. Water Res 31: 455–460. doi:10.1016/S0043-1354(96)00276-X. 33. EPA Guidance Manual, Alternative Disinfectants and Oxidants, 4.4.3.2 Protozoa Inactivation.. Available: http://www.epa.gov/ogwdw/mdbp/pdf/ alter/chapt_4.pdf. Accessed 2013 April 15 34. Loginova IV, Rubtsova SA, Kuchin AV (2008) Oxidation by chlorine dioxide of methionine and cysteine derivatives to sulfoxide. Chem Nat Compd 44: 752–754. doi:10.1007/s10600-009-9182-8.

35. Meister A (1988) Glutathione metabolism and its selective modification.

J Biol Chem 263: 17205–17208. PubMed: 3053703.

36. Nauseef WM (2007) How human neutrophils kill and degrade microbes.

An integrated view. Immunol Rev 219: 88–102. doi:10.1111/j.

1600-065X.2007.00550.x. PubMed: 17850484.

37. Rosen H, Klebanoff SJ, Wang Y, Brot N, Heinecke JW et al. (2009) Methionine oxidation contributes to bacterial killing by the myeloperoxidase system of neutrophils. Proc Natl Acad Sci U S A 106: 18686-18691. doi:10.1073/pnas.0909464106. PubMed: 19833874.

38. Wang L, Bassiri M, Najafi R, Najafi K, Yang J et al. (2007) Hypochlorous acid as a potential wound care agent, Part I. J Burns Wounds 6: 65–79.

39. Robson MC, Payne WG, Ko F, Mentis M, Donati G et al. (2007) Hypochlorous acid as a potential wound care agent, Part II. J Burns Wounds 6: 80–90.

40. Pattison DI, Davies MJ (2001) Absolute rate constants for the reaction of hypochlorous acid with protein side chains and peptide bonds. Chem

PLOS ONE | www.plosone.org

41.

42.

43.

44.

45.

46.

47.

48.

49.

10

Res Toxicol 14: 1453–1464. doi:10.1021/tx0155451. PubMed: 11599938.

Pattison DI, Davies MJ (2006) Reaction of myeloperoxidase-derived oxidants with biological substrates: gaining chemical insight into human inflammatory diseases. Curr Med Chem 13: 3271–3190. doi: 10.2174/092986706778773095. PubMed: 17168851.

Hercegh A, Ghidan Á, Friedreich D, Gyurkovics M, Bedő Zs et al.

(2013) Effectiveness of a high purity chlorine dioxide solution in eliminating intercanal Enterococcus faecialis biofim. Acta microbiol Immunol Hung 60(1): 63–75.

Pullar JM, Vissers MCM, Winterbourn CC (2000) Living with a killer: the effects of hypochlorous acid on mammalian cells. IUBMB Life 50: 259– 266. doi:10.1080/15216540051080958. PubMed: 11327319.

Pullar JM, Vissers MCM, Winterbourn CC (2001) Glutathione oxidation by hypochlorous acid in endothelial cells produces glutathione sulfonamide as a major product but not glutathione disulfide. J Biol Chem 276: 22120–22125. doi:10.1074/jbc.M102088200. PubMed: 11283008.

Müller G, Kramer A (2006) Comparative study of in vitro cytotoxicity of povidone-iodine in solution, in ointment or in a liposomal formulation (Repithel) and selected antiseptics.Dermatology 212 Suppl 1: 91-93. doi:10.1159/000090102. PubMed: 16490982.

Müller G, Kramer A (2007) Revitalisierung von Säugerzellen nach Einwirkung von Antiseptika. (Revitalisation of mammalian cells exposed to antiseptics, in German). GMS Krankenhaushyg Interdiszip 2007 2(2):Doc35. Available: http://www.egms.de/static/en/journals/dgkh/ 2007-2/dgkh000068.shtml. Accessed 2013 April 15.

Harwood DT, Kettle AJ, Winterbourn CC (2006) Production of glutathione sulfonamide and dehydroglutathione from GSH by myeloperoxidase-derived oxidants and detection using a novel LC– MS/MS method. Biochem J 399: 161–168. doi:10.1042/BJ20060978.

PubMed: 16846394.

Ogata N (2007) Denaturation of Protein by Chlorine Dioxide: Oxidative Modification of Tryptophane and Tyrosine Residues. Biochemistry 46: 4898-4911. doi:10.1021/bi061827u. PubMed: 17397139.

KLO2-UCD-HU_2010ssued by the Hungarian National Health and Medical Officer Service (ANTSZ) following the suggestion of the Scientific Committee named ETT TUKEB. Available: http://www.ett.hu/ tukeb.htm Accessed 2013 April 15.

November 2013 | Volume 8 | Issue 11 | e79157

International Journal of

Environmental Research and Public Health Article

Efficacy and Safety Evaluation of a Chlorine Dioxide Solution Jui-Wen Ma 1,2 , Bin-Syuan Huang 1 , Chu-Wei Hsu 1 , Chun-Wei Peng 1 , Ming-Long Cheng 1 , Jung-Yie Kao 2 , Tzong-Der Way 2,3,4 , Hao-Chang Yin 1, * and Shan-Shue Wang 5, * 1

2 3 4 5

*

Unique Biotech Co., Ltd., Rm. 1, 22 F, No. 56, Minsheng 1st Road, Xinxing District, Kaohsiung 800, Taiwan; a26154295@gmail.com (J.-W.M.); chiralrecognize@gmail.com (B.-S.H.); wei751001@gmail.com (C.-W.H.); f08220927@gmail.com (C.-W.P.); great.tree00@msa.hinet.net (M.-L.C.) Institute of Biochemistry, College of Life Science, National Chung Hsing University, No. 145 Xingda Road, South District, Taichung 402, Taiwan; biosjyk@gmail.com (J.-Y.K.); tdway@mail.cmu.edu.tw (T.-D.W.) Department of Health and Nutrition Biotechnology, College of Health Science, Asia University, No. 500, Lioufeng Road, Wufeng District, Taichung 41354, Taiwan Department of Biological Science and Technology, College of Biopharmaceutical and Food Sciences, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan Department of Applied Cosmetology/Degree Program of Cosmetology and Health Care, Kao Yuan University, No. 1821, Zhongshan Road, Lujhu District, Kaohsiung 82151, Taiwan Correspondence: lique.unique@gmail.com (H.-C.Y.); sswang168@gmail.com (S.-S.W.); Tel.: +886-7-222-5711 (H.-C.Y.); +886-7-607-7663 (S.-S.W.); Fax: +886-7-222-5719 (H.-C.Y.); +886-7-607-7050 (S.-S.W.)

Academic Editor: Miklas Scholz Received: 24 February 2017; Accepted: 17 March 2017; Published: 22 March 2017

Abstract: In this study, a chlorine dioxide solution (UC-1) composed of chlorine dioxide was produced using an electrolytic method and subsequently purified using a membrane. UC-1 was determined to contain 2000 ppm of gaseous chlorine dioxide in water. The efficacy and safety of UC-1 were evaluated. The antimicrobial activity was more than 98.2% reduction when UC-1 concentrations were 5 and 20 ppm for bacteria and fungi, respectively. The half maximal inhibitory concentrations (IC50 ) of H1N1, influenza virus B/TW/71718/04, and EV71 were 84.65 ± 0.64, 95.91 ± 11.61, and 46.39 ± 1.97 ppm, respectively. A 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test revealed that the cell viability of mouse lung fibroblast L929 cells was 93.7% at a 200 ppm UC-1 concentration that is over that anticipated in routine use. Moreover, 50 ppm UC-1 showed no significant symptoms in a rabbit ocular irritation test. In an inhalation toxicity test, treatment with 20 ppm UC-1 for 24 h showed no abnormality and no mortality in clinical symptoms and normal functioning of the lung and other organs. A ClO2 concentration of up to 40 ppm in drinking water did not show any toxicity in a subchronic oral toxicity test. Herein, UC-1 showed favorable disinfection activity and a higher safety profile tendency than in previous reports.

Keywords: chlorine dioxide (PubChem CID: 24870); antimicrobial efficacy; antiviral assay; inhalation toxicity; subchronic oral toxicity

1. Introduction Chlorine dioxide, a strong oxidant, can inhibit or destroy microbes [1–5]. Studies have investigated the application of chlorine dioxide in numerous fields such as water or wastewater treatment, environment and food disinfection, and medicine [6–13]. Typically, chlorine dioxide is produced using either an acid-based or an electrolytic method [7,8,10,12]. In the acid-based method, chlorine dioxide is produced by mixing starting materials, such as sodium chlorite and hydrochloric acid, sodium chlorite

Int. J. Environ. Res. Public Health 2017, 14, 329; doi:10.3390/ijerph14030329

www.mdpi.com/journal/ijerph

Int. J. Environ. Res. Public Health 2017, 14, 329

2 of 12

and ferric trichloride, or sodium chlorite and chlorine gas. In the electrolytic method, the reactants are aqueous sodium chloride or saturated saline and sodium hypochlorite.

According to the disinfectants and disinfection byproducts rule (DBPR) of the United States Environmental Protection Agency Microbial and Disinfection Byproduct Rules Simultaneous Compliance Guidance Manual [14], the maximum residual disinfectant level goals (MRDLG) and maximum residual disinfectant levels (MRDL) of chlorine dioxide are 0.8 mg/L [14]. The permissible exposure limits (PELs) for chlorine dioxide defined by the Occupational Safety and Health Administration are as follows: (a) General industry: 0.1 ppm and 0.3 mg/m3 ; (b) Construction industry: 0.1 ppm and 0.3 mg/m3 time weighted average (TWA); (c) American Conference of Governmental Industrial Hygienists threshold limit value: 0.1 ppm and 0.28 mg/m3 TWA; 0.3 ppm and 0.83 mg/m3 short term exposure limit (STEL); (d) National Institute for Occupational Safety and Health recommended exposure limit: 0.1 ppm TWA; 0.3 ppm STEL. The application of chlorine dioxide products or their contact with food or the human body is a serious issue if the products contain high levels of impurities. Impurities are mainly caused by impure reactants such as 10% H2 SO4 and 15% NaClO2 or reaction byproducts such as Cl2 and chloroxy anion.

For example, 10% H2 SO4 and 15% NaClO2 contain 90% and 85% unknown impurities, respectively.

The chlorine dioxide product obtained from a mixture of 10% H2 SO4 and 15% NaClO2 is highly impure. The Cl2 product can react with organic matter to form trihalomethane, which is a carcinogen.

Chloroxy anions, such as ClO2 − or ClO3 − , can be harmful to human health [15].

The domestic and industrial use of chlorine dioxide should be assessed according to product purity, for which the preparation method is an essential step. Low purity starting materials (e.g., 5% HCl and 10% NaClO2 ) have a high impurity content. If the product of these reactions is not further purified, then the chlorine dioxide products produced, which also contain high levels of impurities, are useful only for wastewater treatment and are unsuitable for contact with humans or food because of the harmful impurities. Therefore, a higher percentage of chlorine dioxide gas molecules must be obtained through further chlorine dioxide gas molecule purification.

To increase the safety of chlorine dioxide solution, eliminating or reducing the impurities and increasing the gas chlorine dioxide concentration in a solution is a reasonable approach. Herein, a clean and concentrated process for chlorine dioxide gas production was designed and implemented.

We produced a chlorine dioxide solution (UC-1) containing 2000 ppm chlorine dioxide gas in water through the electrolytic method. The solution was further purified with a film membrane, and subsequently dissolved in reverse osmosis (RO) water. UC-1 was investigated to determine its efficacy, and safety issues such as the antimicrobial activity, in vitro cytotoxicity, in vivo rabbit ocular irritation, in vivo inhalation toxicity, and in vivo subchronic oral toxicity were assessed.

2. Materials and Methods 2.1. Electrolytic Method for Gas Chlorine Dioxide Production The UC-1 solution is produced in an apparatus, the technical details of which will be published later in the form of a patent application (PCT applied PCT/CN2016/080198; PCT applied PCT/CN2016/080199; PCT applied PCT/CN2015/099515; DE202016103175) by an electrochemical method. Briefly, sodium chloride solution was made from 99% (food grade) sodium chloride and RO water and pumped into the electrobath equipment. The electrolysis was operated by 6–12 V and 40–80 A current. After electrolysis, the ClO2 gas was mixed with water using a water-ClO2 mixer which was designed based on the Venturi effect. Mixing of water with ClO2 gas was continued by the cycle till the concentration of ClO2 in water was over 2000 ppm (Figure 1) and pH value was 2.2.

The chlorine dioxide solution produced by this process is named as UC-1.

The chemical composition of the UC1 solution was determined according to a standard method [16]. The following data were obtained: ClO2 : 2120 ppm, free chlorine (Cl2 ): 882 ppm, and total chlorine (Cl2 + HOCl + OCl− ): 900 ppm. The total chlorine concentration is somewhat higher

Int. J. Environ. Res. Public Health 2017, 14, 329 Int. J. Environ. Res. Public Health 2017, 14, 329

3 of 12 3 of 12

than in the case of other ClO2 generators because the electrolyte applied by us does not contain any an obvious advantage. At the same time, despite the higher total chlorine content (which is present NaClO The UC-1 solution wasmostly produced by using only 25% NaCl solution, other additive, 2 . diluted in the UC-1 solutions as HOCl), no detectable adverse effects with were no observed on the which is an obvious advantage.

At the same time, despite the higher total chlorine content (which is test animals or animal tissues. present in the diluted UC-1 solutions mostly as HOCl), no detectable adverse effects were observed on the test animals or animal tissues.

Figure 1. Flowchart of chlorine dioxide solution production.

Figure 1. Flowchart of chlorine dioxide solution production.

2.2. Antimicrobial Efficacy Test 2.2. Antimicrobial Efficacy Test The test was performed following U.S. Pharmacopeia 34 NF29 Microbiological Tests/<51> [17].

The test Effectiveness was performed Testing. following The U.S. Pharmacopeia 34 NF29 Antimicrobial test organisms wereMicrobiological as follows: Tests/<51> Escherichia[17]. coli Antimicrobial Effectiveness Testing.

The test organisms were as follows: Escherichia coliaeruginosa (BCRC (BCRC 11634/ATCC 8739), Staphylococcus aureus (BCRC 10451/ATCC 6538P), Pseudomonas 11634/ATCC 8739), Staphylococcus aureus (BCRC 10451/ATCC 6538P), Pseudomonas aeruginosa (BCRC (BCRC 11633/ATCC 9027), S. aureus subsp. aureus (BCRC 15211/ATCC 33591), Bacillus subtilis 11633/ATCC 9027), S. aureus subsp. aureus (BCRC 15211/ATCC 33591), Bacillus subtilis subspecies. subspecies. (BCRC 10447/ATCC 6633), Listeria monocytogenes (BCRC 14848/ATCC 19114), (BCRC 10447/ATCC 6633), Listeria monocytogenes (BCRC 14848/ATCC 19114), Acinetobacter baumannii Acinetobacter baumannii (BCRC 10591/ATCC 19606), Salmonella enterica subspecies. (BCRC 12947/ATCC (BCRC 10591/ATCC 19606), Salmonella enterica subspecies. (BCRC 12947/ATCC 13311), Klebsiella 13311), Klebsiella pneumoniae (BCRC 16082/ATCC 4352), Penicillium funiculosum (BCRC 30438/ATCC pneumoniae (BCRC 16082/ATCC 4352), Penicillium funiculosum (BCRC 30438/ATCC 11797), and 11797), andalbicans Candida(BCRC albicans (BCRC 21538/ATCC10231).

Candida 21538/ATCC10231).

2.3. Antiviral Assay 2.3. Antiviral Assay Viruses were amplified in MDCK/RD cells. MDCK/RD cells were cultured in 10% fetal bovine Viruses were amplified in MDCK/RD cells. MDCK/RD cells were cultured in 10% fetal bovine serum Dulbecco’s modified When the thecells cellsreached reached90% 90%confluence, confluence, serum Dulbecco’s modifiedEagle’s Eagle’smedium medium(FBS (FBS DMEM).

DMEM). When they were washed with phosphate-buffered at aamultiplicity multiplicityofofinfection infection they were washed with phosphate-bufferedsaline saline(PBS) (PBS) and and infected infected at ofof ◦ C in a 5% 0.01.

Following the infection, 0% FBS DMEM was added, and the cells were incubated at 35 0.01. Following the infection, 0% FBS DMEM was added, and the cells were incubated at 35 °C in a CO5% for 48for h. 48 h.

2 incubator CO2 incubator 5 A 1-mL cellcell suspension into each each well wellof ofaa6-well 6-wellplate, plate,which whichwas was A 1-mL suspension(6(6××10 105cells) cells) was was loaded loaded into ◦ incubated at at 3737C°Cfor PBSwas wasused used dilute UC-1 to final concentrations of 50, 0, 25, incubated for18–24 18–24 h. h. PBS to to dilute UC-1 to final concentrations of 0, 25, 100,50, ◦ C. Following ppm in in wells reacted with cells andand viruses for 2for min at 37at°C. the reaction, the 100,and and200 200 ppm wells reacted with cells viruses 2 min 37Following the reaction, − 8 − 8 −8 −8 thethe 10 10dilution mixture was was incubated at thetotal totalreaction reactionmixture mixturewas wasdiluted dilutedtoto1010 . Subsequently, . Subsequently, dilution mixture incubated 37 ◦°C cells were fixed with 10%10% formalin for 1 for h and with 0.1% crystal at 37 C for for 48–64 48–64h.h.The The cells were fixed with formalin 1 hstained and stained with 0.1% violet crystal for for 5 min.

The The virus-formed plaque number was counted and compared betweenbetween the test and violet 5 min. virus-formed plaque number was counted and compared thecontrol test and groups.

The antiviral activity is shown as the percentage of virus control = plaques in thethe test control groups. The antiviral activity is shown as the percentage of virus control = plaques in test group/plaques in the control group × 100.

The virus control is defined as infected virus with cells group/plaques in the control group × 100. The virus control is defined as infected virus with cells without testing agent and consideredasas100%.

100%. without thethe testing agent and is is considered

Int. J. Environ. Res. Public Health 2017, 14, 329

4 of 12

2.4. In Vitro Cytotoxicity Test (MTT Assay) Mouse lung fibroblast L929 cells were cultured in complete Eagle minimum essential medium (MEM) and incubated at 37 ◦ C ± 1 ◦ C in 5% ± 1% CO2 . Furthermore, 100 µL of L929 cell suspension (1 × 105 cells/mL) was transferred into each well of a 96-well cell culture plate. The cells were subsequently incubated at 37 ◦ C ± 1 ◦ C for 24 h ± 2 h. The culture medium was replaced with 100 µL of the test solution or blank, positive, or negative control. The test solutions contained 0 (control), 200, 400, 600, and 800 ppm UC-1 in MEM. The blank control medium contained 10% horse serum. The cells were incubated for another 24 h. The cells were treated with the solutions in triplicate. After the MTT solution was added to each well, the plate was incubated for 2 h ± 10 min at 37 ◦ C ± 1 ◦ C. The MTT solution was replaced with 100 µL of dimethyl sulfoxide and subsequently subjected to a microplate reader equipped with a 570-nm filter for colorimetric measurement (reference, 650 nm). The triplicate results of the MTT assay are presented as mean ± standard deviation (SD). Cell viability (%) = optical density of the test group/optical density of the control group × 100.

2.5. White Rabbit Ocular Irritation Test Six 2–3-kg female New Zealand white rabbits were purchased from the Taiwan Livestock Research Institute (Xinhua, Tainan, Taiwan); the rabbits were quarantined and acclimatized before treatment.

The animals were fed ad libitum and maintained at 20–26 ◦ C under 30%–70% humidity. Furthermore, 0.1 mL of 50 ppm UC-1 (test solution) was administered to the left eye of the rabbits, and 0.1 mL of 0.9% normal saline (control solution) was administered to the right eye. Subsequently, the eyelids were held together for 1 s for instillation. Each treatment was repeated three times. Ocular irritations were observed for at the 1st, 24th, 48th, and 72nd hour using an ophthalmoscope (Welch Allyn, Skaneateles Falls, NY, USA). Extended observation was necessary in case of persistent lesions to determine the progression or reversal of the lesions. Ocular irritation scores were based on the system for grading ocular lesions (ISO 10993-10). When more than one animal in the test group showed a positive result at any stage of the observations, the test component was considered an eye irritant and further testing was not required or performed. When only one of the test groups showed a mild or moderate reaction that was equivocal, the procedure was conducted on three additional animals. When more than half of the eyes showed a positive result at any stage of the observation, the test component was considered an eye irritant. A severe reaction in only one animal was considered sufficient to label the test component as an eye irritant.

2.6. Inhalation Toxicity Test Fifteen 4-week-old BALB/c male mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan); they were quarantined and acclimatized before treatment in an animal room at China Medical University, Taiwan. The animals were fed ad libitum and maintained at 20–25 ◦ C and 65%–80% humidity. Five mice were housed in one cage and fed with 0 (PBS) and 10 or 20 ppm UC-1 (test solution), which was administered as mist by using a humidifier in an airtight box for 24 h. The clinical symptoms and body weight of the animals were observed; they were subsequently sacrificed to examine their lung sections and organ weight. The experimental animals were observed, and their clinical symptoms were recorded as abnormality (%), defined as the animals behaving abnormally compared with normal animals, and mortality (%), defined as animal death.

2.6.1. Evaluation of the Organ Weight During the experiment, the animals were immediately dissected on death, and a record was made.

All surviving animals were sacrificed and autopsied to observe their appearance and all organs in the mouth, chest, and cranial and abdominal cavities. Subsequently, the organs, including the liver, adrenal glands, kidneys, and gonads, were removed, weighed, and recorded.

Int. J. Environ. Res. Public Health 2017, 14, 329

5 of 12

2.6.2. Hematoxylin and Eosin Staining of Mouse Lung Sections Tissue sections frozen in the optimal cutting temperature compound were fixed in acetone and chloroform; the sections were immersed in filtered Harris hematoxylin (Leica Biosystems Richmond, Inc., Richmond, IL, USA) for 1 min. The slides were rewashed with Tris-buffered saline and Tween 20 (Biokit Biotechnology Inc., Miaoli, Taiwan), and the sections were counterstained with eosin (Leica Biosystems Richmond, Inc., Richmond, IL, USA) for 1–2 min. The sections were dehydrated in ascending alcohol solutions and cleared with xylene. The prepared slides were examined through light microscopy.

2.7. Subchronic Oral Toxicity Test Twenty-five 4-week-old BALB/c male mice were purchased from the National Laboratory Animal Center; they were quarantined and acclimatized before treatment in an animal room at China Medical University. The animals were fed ad libitum and maintained at 20–25 ◦ C under 65%–80% humidity.

Five mice were housed in one cage and fed 0 (PBS; control), 5, 10, 20, and 40 ppm UC-1 (test solutions) continuously for 90 days. PBS or test solutions fed as drinking water were freshly prepared daily before treatments.

2.7.1. Evaluation of the Clinical Symptoms The experimental animals were observed, and their clinical symptoms were recorded as abnormality (%), defined as the animals behaving abnormally compared with normal animals, and mortality (%), defined as animal death.

2.7.2. Body Weight The body weight of the experimental animals was recorded at treatment initiation and once per week during the experimental period using an electronic balance (AND, FX-2000i, Tokyo, Japan).

2.7.3. Necropsy, Gross Examination, and Organ Weighing During the experiment, the animals were dissected immediately on death, and a record was made. All surviving animals were sacrificed and autopsied to observe their appearance, and all organs in the mouth, chest, and cranial and abdominal cavities were analyzed. Subsequently, the organs, including the liver, adrenal glands, kidneys, and gonads, were removed, weighed, and recorded.

2.8. Statistical Analysis The results were analyzed using SPSS Version 20.0 (IBM Corp., Armonk, NY, USA) with one-way analysis of variance, F-test, and Duncan’s new multiple range test for comparing more than two mean values; results with p < 0.05 indicated significant differences. The results represent at least 3 independent experiments and are shown as the mean ± SD. 2.9. Ethical Statement This research was approved by the China Medical University Laboratory Animal Service Center.

Program Number: 10442699 (for the white rabbit ocular irritation test) and 10442686 (for the inhalation toxicity and subchronic oral toxicity tests).

3. Results In this study, a UC-1 containing 2000 ppm chlorine dioxide in water was produced through the electrolytic method with food-grade salt (99% NaCl) and RO water as the starting reactants.

Subsequently, the chlorine dioxide was purified through a film and dissolved in RO water. Because a chlorine dioxide solution can be directly applied to food or human hygiene or preventative health measures, its safety and efficacy were investigated.

Int. J. Environ. Res. Public Health 2017, 14, 329

6 of 12

Int. J. Environ. Res. Public Health 2017, 14, 329

6 of 12

3.1. Antimicrobial Efficacy Test 3.1. Antimicrobial Efficacy Test The in vitro antimicrobial activity of UC-1 was examined. The in vitro antimicrobial activity Thethan in vitro antimicrobial of UC-1 examined.

The in vitro antimicrobial was was more 98.2% reduction activity for bacteria andwas fungi (Table 1); excellent antimicrobialactivity activity was more than 98.2% reduction for bacteria and fungi (Table 1); excellent antimicrobial activity was observed at low concentrations of 5 and 20 ppm UC-1 for bacteria and fungi, respectively. observed at low concentrations of 5 and 20 ppm UC-1 for bacteria and fungi, respectively.

Table 1. Antimicrobial efficacy of UC-1.

Table 1. Antimicrobial efficacy of UC-1.

Original Original Inoculum Inoculum (CFU/mL) (CFU/mL) c, * c, Escherichia colicoli 2.55 1055 Escherichia * 2.55× × 10 c, * c, Staphylococcus aureus 3.15 × 1055 Staphylococcus aureus * 3.15 × 10 c, Pseudomonas aeruginosa 2.55 1055 Pseudomonas aeruginosa* c,* 2.55× × 10 c, Staphylococcus aureus subsp.

Aureus 2.60 1055 2.60× × 10 Staphylococcus aureus subsp.

Aureus* c,* c, Bacillus subtilis subspecies 3.75 1055 Bacillus subtilis subspecies* c,* 3.75× × 10 c, Listeria monocytogenes 8.20 1055 8.20× × 10 Listeria monocytogenes* c,* c, * Acinetobacter baumannii 5.40 × 1055 5.40 × 10 Acinetobacter baumannii c,* c, * Salmonella enterica subspecies 4.80 × 1055 c, Salmonella enterica subspecies * 4.80 × 10 c, * Klebsiella pneumoniae 9.30 × 1055 c, 9.30 × 10 Klebsiella pneumoniae * ,c∆ 3.70 1055 Penicillium funiculosum 3.70× × 10 Penicillium funiculosum ,cΔ c,∆ 3.20 1055 Candida albicans Candida albicans c,Δ 3.20× × 10

Organisms Organisms

a

Counts of UC-1 at Counts of UC-1 at Contact Time a Contact Time a (CFU/mL) (CFU/mL) <1 <1 <1 <1 <1 <1 <1 <1 3 1.35 ××1010 3 1.35 <1 <1 <1 <1 <1 <1 <1 <1 3 6.70 3 6.70 ××1010 <1 <1

Percent Percent Reductions Reductions(R) (R) b b

>99.9 >99.9 >99.9 >99.9 >99.9 >99.9 >99.9 >99.9 99.6 99.6 >99.9 >99.9 >99.9 >99.9 >99.9 >99.9 >99.9 >99.9 98.2 98.2 >99.9 >99.9

b

The contact time was 1010 min. no significant significantbacteriostasis bacteriostasis fungistasis. a The b Percentreductions contact time was min. Percent reductionsof of <1% <1% represent represent no oror fungistasis.

UC-1 concentrations were 5 and 20 ppm for bacteria and fungi, respectively. * presented as bacteria; ∆ presented as c UC-1 concentrations were 5 and 20 ppm for bacteria and fungi, respectively. * presented as bacteria; Δ presented fungi. CFU: colony-forming unit. as fungi. CFU: colony-forming unit. c

3.2. Antiviral Assay 3.2. Antiviral Assay TheThe antiviral activity of 0, 25, 50, 100, and 200 ppm UC-1 after 2 min of reaction is shown in antiviral activity of 0, 25, 50, 100, and 200 ppm UC-1 after 2 min of reaction is shown in Figure 2.

For H1N1 virusB/TW/71718/04, B/TW/71718/04, 200 ppm had thesignificant most significant Figure 2. For H1N1and and influenza influenza virus 200 ppm UC-1 UC-1 had the most effect effect in inhibiting viral plaque formation.

The half maximal inhibitory concentration (ICH1N1 50 ) of H1N1 in inhibiting viral plaque formation. The half maximal inhibitory concentration (IC50) of was was84.65 84.65 ± 0.64 ppm of influenza B/TW/71718/04 was 95.91ppm. ± 11.61 EV71, ± 0.64 ppm andand thatthat of influenza virusvirus B/TW/71718/04 was 95.91 ± 11.61 For ppm.

EV71, For 50 ppm 50 UC-1 ppm showed UC-1 showed significant inhibition activity, with an IC of 46.39 ± 1.97 ppm at 2 min. significant inhibition activity, with an IC50 of 46.39 50 ± 1.97 ppm at 2 min. The results Theshowing results showing significance (p < 0.05) are Bars presented.

Barsas are plotted as means ± SD. statisticalstatistical significance (p < 0.05) are presented. are plotted means ± SD.

Figure 2. Antiviral efficacy against influenza virus A/WSN/33, influenza virus B/TW/71718/04, and Figure 2. Antiviral efficacy against influenza virus A/WSN/33, influenza virus B/TW/71718/04, enterovirus 71. Bars are plotted as means ± standard deviation (SD). Means with the same letter did and enterovirus 71. Bars are plotted as means ± standard deviation (SD). Means with the same letter not differ significantly at p < 0.05 according to the ANOVA (Analysis of Variance) F-test and Duncan’s did not differ significantly at p < 0.05 according to the ANOVA (Analysis of Variance) F-test and new multiple range test.

Duncan’s new multiple range test.

Int. J. Environ. Res. Public Health 2017, 14, 329

7 of 12

Int. J. Environ. Res. Public Health 2017, 14, 329

7 of 12

3.3. In Vitro Cytotoxicity Test (MTT Assay) 3.3. In Vitro Cytotoxicity Test (MTT Assay) The cytotoxic ppm UC-1 UC-1against againstL929 L929lung lungfibroblast fibroblast cells was The cytotoxiceffect effectofof0,0,200, 200,400, 400,600, 600, and and 800 800 ppm cells was analyzed.

The cell viability was 74.0%–100.0% at UC-1 concentrations below 600 ppm.

L929 analyzed. The cell viability was 74.0%–100.0% at UC-1 concentrations below 600 ppm. L929 cellcell viability was reduced viability was reducedtoto40.3% 40.3%atat800 800ppm ppmUC-1 UC-1 (Figure (Figure 3).

3).

Figure 3. Cytotoxic effects of various UC-1 concentrations on L929 cells.

Figure 3. Cytotoxic effects of various UC-1 concentrations on L929 cells.

3.4. Ocular Irritation Test 3.4. Ocular Irritation Test The cornea, iris, and conjunctivae were evaluated in a rabbit ocular irritation test. The 50 ppm The cornea,induced iris, andneither conjunctivae were evaluated in a rabbit ocular irritation Theat50 ppm UC-1 solution significant clinical signs nor ocular gross changes in thetest. rabbits each UC-1 solution induced neither significant clinical signs nor ocular gross changes in the rabbits at each time point (Table 2). Therefore, single ocular applications with 0.1 mL of 50 ppm UC-1 did not cause time point (Table 2).

Therefore, single ocular applications with 0.1 mL of 50 ppm UC-1 did not cause ocular irritation in rabbits. ocular irritation in rabbits.

Table 2. Grades in the clinical observation of individual rabbits for the ocular irritation test.

Table 2. Grades in the clinical observation of individual rabbits for the ocular irritation test.

Applied Regions

Left Eye

Right Eye

Clinical Observation Items for (Time point/h) Grading Clinical Observation (Time point/h) Items for 1 24 48 72 Animal No. Component Grading 1 0 0 24 72 Cornea 0 0 48 0 0 RB-160114-01Cornea Iris 00 0 0 0 0 0 IrisConjunctivae 0 0 1 0 0 RB-160114-01 Conjunctivae 00 0 10 0 Cornea 0 0 Test RB-160114-03Cornea Iris Left Eye 0 0 0 0 0 0 0 0 Test (50 ppm UC-1) 0 0 IrisConjunctivae 0 0 0 00 0 RB-160114-03 (50 ppm UC-1) Cornea 0 0 Conjunctivae 00 0 0 0 RB-160114-06 Iris 0 0 0 0 Cornea 0 0 0 0 0 0 IrisConjunctivae 0 0 0 00 0 RB-160114-06 Cornea 0 0 Conjunctivae 00 0 00 0 RB-160114-01 Iris 0 0 0 0 Cornea 0 0 0 0 Conjunctivae 0 0 0 0 Iris 0 0 0 0 RB-160114-01 Cornea 0 0 Conjunctivae 00 0 00 0 Control Right Eye RB-160114-03 Iris 0 0 0 0 (0.9% Saline) Cornea 00 0 Control Conjunctivae 0 0 0 0 0 Iris 0 0 0 0 RB-160114-03 (0.9% Saline) Cornea 0 0 0 0 Conjunctivae 0 0 0 0 RB-160114-06 Iris 0 0 0 0 Cornea 00 0 Conjunctivae 0 0 0 0 0 Applied Test Component Regions Test

Animal No.

RB-160114-06

3.5. Inhalation Toxicity Test

Iris Conjunctivae

0 0

0 0

0 0

0 0

In an inhalation toxicity test, we used 0, 10, and 20 ppm UC-1, which was administered as mist 3.5. Inhalation Toxicity Test by using a humidifier in an airtight box containing five mice. The test showed no abnormality and In an inhalation toxicity test, we used 0, 10, and 20 ppm UC-1, which was administered as mist no mortality for the control and test components within 24 h (Table 3). The weights of the heart, liver, by using a humidifier in an airtight box containing five mice. The test showed no abnormality and

Int. J. Environ. Res. Public Health 2017, 14, 329

8 of 12

no mortality for the control and test components within 24 h (Table 3). The weights of the heart, liver, spleen, and kidney of the test group did not differ significantly from those in the control group (Table 4). Hematoxylin and eosin staining of the mice lung sections (Figure 4) showed that 10 and 20 ppm UC-1 did not induce significant clinical signs of changes in the mouse lung cells at 24 h.

Therefore, inhalation of 10 and 20 ppm UC-1 did not cause irritation in the mice.

Int. J. Environ. Res. Public 2017, 14, 329 Table 3.Health Evaluation of

clinical symptoms in the inhalation toxicity test.

8 of 12

spleen, and kidney of the test group did not differ significantly from those in the control group (Table Test Component Animal No. Abnormality Rate (%) Mortality (%) 4). Hematoxylin and eosin staining of the mice lung sections (Figure 4) showed that 10 and 20 ppm Control: 5 signs of changes in the 0 mouse lung cells at 24 0 h. Therefore, UC-1 did not inducePBS significant clinical ppm 0 mice.

0 inhalation 10 of 10 andUC-1 20 ppm UC-1 did 5not cause irritation in the

20 ppm UC-1

5

0

0

of clinical symptoms in the inhalation toxicity test.

Table 3. Evaluation PBS: phosphate-buffered saline.

Test Component

Animal No.

Abnormality Rate (%)

Mortality (%)

Control: PBS 5 0 Table 4. Evaluation of organ weight for 0the inhalation toxicity test.

10 ppm UC-1 20 ppm UC-1

Test Component

5 5

0 0

0 0

PBS: phosphate-buffered Heart (g) Liver (g) saline.

Spleen (g)

Kidney (g)

Control: PBS 0.3 ± 0.1 0.7 ± 0.2 0.5 ± 0.1 0.6 ± 0.1 Table 4. Evaluation of organ weight for the inhalation toxicity test.

10 ppm UC-1 0.4 ± 0.1 0.7 ± 0.2 0.5 ± 0.2 0.6 ± 0.2 Test Component Heart Liver (g) 0.1 Spleen (g) ± 0.2 20 ppm UC-1 0.3 ±(g) 0.2 0.8 ± 0.6(g) ± 0.1 Kidney0.6 Control: PBS

0.3 ± 0.1

0.7 ± 0.2

0.5 ± 0.1

0.6 ± 0.1

Statistical analyses of UC-1 the presented performed at the 10 ppm 0.4 ±data 0.1 were 0.7 ± 0.2 0.5 ±95% 0.2 significance 0.6 ± 0.2level (p < 0.05).

20 ppm UC-1

0.3 ± 0.2

0.8 ± 0.1

0.6 ± 0.1

0.6 ± 0.2

Statistical analyses of the presented data were performed at the 95% significance level (p < 0.05).

Figure 4. Hematoxylin and eosin staining of mouse lung sections in the inhalation toxicity test. The

Figure 4.Scale Hematoxylin and eosin staining of mouse lung sections in the inhalation toxicity test. bar labeled in this figure was 100 μm.

The Scale bar labeled in this figure was 100 µm.

Int. Int. J. Environ.

Res. Public Health J. Environ.

Res. Public Health2017, 2017,14, 14,329 329 Int. J. Environ. Res. Public Health 2017, 14, 329

of 12 9 of9 12 9 of 12

3.6. Subchronic Oral Toxicity Test Subchronic OralToxicity ToxicityTest Test 3.6.3.6.

Subchronic Oral In the subchronic oral toxicity test, 0, 5, 10, 20, and 40 ppm UC-1 was prepared to feed the mice.

In the subchronic oral toxicity test, 0, 5, 10, 20, and 40 ppm UC-1 was prepared to feed the mice.

In the observations subchronic oral toxicity 0, 5, no 10, abnormality 20, and 40 ppm was prepared feed for the the mice.

Clinical of the mice test, showed and UC-1 no mortality after 90todays Clinical observations of the mice showed no abnormality and no mortality after 90 days for the control and test groups 5). Theno mouse weight and wasno not influenced Clinical observations of the (Table mice showed abnormality mortality after(Figure 90 days5).forMoreover, the control control and test groups (Table 5). The mouse weight was not influenced (Figure 5). Moreover, and gross did weight not show anynot pathological (Figure 6). Thenecropsy weights of andnecropsy test groups (Tableexamination 5). The mouse was influencedsymptoms (Figure 5).

Moreover, and necropsy and gross examination did not show any pathological symptoms (Figure 6). The weights of theexamination heart, liver, spleen, kidney the test groups did not (Figure differ significantly compared those gross did notand show any of pathological symptoms 6). The weights of thewith heart, liver, the heart, liver, spleen, and kidney of the test groups did not differ significantly compared with those in the control group (Table 6).groups Therefore, administration of up to 40 ppm UC-1 to mice for 90 days is spleen, kidney of (Table the test6). did administration not differ significantly compared with thosefor in90 the control in theand control group Therefore, of up to 40 ppm UC-1 to mice days is nontoxic. group (Table 6). Therefore, administration of up to 40 ppm UC-1 to mice for 90 days is nontoxic. nontoxic.

Figure 5.

Mouse weighttrend trend chartin in the subchronic subchronic oral toxicity test.

CTL: control.

Figure 5.5.

Mouse oraltoxicity toxicitytest. test.CTL: CTL:control. control.

Figure Mouseweight weight trendchart chart in the the subchronic oral

Figure 6. Observation of mouse lungs and organs in the subchronic oral toxicity test.

Figure 6. Observation of mouse lungs and organs in the subchronic oral toxicity test.

Figure 6. Observation of mouse lungs and organs in the subchronic oral toxicity test.

Table 5. Evaluation of clinical symptoms in the subchronic oral toxicity test.

Table 5. Evaluation of clinical symptoms in the subchronic oral toxicity test.

Table Evaluation of clinical in theRate subchronic oral toxicity Test5.

Component Animal No.symptoms Abnormality (%) Mortality (%)test.

Test Component Control: PBS Control: PBS 5 ppm UC-1 Test Component 5 ppm 10 ppmUC-1 UC-1 Control: PBS 10 20 ppm ppm UC-1 UC-1 20 UC-1 5 ppm UC-1 40 ppm ppm UC-1 40 ppm UC-1 10 ppm UC-1

Animal No. 5 55 Animal No. 55 555 555 55

Abnormality Rate (%) Mortality (%) 0 0 00 00 Abnormality Rate (%) Mortality (%) 00 00 0 00 0 00 00 0 00 0 0 0 0 0

20 ppm UC-1 5 0 0 Table 6. Evaluation of organ weight in the subchronic oral toxicity test.

40 ppm UC-1 0 0 Table 6. Evaluation of5organ weight in the subchronic oral toxicity test.

Test Component Heart (g) Liver (g) Spleen (g) Kidney (g) Test Component Heart (g) Liver (g) Spleen (g) Kidney (g) Control: PBS 0.4 ± 0.1 0.8 ± 0.2 0.5 ± 0.1 0.7 ± 0.1 Table 6. Evaluation of organ weight in the subchronic oral toxicity test.

Control: PBS 0.4 0.8 0.5 0.7 5 ppm UC-1 0.5 ±± 0.1 0.2 0.8 ±± 0.2 0.3 0.6 ±± 0.1 0.1 0.6 ±± 0.1 0.2 5 ppm UC-1 0.5 ± 0.2 0.8 ± 0.3 0.6 ± 0.1 0.6 ± 0.2 10 ppm UC-1 0.4 ± 0.2 0.9 ± 0.1 0.5 ± 0.3 0.7 ± 0.2 10 ppm 0.4 ±± 0.2 0.9 ±± 0.1 0.5 0.7 Test Heart Liver Spleen Kidney 20Component ppm UC-1 UC-1 0.4 0.1 (g) 0.8 0.2(g) 0.5 ±± 0.3 0.1 (g) 0.7 ±± 0.2 0.1 (g) 20 0.4 0.8 0.5 0.7 40 ppm ppm UC-1 UC-1 0.5 ±± 0.1 0.1 0.8 ±± 0.2 0.3 0.5 ±± 0.1 0.1 0.6 ±± 0.1 0.1 Control: PBS 0.4± 0.1 ± 0.1 0.8± ± 0.5±± 0.7± ± 40 ppm UC-1 0.5 0.8 0.30.2 0.5 0.10.1 0.6 0.10.1

Statistical analyses of the presented data were0.8 performed at the 95% significance level (p < 0.05).

5 ppm UC-1 0.5 ± 0.2were ± 0.3 at 0.695% ± 0.1 0.6 ± 0.2 (p Statistical analyses of the presented data performed the significance level < 0.05).

10 ppm UC-1 0.4 ± 0.2 0.9 ± 0.1 0.5 ± 0.3 0.7 ± 0.2 20 ppm UC-1 0.4 ± 0.1 0.8 ± 0.2 0.5 ± 0.1 0.7 ± 0.1 40 ppm UC-1 0.5 ± 0.1 0.8 ± 0.3 0.5 ± 0.1 0.6 ± 0.1 Statistical analyses of the presented data were performed at the 95% significance level (p < 0.05).

Int. J. Environ. Res. Public Health 2017, 14, 329

10 of 12

4. Discussion UC-1 with gas chlorine dioxide and fewer impurities was prepared using a patented green process design, and the efficacy and safety of UC-1 were evaluated. Many studies have reported the potent oxidant and antimicrobial activity of chlorine dioxide in vitro. Recent reports have addressed concerns related to microbial decontamination of food by chlorine dioxide [6,9,18–21]. In these studies, chlorine dioxide was produced using various methods (e.g., 2% NaClO2 with H3 PO4 , 4% Cl2 with 80% NaClO2, and by using an electrogenerator); the antimicrobial activity was more than 2% for 5–75 mg/L ClO2 within 5–30 min. Here, the antimicrobial activity was more than 98.2% reduction at UC-1 concentrations of 5 and 20 ppm for bacteria and fungi, respectively. In the MTT test, the viability of L929 cells was 93.7% at 200 ppm UC-1 that a concentration is over routine use.

No significant symptoms were observed with 50 ppm UC-1 in the ocular irritation test.

No abnormality or mortality was observed in clinical symptoms, lungs, and other organs at 10 ppm or 20 ppm UC-1 in the inhalation toxicity test. Paulet and Desbrousses [22] administered 2.5, 5, and 10 ppm chlorine dioxide to rats and rabbits in 1970; they reported that 2.5 ppm chlorine dioxide had the lowest-observed-adverse-effect level (LOAEL), causing thoracic effects in rats at 7 h/day for 30 days and pulmonary effects in rabbits at 4 h/day for 45 days. Paulet and Desbrousses [23] increased the test concentration to 5, 10, and 15 ppm chlorine dioxide and reduced the dose time to 15 min per dose, 2–4 times per day, for 4 weeks in rats. The results showed a no-observed-averse-effect level (NOAEL) of 5 ppm and an LOAEL of 10 ppm for lung damage.

The mice were fed drinking water containing up to 40 ppm UC-1 for 90 days; the concentration showed no toxicity in the sub-chronic orally toxicity test. Daniel et al. [24] reported the oral exposed toxicity of chlorine dioxide in drinking water administered to Sprague–Dawley rats for 90 days; they used different concentrations of chlorine dioxide (0, 25, 50, 100, and 200 mg/L corresponding to doses of 0, 2, 5, 8, and 15 mg/kg·day). The spleen and liver weight decreased significantly at 25 and 50 mg/L, respectively. They showed nasal lesions caused by 25 mg/L chlorine dioxide vapors in drinking water. In that study, the LOAEL was 25 mg/L. Bercz et al. [25] conducted a similar test in African green monkeys (Cercopithecus aethiops) by using 0, 30, 100, and 200 mg/L chlorine dioxide for 4–6 weeks. Furthermore, 200 mg/L chlorine dioxide caused erythema and ulceration of the oral mucosa after 1 week, and 100 mg/L chlorine dioxide reduced the serum thyroxine (T4) levels after 6 weeks; in that study, the NOAEL was 30 mg/L and LOAEL was 100 mg/L for the oral exposure of the monkeys.

5. Conclusions UC-1 was produced through a green process with clean starting materials and procedures.

UC-1 solution demonstrated satisfactory antibacterial, antifungal, and antiviral activity. Low toxicity was demonstrated through an in vitro cytoxicity test (high IC50 765 ± 18 ppm), 50 ppm ClO2 did not cause eye irradiation in an ocular irritation test, mice did not exhibit abnormality and mortality in a 20 ppm ClO2 inhalation toxicity test, and concentrations of UC-1 up to 40 ppm were nontoxic to mice for 90 days in subchronic oral toxicity test. Therefore, a higher safety profile for UC-1 than those yielded in previous studies was demonstrated.

Acknowledgments: We thank Unique Biotech Co. Ltd. for providing UC-1 solution support. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author Contributions: Hao-Chang Yin and Shan-Shue Wang were involved in research design, discussion, analysis, decision, revision, and funds. Jui-Wen Ma and Bin-Syuan Huang processed the data, implemented most experiments, and wrote the manuscript. Chu-Wei Hsu, Chun-Wei Peng, and Ming-Long Cheng helped execute detailed experiments as well as collect and manage data. Jung-Yie Kao and Tzong-Der Way were technical advisors and study coordinators and provided helpful suggestions for experimental design, novel opinions for the study, and collaboration with other laboratories.

Conflicts of Interest: The authors declare no conflict of interest.

Int. J. Environ. Res. Public Health 2017, 14, 329

11 of 12

References 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

Sanekata, T.; Fukuda, T.; Miura, T.; Morino, H.; Lee, C.; Maeda, K.; Araki, K.; Otake, T.; Kawahata, T.; Shibata, T. Evaluation of the antiviral activity of chlorine dioxide and sodium hypochlorite against feline calicivirus, human influenza virus, measles virus, canine distemper virus, human herpesvirus, human adenovirus, canine adenovirus and canine parvovirus. Biocontrol Sci. 2010, 15, 45–49. [CrossRef] [PubMed] Ogata, N.; Shibata, T. Protective effect of low-concentration chlorine dioxide gas against influenza a virus infection. J. Gen. Virol. 2008, 89, 60–67. [CrossRef] [PubMed] Morino, H.; Fukuda, T.; Miura, T.; Lee, C.; Shibata, T.; Sanekata, T. Inactivation of feline calicivirus, a norovirus surrogate, by chlorine dioxide gas. Biocontrol Sci. 2009, 14, 147–153. [CrossRef] [PubMed] Tanner, R.S. Comparative testing and evaluation of hard-surface disinfectants. J. Ind. Microbiol. 1989, 4, 145–154. [CrossRef] Junli, H.; Li, W.; Nenqi, R.; Li, L.X.; Fun, S.R.; Guanle, Y. Disinfection effect of chlorine dioxide on viruses, algae and animal planktons in water. Water Res. 1997, 31, 455–460. [CrossRef] Kim, J.; Marshall, M.R.; Du, W.X.; Otwell, W.S.; Wei, C.I. Determination of chlorate and chlorite and mutagenicity of seafood treated with aqueous chlorine dioxide. J. Agric. Food Chem. 1999, 47, 3586–3591.

[CrossRef] [PubMed] Hubbard, H.; Poppendieck, D.; Corsi, R.L. Chlorine dioxide reactions with indoor materials during building disinfection: Surface uptake. Environ. Sci. Technol. 2009, 43, 1329–1335. [CrossRef] [PubMed] Yu, C.H.; Huang, T.C.; Chung, C.C.; Huang, H.H.; Chen, H.H. Application of highly purified electrolyzed chlorine dioxide for Tilapia fillet disinfection. Sci. World J. 2014. [CrossRef] [PubMed] Choi, S.; Park, S.; Kim, Y.; Kim, B.S.; Beuchat, L.R.; Hoikyung, K.; Ryu, J.H. Reduction of Salmonella enterica on the surface of eggshells by sequential treatment with aqueous chlorine dioxide and drying. Int. J. Food Microbiol. 2015, 210, 84–87. [CrossRef] [PubMed] Smith, D.J.; Ernst, W.; Herges, G.R. Chloroxyanion residues in cantaloupe and tomatoes after chlorine dioxide gas sanitation. J. Agric. Food Chem. 2015, 63, 9640–9649. [CrossRef] [PubMed] Hsu, C.S.; Chen, I.M.; Liang, C.K.; Shih, C.H. Evaluation of disinfection efficiency in pet’s hospital by using chlorine dioxide. Sustain. Environ. Res. 2016, 26, 191–195. [CrossRef] Hsu, M.S.; Wu, M.Y.; Huang, Y.T.; Liao, C.H. Efficacy of chlorine dioxide disinfection to non-fermentative Gram-negative bacilli and non-tuberculous mycobacteria in a hospital water system. J. Hosp. Infect. 2016, 93, 22–28. [CrossRef] [PubMed] Yeturu, S.K.; Acharya, S.; Urala, A.S.; Pentapati, K.C. Effect of Aloe vera, chlorine dioxide, and chlorhexidine mouth rinses on plaque and gingivitis: A randomized controlled trial. J. Oral Biol. Craniofac. Res. 2016, 6, 54–58. [CrossRef] [PubMed] U.S. Environmental Protection Agency. Microbial and Disinfection Byproduct Rules Simultaneous Compliance Guidance Manual; U.S. Environmental Protection: Washington, DC, USA, 1999.

Couri, D.; Abdel-Rahman, M.S.; Bull, R.J. Toxicological effects of chlorine dioxide, chlorite and chlorate.

Environ. Health Perspect. 1982, 46, 13–17. [CrossRef] [PubMed] Standard Methods for the Examination of Water and Wastewater, 19th ed.; American Public Health Association: Washington, DC, USA; American Water Works Association: Washington, DC, USA; Water Environment Federation: Alexandria, VA, USA, 1995.

U.S. Pharmacopeia. 34 NF29 Microbiological Tests/<51> Antimicrobial Effectiveness Testing; U.S. Pharmacopeial Convention: Rockville, MD, USA. Trinetta, V.; Morgan, M.; Linton, R.; Dermirci, A.; Ngadi, M.O. Chlorine dioxide for microbial decontamination of food. In Microbial Decontamination in the Food Industry; Demirci, A., Ngadi, M.O., Eds.; Woodhead Publishing: Cambridge, UK, 2012; pp. 533–562.

Sorlini, S.; Gialdini, F.; Biasibetti, M.; Collivignarelli, C. Influence of drinking water treatments on chlorine dioxide consumption and chlorite/chlorate formation. Water Res. 2014, 54, 44–52. [CrossRef] [PubMed] Park, S.H.; Kang, D.H. Antimicrobial effect of chlorine dioxide gas against foodborne pathogens under differing conditions of relative humidity. LWT- Food Sci. Technol. 2015, 60, 186–191. [CrossRef] Arango, J.; Rubino, M.; Auras, R.; Gillett, J.; Schilder, A.; Grzesiak, A.L. Evaluation of chlorine dioxide as an antimicrobial against Botrytis cinerea in California strawberries. Food Packag. Shelf Life 2016, 9, 45–54.

[CrossRef]

Int. J. Environ. Res. Public Health 2017, 14, 329

22.

23.

24.

25.

12 of 12

Paulet, G.D.; Desbrousses, S. On the action of ClO2 at low concentrations on laboratory animals.

Arch. Mal. Prof. 1970, 31, 97–106. [PubMed] Paulet, G.D.; Desbrousses, S. Actions of discontinuous exposure to chlorine dioxide (ClO2 ) on the rat.

Arch. Mal. Prof. 1974, 35, 797–804.

Daniel, F.B.; Condie, L.W.; Robinson, M.; Stober, J.A.; York, R.G.; Olson, G.R.; Wang, S.R. Comparative subchronic toxicity studies of three disinfectants. J. Am. Water Works Assn. 1990, 82, 61–69.

Bercz, J.P.; Jones, L.; Garner, L.; Murray, D.; Ludwig, D.A.; Boston, J. Subchronic toxicity of chlorine dioxide and related compounds in drinking water in the nonhuman primate. Environ. Health Perspect. 1982, 46, 47–55. [CrossRef] [PubMed] © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Environmental Health Perspectives Vol. 46, pp. 57-62, 1982, PDF

Kontrollierte klinische Evaluierungen von Chlordioxid, Chlorit und Chlorat am Menschen von Judith R. Lubbers, Sudha Chauan, und Joseph, R. Bianchine1 Um die relative Sicherheit von chronisch verabreichten Chlorwasser-Desinfektionsmitteln beim Menschen zu untersuchen, wurde eine kontrollierte Studie durchgeführt. Die klinische Bewertung wurde in den drei Phasen durchgeführt, die für Arzneimitteluntersuchungen üblich sind. In Phase I, einer Untersuchung zur Erhöhung der Toleranz, wurden die akuten Auswirkungen einer progressiven Erhöhung der Einzeldosen von Chlordesinfektionsmitteln auf normale gesunde erwachsene männliche Freiwillige untersucht. In Phase II wurden die Auswirkungen der täglichen Einnahme der Desinfektionsmittel in einer Konzentration von 5 mg/I. über zwölf aufeinanderfolgende Wochen auf Normalprobanden untersucht. Bei Personen mit einem niedrigen Glukose-6-Phosphat-Dehydrogenase-Spiegel ist zu erwarten, dass sie besonders anfällig für oxidative Bereiche sind; daher wurde in Phase III Chlorit in einer Konzentration von 5 mg/1. zwölf aufeinanderfolgende Wochen lang täglich einer kleinen Gruppe potenziell gefährdeter Personen mit einem Glukose-6-Phosphat-Dehydrogenase-Mangel verabreicht. Die physiologischen folgt bewertet: Auswertung einer Reihe von qualitativen und quantitativen Tests. Die drei Phasen dieser kontrollierten doppelblinden klinischen Bewertung von Chlordioxid und potenziellen Metaboliten in menschlichen männlichen Probanden wurden ereignislos abgeschlossen. Es gab keine offensichtlichen unerwünschten klinischen Folgeerscheinungen, die von einer der teilnehmenden Versuchspersonen oder von dem sie betreuenden Ärzteteam festgestellt wurden. In mehreren Fällen waren statistisch signifikante Trends bei bestimmten biochemischen oder physiologischen Parametern mit der Behandlung verbunden; Keiner dieser Trends wurde jedoch als physiologisch relevant eingestuft. Es ist nicht auszuschließen, dass diese Tendenzen über einen längeren Behandlungszeitraum tatsächlich Anteile von klinischer Bedeutung erreichen. Durch das Fehlen schädlicher physiologischer Reaktionen innerhalb der Grenzen der Studie wurde jedoch die relative Sicherheit der oralen Aufnahme von Chlordioxid und seinen Metaboliten Chlorit und Chlorat nachgewiesen.

Einführung Chlordioxid wird derzeit in den Vereinigten Staaten als Alternative zur Chlorwasseraufbereitung ernsthaft in Betracht gezogen.

Bevor Chlordioxid routinemäßig als Wasserdesinfektionsmittel verwendet werden darf, muss die Sicherheit der oralen Einnahme von Chlordioxid und seinen Nebenprodukten durch den Menschen bewertet werden. Zu diesem Zweck wurde unter der Schirmherrschaft von USEPA HERL #CR805643 eine kontrollierte klinische Bewertung von Chlordioxid, Chlorit und Chlorat durchgeführt.

Die Studie wurde in drei Teilen durchgeführt. Phase I war für die Bewertung der akuten physiologischen Auswirkungen der progressiv ansteigenden Dosen von Desinfektionsmitteln, die normalen gesunden erwachsenen Männern verabreicht werden.

Die chronische Einnahme durch normale männliche Probanden wurde in Phase II untersucht. In Phase III wurde die

1

physiologisch-logische Reaktion einer kleinen Gruppe potenziell anfälliger Personen, die einen Mangel an Glukose-6Phosphatdehydrogenase aufweisen, auf die chronische Einnahme von Chlorit untersucht.

Methoden Themenauswahl Für Phase I und für Phase II wurden normale gesunde erwachsene männliche Freiwillige ausgewählt.

Kein prospektiver Studienteilnehmer, der bei der routinemäßigen klinischen Serumanalyse eine signifikante Anomalie aufwies, wurde ein Blutbild, eine Urinanalyse oder ein Elektrokardiogramm gewählt.

Die Probanden wiesen bei der Voruntersuchung keine körperlichen Abnormalitäten auf, waren 21 bis 35 Jahre alt und wogen nicht mehr oder weniger als ± 10% vom normalen

Die Abteilung für Pharmakologie, Ohio State University, College of Medicine, 338 W. 10th Avenue, Columbus, OH 43210, USA

Körpergewicht für ihren Körperbau und ihre Statur. Eine Krankheitsgeschichte oder eine medizinische oder chirurgische Bedingung, welche die Absorption, Ausscheidung oder den Stoffwechsel von Substanzen durch den Körper beeinträchtigen könnte, schloss eine Aufnahme aus. Die regelmäßige Einnahme von Medikamenten vor Beginn der Untersuchung, sei es zu therapeutischen oder Freizeitzwecken, führte zum Ausschluss aus der Studie.

Normale Methämoglobinspiegel, Schilddrüsenfunktion und Glutathionspiegel waren obligatorisch.

Von jedem Probanden wurde vor Beginn der Behandlung eine schriftliche Einwilligung nach Aufklärung eingeholt.

Für die Phase III wurden die Probanden als Glucose-6Phosphatdehydrogenase (G-6-PD) - defizient definiert, und zwar auf der Grundlage eines Hämoglobin-G-6-PD-Spiegels von weniger als 5,0 IU/GM-Hämoglobin im Screening vor der Studie.

Die Phase-III-Probanden waren in allen anderen Aspekten normal.

WasserdesinfektionsmittelAufbereitung Eine detaillierte Beschreibung der WasserdesinfektionsmittelAufbereitungstechniken wurde von Lubbers und Bianchine 1 vorgelegt. Im Allgemeinen wurden frisch vorgefertigte Stammlösungen von Chlordioxid, Natriumchlorit, Natriumchlorat, Chlor und Chloramin mit den kolorimetrischen Techniken von Palin2 untersucht und dann mit organisch-freiem entmineralisiertem deionisiertem Wasser auf geeignete Konzentrationen verdünnt. Einzelne Flaschen wurden verschlossen und bis zur Verwendung im Dunkeln unter Kühlung gelagert. Alle Flaschen wurden von einem unabhängigen Beobachter kodiert, und die Identität jeder Flasche blieb sowohl für das Untersuchungspersonal als auch für die freiwilligen Versuchspersonen "doppelblind".

Studiendesign: Phase I Die 60 Freiwilligen in Phase I wurden nach dem Zufallsprinzip in sechs Behandlungsgruppen aufgeteilt1. Zehn Personen wurden für jede der Desinfektionsmittel eingeteilt; die zehn Mitglieder der Kontrollgruppe erhielten unbehandeltes Wasser. Die Studie umfasste eine Serie von sechs Sequenzen von jeweils drei Tagen.

Die Behandlungskonzentrationen wurden für jede Behandlung erhöht.

Die spezifischen Konzentrationen oder Desinfektionsmittel, die den Studienteilnehmern verabreicht wurden, sind in Tabelle 1 aufgeführt. Der ersten Behandlung ging eine klinische Auswertung der Entnahme von Blut- und Urinproben zur Bestimmung der Laborgrundwerte der Vorbehandlung voraus. Am ersten Tag jeder dreitägigen Behandlungssequenz nahm jeder Freiwillige 1000 ml des Wassers in zwei Portionen zu sich. Die zweite 500-ml-Portion Aliquot wurde 4 Stunden nach der ersten verabreicht. Jede 500-ml-Portion wurde innerhalb von 15 Minuten verzehrt. Nur am ersten Tag jeder Behandlungssequenz wurden zwei Dosen Desinfektionsmittel verabreicht. Am zweiten und dritten Tag jeder Sequenz wurde kein Desinfektionsmittel verabreicht, da diese

beiden Tage als Nachbeobachtungstage dienen sollten. Der zweite Tag der Behandlungssequenz bestand aus einer körperlichen Untersuchung und der Entnahme von Blut- und Urinproben zur Bestimmung der Laborwerte nach der Behandlung. Am dritten Tag wurde jeder Freiwillige einer körperlichen Untersuchung unterzogen, um die Restwirkung der Behandlung mit den Wasserdesinfektionsmitteln und Nebenprodukten zu bestimmen.

Bei jedem Niveau der Dosierung wurde eine Geschmacksbewertungen erhalten. Die Studienteilnehmer wurden gebeten, die behandelten Wasser als sehr unangenehm, leicht unangenehm, nicht angenehm, angenehm oder geschmacklos zu bewerten.

Studiendesign: Phase II Die sechzig Freiwilligen der Phase II wurden nach dem Zufallsprinzip in sechs Behandlungsgruppen zu je zehn Probanden aufgeteilt3. Um ein effizientes Management der 60 Probanden zu gewährleisten, wurden sie nach dem Zufallsprinzip in drei Untergruppen eingeteilt. Diese Untergruppen wurden nacheinander an drei aufeinanderfolgenden Tagen in die Studie aufgenommen und in ähnlicher Weise wieder aus der Studie entlassen. Für alle Behandlungsgruppen betrug die Konzentration der aufgenommenen Desinfektionsmittel 5 mg/l.

Die Kontrollgruppe erhielt unbehandeltes Wasser. Jeder Proband erhielt 12 Wochen lang 500 ml täglich. Physikalische Untersuchungen, die Entnahme von Blut- und Urinproben für Laboruntersuchungen und geschmackliche Beurteilungen wurden wöchentlich während der Behandlungszeit und für 8 Wochen nach Beendigung der Behandlung durchgeführt.

Studiendesign: Phase III Die drei Probanden mit Glucose-6-phosphat-Dehydrogenmangel der Phase III erhielten Natriumchlorit in einer Konzentration von 5 mg/l Chlorit4. Das Behandlungsprotokoll war identisch mit dem der Phase II, wobei jedem Freiwilligen täglich 500 ml Lösung verabreicht wurden.

Verfahren zur Bewertung Eine umfangreiche Reihe von Parametern wurde überwacht, um die biochemischen und physiologischen Faktoren auf die orale Einnahme von Wasserdesinfektionsmitteln und Nebenprodukten der Wasseraufbereitung zu bewerten (Tabelle 2). Alle Laborbestimmungen der biochemischen Parameter wurden von einem lizenzierten medizinischen Labor, Consolidated Biomedical Laboratories, Inc. (CBL), Columbus, Ohio, HEWLizenznummer 34-1030, durchgeführt. Für jeden Probanden wurden die Ausgangswerte vor der Behandlung und sechs Sätze von Nachbehandlungswerten zusammengestellt. Die Labortests wurden sorgfältig ausgewählt.

Orale Aufnahme von Chlordesinfektionsmitteln beim Menschen Tabelle 1. Konzentration der Desinfektionsmittel in Phase I: akute Toleranz bei steigender Dosis.* Desinfektionsmittelkonzentration, mg/l Desinfektionsmittel für Wasser

Tag 1

Tag 4

Tag 7

Tag 10

Tag 13

Tag 16

Chlorat 0.01

0.01

0.1

0.5

1.0

1.8

2.4

Wasserkontrolle

0

0

0

0

0

0

Chlordioxid

0.1

1.0

5.0

10.0

18.0

24.0

Chlorit

0.01

0. 1

0.5

1.0

1.8

2.4

Chlor

0.1

1.0

5.0

10.0

18.0

24.0

Chloramin

0.01

1.0

8.0

18.0

24.0

*Für jede Dosis wurden zwei Portionen von jeweils 500 ml in Intervallen von 4 Stunden verabreicht.

Tabelle 2. Biochemische Parameter, die bei der kontrollierten klinischen Bewertung von Chlordioxid, Chlorit und Chlorat beim Menschen untersucht wurden.

Serum-Chemie

Plasma-Glukose, Natrium, Kalium, Chlorid, Harnstoff-Stickstoff, Kreatinin, BUN/KreatininVerhältnis, Harnsäure, Kalzium, Phosphor, alkalische Phosphatase, Gamma-GlutamylTransferase, Gesamtbilirubin, Serum-Glutaminsäure-oxalessigsäure-Transaminase, SerumGlutaminsäure-Pyruvat-Transaminase, Milchsäuredehydrogenase, Cholesterin, Triglyceride, Gesamtprotein-Albumin, Globulin, Albumin/Globulin-Verhältnis, Eisen

Blutbild

Thrombozytenzahl, Anzahl der weißen Blutkörperchen, Anzahl der roten Blutkörperchen, Hämoglobin, Hämatokrit, mittleres eorpuskuläres Volumen, mittleres korpuskuläres Volumen, mittleres korpuskuläres Hämoglobin, mittlere korpuskuläre Hämoglobinkonzentration, hohe Peroxidaseaktivität, Neutrophile, Lymphozyten, Monozyten, Eosinophilen, Basophile, große ungefärbte Zellen

Urin Analyse

Farbe*, Aussehen*, spezifisches Gewicht, pH-Wert, Protein, Zucker*, Aceton, Blut*, weißes Blutbild, rotes Blutbild, Abgüsse*, Kristalle*, Bakterien*, Schleim*, amorphe Ce1ls*, Epithelzellen Serum-Haptoglobin, Sichelzelle*, Methämoglobin, Glukose-6-Phosphat-Dehydrogenase, Coombs-Test*, Hämoglobin-Elektrophorese*, T-3 (Aufnahme), T-4 (RIA), freier ThyroxinIndex, Elektrokardiogramm*

Spezielle Tests

Körperliche Untersuchung

systolischer Blutdruck, diastolischer Blutdruck, Atemfrequenz, Pulsfrequenz, Mundtemperatur

* Diese Parameter ergaben nur qualitative Daten; Es wurde keine statistische Analyse durchgeführt.

Auf der Grundlage der Literatur (5-8) wurden Bereiche der zu erwartenden biochemischen Reaktion auf die Einnahme von Chlor Oxidationsmittel definiert; ein Teil der Testreihe wurde speziell der Überwachung dieser Reaktion gewidmet. Die Bildung von Antikörpern an der Oberfläche der roten Blutkörperchen wurde klinisch durch den qualitativen Coombs-Test überwacht; die Schilddrüsenfunktion durch T-3 (Aufnahme), T-4 (RIA) und freies Thyroxin; und die Reaktion auf oxidativen Stress durch Glukose-6-Phosphatdehydrogenase, Methämoglobinund Glutathions-Spiegel. Die Hämoglobin-Elektrophorese wurde zum Nachweis möglicher abnormaler Hämoglobin-Bindungen eingesetzt. Eine Reihe von peripheren Parametern wurde untersucht, um zusätzliche Informationen zu erhalten und die Beurteilung des allgemeinen physiologischen Wohlbefindens zu

unterstützen. Die untersuchten spezifischen Serum-, Blut- und Urinparameter wurden von Lubbers und Bianchine diskutiert1.

Die erhaltenen numerischen Werte wurden gesammelt und analysiert, indem die Einrichtungen der Ohio State University Division of Computing Services for Medical Education and Research genutzt wurden. Speziell entwickelte Programme ermöglichten eine schnelle klinische Rückmeldung. Jeder Wert für einen einzelnen Probanden, der sich vom Gruppenmittelwert um mehr als zwei Standardabweichungen unterschied, wurde notiert. Außerdem wurde jeder einzelne Wert, der außerhalb des normalen Laborbereichs für diesen Parameter lag, als abnormal bezeichnet. Chemische Parameter für Freiwillige, die abnormale Werte aufwiesen, wurden einer sorgfältigen Untersuchung

unterzogen; die Sicherheit und die Möglichkeit einer Überempfindlichkeit gegenüber den Desinfektionsmitteln wurden für jede dieser Personen während der gesamten Studie kontinuierlich bewertet.

Für statistische Analysen wurden kommerziell erhältliche Computerpakete verwendet, insbesondere die Biomedizinischen Computerprogramme (BMDP) und das Statistikpaket für die Sozialwissenschaften (SPSS). Für zweiseitige Varianzanalysen mit wiederholten Messungen wurden die BMDP2V verwendet.

BMDP1R wurde zur Durchführung multipler linearer Regressionsanalysen verwendet. Für paarweise t-Tests und einfache t-Tests wurde der SPSS-T-Test verwendet.

Ergebnisse Qualitative Ein wichtiger Aspekt dieser Studie war die sorgfältige und kontinuierliche medizinische Beobachtung aller Probanden. Die allgemeine klinische Anamnese und die körperliche Untersuchung allein mit subjektiven Beobachtungen und qualitativen Labortests während dieser Studie wurden in der medizinischen Datei jedes Probanden gesammelt. Eine sorgfältige Die Einsichtnahme in jede dieser medizinischen Akten ergab einen Überblick über den allgemeinen klinischen Gesundheitszustand jedes Probanden. Die sorgfältige klinische Bewertung jedes Probanden in den Phasen I, II und III ergab keine klinisch wichtige Auswirkung auf das medizinische Wohlbefinden eines jeden Probanden infolge der Einnahme von Desinfektionsmitteln. Ferner gab es keine offensichtliche Gruppierung der geringfügigen subjektiven Symptome und objektiven Anzeichen, die während der gesamten Studie festgestellt wurden; die episodisch festgestellten "Erkältungen", "Lymphadenopathien", "Halsschmerzen" und "Grippe"-Probleme scheinen zufällig auf die Behandlungsgruppen verteilt zu sein.

Alle Testpersonen blieben während der Untersuchung negativ in Bezug auf die Coombs-Tests und die Sichelzell-Tests. Die Ergebnisse der Hämoglobin-Elektrophorese deuteten darauf hin, dass in Phase II bei einer kleinen Anzahl von Testpersonen abnorme Hämoglobinverteilungen auftraten, aber es zeigte sich, dass diese Personen sowohl in den Behandlungsgruppen als auch in der Kontrollgruppe zufällig verteilt waren.

Die Vitalfunktionen (Blutdruck, Pulsfrequenz, Atemfrequenz und Körpertemperatur) wurden regelmäßig gemessen, um dem überwachenden Arzt eine sofortige Rückmeldung über die akute physiologische Reaktion der Studienteilnehmer auf die Behandlung zu geben. Die statistische Analyse der Vitalfunktionen beschränkte sich auf die Berechnung von arithmetischen Gruppenmitteln und Standardabweichungen vom Mittelwert. Die zusammengestellten Vitalfunktionen wurden auf Anzeichen eines konsistenten Ansprechens auf die Behandlung untersucht. Es wurden keine derartigen Beweise gefunden.

Die subjektiven Bewertungen der Schmackhaftigkeit zeigten, dass nur wenige Probanden fanden, dass die Testsubstanzen bei Konzentrationen von bis zu 24 mg/l einen unangenehmen Geschmack hatten.

Quantitative

Für die Phase-I-Studie zur akuten Steigdosis-Toleranz wurde eine zweiseitige Varianzanalyse mit wiederholten Messungen durchgeführt, um die Werte der Behandlungsgruppe für jeden biochemischen Parameter mit den entsprechenden Werten der Kontrollgruppe zu vergleichen. Die Varianzanalyse ermöglichte eine Unterscheidung zwischen den möglichen Variationsquellen.

Unterschiede zwischen zwei Gruppen, die bereits vor der Behandlung bestanden, parallele Variationen in den quantitativen chemischen Werten aufgrund von Labordrift und authentische behandlungsbedingte Veränderungen der physiologischen Parameter konnten unterschieden werden. Für jeden Fall wurden drei Wahrscheinlichkeiten berechnet: die Gruppen-Hauptwirkung (G), die Zeit-Hauptwirkung (R) und die Gruppen-ZeitWechselwirkung (RG). Die Behandlungsgruppen und die entsprechenden biochemischen Parameter, für die eine starke Wahrscheinlichkeit einer behandlungsbedingten Veränderung berechnet wurde (d.h. RG 0,05), sind in der ersten Spalte von Tabelle 3 aufgeführt.

Um die klinische Bedeutung der statistisch signifikanten Gruppenzeitwechselwirkungen zu bestimmen, wurden die Gruppen-, Mittelwert- und Standardabweichungen vom Mittelwert für den Vorbehandlung Basis Assay und jeden Nachbehandlung Assay für jede der Behandlungsgruppen untersucht. In allen Fällen bleiben die Gruppenmittelwerte deutlich innerhalb der festgelegten Normalbereiche.

Aufgrund des geringen Ausmaßes der Veränderung innerhalb des Normalbereichs und der Dauer der Studie kam man zu dem Schluss, dass die durch die Varianzanalyse identifizierten Trends wahrscheinlich keine klinische Bedeutung haben werden. Die Möglichkeit, dass die Trends mit zunehmender Exposition klinisch wichtig werden, kann nicht ausgeschlossen werden.

Für Phase II wurden alternative statistische Techniken eingesetzt.

Zunächst wurde eine Omnibustesttechnik verwendet. Um die Hypothese zu testen, dass die Reaktion einer oder mehrerer der Gruppen sich von der der übrigen Gruppen unterschied, wurde eine Varianzanalyse mit wiederholten Messungen durchgeführt, in die Werte für alle sechs Behandlungsgruppen einbezogen wurden. Für die Parameter Harnstoff-Stickstoff und mittleres korpuskuläres Hämoglobin wurden RG-Werte < 0,05 erhalten. Es wurden ergänzende Tests durchgeführt. Es wurden Varianzanalysen mit wiederholten Messungen durchgeführt, bei denen die Werte jeder Behandlungsgruppe mit den korpuskulären Ansprechwerten der Kontrollgruppe verglichen wurden. Die Verwendung der Varianzanalyse auf diese Weise ist bei der gemeinsamen Kontrollgruppe fehlerhaft. Die Ergebnisse der Analysen können jedoch mit Vorsicht verwendet werden. Die Varianzanalyse ergab statistisch signifikante RG-Werte beim Vergleich der Gruppenmittelwerte des Korpuskularhämoglobins für die Chlorit- und die Chloratgruppe und der Gruppenmittelwerte des Harnstoffstickstoffs der Chlorat- und Chlordioxid Behandlungsgruppen mit den korrespondierenden Kontrollgruppenwerten, wie in Tabelle 3 dargestellt. Bei der linearen Regressionsanalyse der mittleren korpuskularen Hämoglobinwerte der Chloritgruppe, der Harnstoffstickstoffwerte der Chloratgruppe und der Harnstoffstickstoffwerte der Chloritgruppe wurden keine linearen Trends festgestellt.

Die mittleren korpuskulären Hämoglobinwerte in der ChloratGruppe ergaben bei linearer Regressionsanalyse eine Wahrscheinlichkeit von 0,01. Die relative Steigung, die mit der

Veränderung während der 12-wöchigen Behandlungsperiode verbunden war, betrug etwa 1% des normalen physiologischen Bereichs pro Woche. Wir glauben, dass der Variation keine physiologische Bedeutung mit Sicherheit zugeschrieben werden kann. Auf der Grundlage dieser Studie ist es jedoch unmöglich, die potenzielle physiologische Bedeutung des Trends auszuschließen. Weitere Studien sind gerechtfertigt.

Die geringe Anzahl von Probanden (drei) in Phase III negierte den Wert vieler statistischer Verfahren. Es wurden lineare Regressionsanalysen gewählt. In der dritten Spalte von Tabelle 3 sind die biochemischen Parameter aufgeführt, für die eine hohe Wahrscheinlichkeit einer zeitlichen Veränderung berechnet wurde. Die p-Werte, die berechnet wurden durch die lineare Regressionsanalyse für vier biochemische Parameter weniger als

0,05 betrug. Um die relative Größe der Veränderung zu bestimmen, wurde die prozentuale Veränderung des Normalbereichs pro Woche berechnet. Diese statistischen Analysen zeigen eine gute Wahrscheinlichkeit, dass für das A/GVerhältnis, T-4 (RIA), freies Thyroxin, die mittlere korpuskuläre Hämoglobinkonzentration und die Methämoglobinwerte während der 12-wöchigen Behandlungsdauer eine zeitliche Veränderung auftritt. In Ermangelung einer gleichzeitigen Kontrollgruppe und unter Berücksichtigung der kleinen Gruppengröße und der Möglichkeit einer Labordrift ist jedoch Vorsicht im Umgang mit den Ergebnissen geboten. Wir können mit Zuversicht nur sagen, dass Trends angezeigt wurden. Wir können weder sagen, dass diese Trends physiologischen Ursprungs waren, noch können wir ihnen physiologische Konsequenzen zuschreiben.

Orale Aufnahme von Chlordesinfektionsmitteln beim Menschen Tabelle 3. Biochemische Parameter und Behandlungsgruppen, in denen statistische Analysen eine hohe Änderungswahrscheinlichkeit zeigten, die auf die Einnahme von Desinfektionsmittel zurückzuführen war.

Test

Phase I (a)

Phase II (b)

Harnstoffstickstoff (BUN)

Chlorit

Chlorat Chlordioxid

Kreatinin

Chlorit Chlor Chlorit

BUN / Kreatinin Verhältnis Harnsäure Kalzium Gamma-Glutamyltransferase Gesamt-Bilirubin Albumin / Globulin-Verhältnis Eisen Methämoglobin T-4 (RIA) Freier Thyroxin-Index Mittleres korpuskuläres Hämoglobin Mittlere korpuskuläre Hämoglobinkonzentration Lymphozyten

Phase III (c)

Chlordioxid Chlor Chlor Chlorat Chlorit Chlorat Chlorat

Chlorit Chlorit Chlorit Chlorit Chlorat

Chlor

a) Die Zweiwegevarianzanalyse ergab Gruppen-Zeit-Wechselwirkungen (RG-Werte) < 0,05 bei Vergleichen der Behandlungsgruppenwerte mit die der Kontrollgruppe. b) Die Zwei-Wege-Varianzanalyse ergab Gruppen-Zeit-Wechselwirkungen (RG-Werte) < 0,05 sowohl in der Omnibus- als auch in der Behandlungsgruppenkontrolle Gruppenvergleiche. c) Die lineare Regressionsanalyse zeigte eine starke Wahrscheinlichkeit einer zeitlichen Änderung. p-Werte < 0,05.

Diskussion Mehrere Forscher haben sich mit den physiologisch-logischen Auswirkungen der oralen Einnahme der Oxidationsmittel Chlordioxid, Chlorit und Chlorat befasst. Musil et al. 5 assoziierten die orale Einnahme von Chlorit mit der Bildung von Methämoglobin. In Studien von Heffernan et al. 6,7 Abdel-Rahman et a1.8 und Court et al.9 wurden bei Tieren, die mit Chlorit behandelt wurden, eine hämolytische Anämie und unterdrückte Glutathionspiegel beobachtet. Es wurde gezeigt, dass die orale

Verabreichung von Chlorat an Labortiere oxidativ induziert zu der Zerstörung der Hämoglobin- und Methämoglobinbildung führt10,11. Die Möglichkeit einer renalen Toxizität bei hohen Chlorit-Aufnahmemengen wurde durch das von Heffernan et al. berichtete erhöhte Nieren/Körpergewichtsverhältnis angedeutet 7.

Haller und Northgraves12 und Fridlyand und Kagan13 untersuchten die chronische Toxizität von oral verzehrtem Chlordioxid bei Ratten; es wurde eine leicht erhöhte Zweijahresmortalitätsrate und eine verringerte Gewichtszunahme beobachtet. Die orale Verabreichung von Chlorit14,15,16 an Mäuse erhöhte nachweislich

das mittlere Korpuskularvolumen, die osmotische Fragilität und die Glukose-6-Phosphatdehydrogenase-Aktivität der Erythrozyten; es wurde über morphologische Veränderungen berichtet. Beim Afrikanischen Grünen Meerkatzen beeinträchtigte Chlordioxid die Schilddrüsenfunktion; die Einnahme von Chlorit führte zu vorübergehenden Veränderungen des Hämoglobinspiegels und der Erythrozyten Zahl 17.

Die mütterliche Toxizität, die embryonale Toxizität und das teratogene Potenzial von Natriumchlorit Konzentrationen wurden bei Ratten untersucht18.

Leider sind die verfügbaren Informationen über die Auswirkungen der Einnahme von Chlordioxid, Chlorit und Chlorat beim Menschen sehr begrenzt. Epidemiologische Studien19,20 konnten keine signifikanten expositionsbedingten Auswirkungen eindeutig identifizieren.

Die in diesem Bericht beschriebene klinische Bewertung war ein Versuch, die Wirkungen von Chlorit, Chlordioxid und Chlorat beim Menschen unter kontrollierten klinischen Bedingungen zu klären.

Im Verlauf der dreiphasigen Studie wurde ein riesiges Volumen an Rohdaten erfasst. Routinemäßige Urinuntersuchungen wurden durchgeführt und eine sorgfältige Untersuchung dieser Informationen durchgeführt. In keiner der drei Phasen dieser menschlichen Untersuchung der relativen Sicherheit und Verträglichkeit der oralen Einnahme von Chlordesinfektionsmitteln wurde ein endgültiger Befund über schädliche physiologische Auswirkungen festgestellt. In mehreren Fällen waren statistisch signifikante Trends mit der Behandlung verbunden; Es wurde jedoch festgestellt, dass keiner dieser Trends unmittelbare physiologische Konsequenzen hat.

Man kann nicht ausschließen, dass diese Trends über einen längeren Behandlungszeitraum tatsächlich auftreten könnten und Proportionen von klinischer Bedeutung erreichen. Im Rahmen der Studie liegt jedoch die relative Sicherheit.

Die orale Aufnahme von Chlordioxid und seinen Metaboliten Chlorit und Chlorat wurde durch das Fehlen einer schädlichen physiologischen Reaktion nachgewiesen.

REFERENCES 1

Lubbers, J. R., and Bianchine, J. R. The effects of the acute rising dose administration of chlorine dioxide, chlorate and chlorite to normal healthy adult male volunteers. J. Environ.

Pathol. Toxicol. 5 (2, 3): 865-878 (1982).

2 Palin, A. T. Methods for the determination, in water of free and combined available chlorine, chlorine dioxide and chlorite, bromine, iodine and ozone, using diethyl-p-phenylene diamine (DPD). J. Inst. Water Engr. 21: 537-549 (1976).

3 Lubbers, J. R., Chauhan, S., Miller, J. K., and Bianchine, J. R. The effects of chronic administration of chlorine dioxide, chlorite and chlorate to normal healthy adult male volunteers. J. Environ. Pathol. Toxicol. 5 (2, 3): 879-888 (1982).

4 Lubbers, J. R., Chauhan, S., Miller, J. K. and Bianchine, J. R. The effects of chronic administration of chlorite to glucose-6phosphate dehydrogenase deficient healthy adult male volunteers. J. Environ. Pathol. Toxicol. 5 (2, 3): 889-892 (1982).

5 Musil, J., Kontek, Z., Chalupa, J., and Schmidt, P. Toxicological aspects of chlorine dioxide application for the treatment of water containing phenol. Chem. Technol. Praze. 8: 327-345 (1964).

6 Heffernan, W. P., Guion, C., and Bull, R. J. Oxidative damage to the erythrocyte induced by sodium chlorite in vitro. J. Environ.

Pathol. Toxicol. 2(6): 1487-1499 (1979).

7 Heffernan, W. P., Guion, C., and Bull, R. J. Oxidative damage to the erythrocyte induced by sodium chlorite in vitro. J. Environ.

Pathol. Toxicol. 2(6): 1501-1510 (1979).

8 Abdel-Rhaman, M. S., Couri, D., and Bull, R.J. Kinetics of C102 and effects of C102, and C102, and C103 in drinking water on blood glutathione and hemolysis in rat and chicken. J. Environ.

Path. Toxicol. 3(1,2): 431-449 (1979).

9 Couri, D., and Abdel-Rahman, M.S. Effect of chlorine dioxide and metabolites on glutathione dependent system in rat, mouse and chicken blood. J. Environ. Pathol. Toxicol. 3(1,2): 451-460 (1979).

10

Richardson, A. P. Toxic potentialities of continued administration of chlorate for blood and tissues. J. Pharmacol.

Exptl. Therap. 59: 101-103, (1937).

11 Jung, F., and Kuon, R. Zum inaktiven hemoglobin das Bluter.

Naunyn-Schmiedebergs Arch. Exptl. Pathol. Pharmakol. 216: 103-111 (1951).

12 Haller, S. F., and Northgraves, W.W. Chlorine dioxide and safety. TAPPI 33: 199-202 (1955).

13 Fridyland, S. A., and Kagan, G. Z. Experimental validation of standards for residual chlorine dioxide in drinking water.

Hygiene Sanitation 36: 18-21 (1971).

14 Moore, G. S., and Calabrese, E. J. The effects of chlorine dioxide and sodium chlorite on erythrocytes of A-J and C-57L-J mice. J. Environ. Pathol. Toxicol. 4(2, 3): 513-524 (1980).

15 Moore, G. S. and Calabrese, E. J. G-6-PD-deficiency-a potential high-risk group to copper and chlorite ingestion. J. Environ.

Pathol. Toxicol. 4(2, 3): 271-279 (1980).

16 Moore, G. S., Calabrese, E. J. and Ho, S. C. Groups at potentially high-risk from chlorine dioxide treated water. J. Environ. Pathol. Toxicol. 4(2, 3): 465-470 (1980).

17 Berez, J. P., DiBiasi, D. L., Jones, L., Murray, D., and Boston, J. Subchronic toxicity of alternate disinfectants and related compounds in the non-human primate. Environ. Health Perspect. 46: 47-55 (1982).

18 Couri, D., Miller, C. H., Bull, R. J., Delphia, J. M., and Ammar, E. M. Assessment of maternal toxicity, embryotoxicity and teratogenic potential of sodium chlorite in SpragueDawley rats.

Environ. Health Perspect. 46: 25-29 (1982).

19 Haring, B. J., and Zoetman, B. C. Corrosiveness of drinking water and cardiovascular diesase mortality. Bull. Environ.

Contam. Toxicol. 25: 658-662 (1981).

20 Michael, G. E., Miday, R. K., Bercz, J. P., Miller, R. G., Greathouse, D. G., Kraemer, D. F., and Lucas, J. B. Chlorine dioxide water disinfection: a prospective epidemiology. Arch.

Environ. Health 36(1): 20-27 (1981).

Environmental Health Perspectives Vol. 46, pp. 57-62, 1982

Controlled Clinical Evaluations of Chlorine Dioxide, Chlorite and Chlorate in Man

by Judith R. Lubbers,* Sudha Chauan,* and Joseph R. Bianchine* To assess the relative safety of chronically administered chlorine water disinfectants in man, a controlled study was undertaken. The clinical evaluation was conducted in the three phases common to investigational drug studies. Phase I, a rising does tolerance investigation, examined the acute effects of progressively increasing single doses of chlorine disinfectants to normal healthy adult male volunteers. Phase II considered the impact on normal subjects of daily ingestion of the disinfectants at a concentration of 5 mg/l. for twelve consecutive weeks. Persons with a low level of glucose-6-phosphate dehydrogenase may be expected to be especially susceptible to oxidative stress; therefore, in Phase III, chlorite at a concentration of 5 mg/l. was administered daily for twelve consecutive weeks to a small group of potentially at-risk glucose-6-phosphate dehydrogenase-deficient subjects. Physiological impact was assessed by evaluation of a battery of qualitative and quantitative tests. The three phases of this controlled double-blind clinical evaluation of chlorine dioxide and its potential metabolites in human male volunteer subjects were completed uneventfully. There were no obvious undesirable clinical sequellae noted by any of the participating subjects or by the observing medical team. In several cases, statistically significant trends in certain biochemical or physiological parameters were associated with treatment; however, none of these trends was judged to have physiological consequence. One cannot rule out the possibility that, over a longer treatment period, these trends might indeed achieve proportions of clinical importance. However, by the absence of detrimental physiological responses within the limits of the study, the relative safety of oral ingestion of chlorine dioxide and its metabolites, chlorite and chlorate, was demonstrated.

Introduction Chlorine dioxide is currently under serious consideration in the United States as an alternative to chlorine water treatment. Before chlorine dioxide may be used routinely as a water disinfectant, the safety of oral human ingestion of chlorine dioxide and its by-products must be assessed. For this purpose, a controlled clinical evaluation of chlorine dioxide, chlorite and chlorate was undertaken under the auspices of USEPA HERL #CR805643.

The study was conducted in three parts. Phase I was designed to evaluate the acute physiological The Department of Pharmacology, The Ohio State University, College of Medicine, 333 W. 10th Avenue, Columbus, *

OH 43210

effects of progressively increasing doses of disinfectants administered to normal healthy adult males.

Chronic ingestion by normal male volunteers was studied in Phase II. Phase III assessed the physiological response of a small group of potentially susceptible individuals, those deficient in glucose-6phosphate dehydrogenase, to chronic ingestion of chlorite.

Methods Subject Selection For Phase I and for Phase II, normal healthy adult male volunteers were selected. No prospective study participant who exhibited a significant abnormality in the routine clinical serum analysis,

58

blood count, urinalysis, or electrocardiogram was selected. Subjects manifested no physical abnormalities at the pretreatment examination, were 21 to 35 years of age, and weighed within ± 10% of normal body weight for their frame and stature. A history of disease or any medical or surgical condition which might interfere with the absorption, excretion, or metabolism of substances by the body precluded inclusion. Regular drug intake prior to the start of the investigation, either therapeutic or recreational, resulted in exclusion from the study.

Normal methemoglobin levels, thyroid function, and glutathione levels were mandatory. Written informed consent was obtained from each subject prior to initiation of treatment.

For Phase III, volunteers were defined as glucose6-phosphate dehydrogenase (G-6-PD)-deficient on the basis of a hemoglobin G-6-PD level of less than 5.0 IU/GM hemoglobin in the pre-study screening.

Phase III subjects were normal in all other respects.

Water Disinfectant Preparation A detailed description of the water disinfectant preparation techniques has been presented by Lubbers and Bianchine (1). In general, freshly prepared stock solutions of chlorine dioxide, sodium chlorite, sodium chlorate, chlorine and chloramine were assayed by the colorimetric techniques of Palin (2) then diluted with organic-free demineralized deionized water to appropriate concentrations.

Individual bottles were capped and stored in the dark under refrigeration until use. All bottles were coded by an independent observer and the identity of each bottle remained "double-blind" to both the investigative staff and the volunteer subjects.

Study Design: Phase I The 60 volunteers in Phase I were divided at random into six treatment groups (1). Ten persons were assigned to receive each of the disinfectants; the ten members of the control group received untreated water. The study involved a series of six sequences of three days each. Treatment concentrations were increased for each treatment. The specific concentrations or disinfectant administered to the study participants are listed in Table 1. A clinical evaluation of the collection of blood and urine samples for determination of pretreatment baseline laboratory values preceded the first treatment. On the first day of each three day treatment sequence, each volunteer ingested 1000 ml of the water in two portions. The second 500 ml portion aliquot was administered 4 hr after the first. Each 500 ml portion was consumed within 15 min. Only two doses of disinfectant were administered on the

LUBBERS, CHAUAN AND BIANCHINE

first day of each treatment sequence. No disinfectant was administered on the second and third day of each sequence, since these two days were to serve as followup observation days. The second day of the treatment sequence consisted of a physical examination and collection of blood and urine samples for determination of posttreatment laboratory values. On the third day, each volunteer was given a physical examination to determine residual effects of treatment with the water disinfectants and byproducts.

Taste evaluations were obtained at each dose level. Study participants were asked to rate the treated water as very unpleasant, slightly unpleasant, not pleasant, pleasant, or tasteless.

Study Design: Phase II The sixty volunteers of Phase II were divided at random into six treatment groups of ten subjects each (3). In order to assure efficient management of the 60 subjects, they were randomly assigned to three subsets. These subsets were sequentially entered into the study on three successive days and exited from this study in a similar fashion. For all of the treatment groups, the concentration of disinfectants ingested was 5 mg/l. The control group received untreated water. Each subject received 500 ml daily for 12 weeks. Physicals, collection of blood and urine samples for laboratory assays, and taste evaluations were conducted on a weekly basis during the treatment period and for 8 weeks following cessation of treatment.

Study Design: Phase III The three glucose-6-phosphate dehydrogenasedeficient subjects of Phase III were given sodium chlorite at a concentration of 5 mg/I. chlorite (4).

The treatment protocol was identical to that of Phase II, with daily administration of 500 ml of solution to each volunteer.

Evaluation Procedures An extensive battery of parameters was monitored to assess the biochemical and physiological response to the oral ingestion of the water disinfectants and water treatment by-products (Table 2).

All laboratory determninations of biochemical parameters were conducted by a licensed medical laboratory, Consolidated Biomedical Laboratories, Inc. (CBL), Columbus, Ohio, HEW license number 34-1030. For each volunteer, pretreatment baseline values and six sets of posttreatment values were compiled. Laboratory tests were carefully chosen.

ORAL INTAKE OF CHLORINE DISINFECTANTS IN MAN

59

Table 1. Concentration of disinfectants in phase I: acute rising dose tolerance.a

Disinfectant concentration, mg/l.

Water disinfectant Day 1 Day 4 Day 7 Day 10 Chlorate 0.01 0.1 0.5 1.0 Water control 0 0 0 0 Chlorine dioxide 5.0 10.0 0.1 1.0 Chlorite 1.0 0.01 0.1 0.5 Chlorine 0.1 5.0 10.0 1.0 Chloramine 8.0 18.0 0.01 1.0 aFor each dose, two portions of 500 ml each were administered at 4-hr intervals.

Day 13

Day 16

1.8 0 18.0 1.8 18.0 24.0

2.4 0 24.0 2.4 24.0

Table 2. Biochemical parameters assayed in the controlled clinical evaluation of chlorine dioxide, chlorite and chlorate in man.

Serum chemistry

Plasma glucose, sodium, potassium, chloride, urea nitrogen, creatinine, BUN/creatinine ratio, uric acid, calcium, phosphorus, alkaline phosphatase, gamma glutamyl transferase, total bilirubin, serum glutamic-oxaloacetic transaminase, serum glutamic-pyruvic transaminase, lactic dehydrogenase, cholesterol, triglycerides, total protein albumin, globulin, albumin/globulin ratio, iron Blood count Platelet count, white blood cell count, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, high peroxidase activity, neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells Urinalysis Color,a appearance,a specific gravity, pH, protein, sugar,a acetone, blood,a white blood count, red blood count, casts,a crystals,a bacteria,a mucus*, amorphous cells,a epithelial cells Special tests Serum haptoglobin, sickle cell,a methemoglobin, glucose-6-phosphate dehydrogenase, Coombs test,a hemoglobin electrophoresis,a T-3 (uptake), T-4 (RIA), free thyroxine index, electrocardiograma Physical exam Systolic blood pressure, diastolic blood pressure, respiration rate, pulse rate, oral temperature aThese parameters yielded qualitative data only; no statistical analysis was performed.

On the basis of the literature (5-8), areas of suspected biochemical response to ingestion of chlorine oxidants were defined; a portion of the test battery was specifically devoted to monitoring this response.

Red blood cell surface antibody formation was clinically monitored by the qualitative Coombs test; thyroid function by T-3 (uptake), T-4 (RIA), and free thyroxine; and response to oxidative stress by glucose-6-phosphate dehydrogenase, methemoglobin and glutathione levels. Hemoglobin electrophoresis was used to detect possible hemoglobin abnormalities. A battery of peripheral parameters was assayed to provide supplementary information and to assist in evaluation of overall physiological well-being.

The specific serum, blood and urine parameters assayed have been discussed by Lubbers and Bianchine (1).

The numerical values obtained were collected and analyzed by utilizing the facilities of The Ohio State University Division of Computing Services for Medical Education and Research. Specially designed programs facilitated rapid clinical feedback. Any value for an individual subject which differed from the group mean by more than two standard deviations was noted. In addition, every individual value which fell outside normal labora-

tory ranges for that parameter was designated as abnormal. Chemical parameters for volunteers who exhibited abnormal values were subjected to careful scrutiny; the safety and the possibility of hypersensitivity to the disinfectant agents were evaluated for each of these individuals on a continuing basis throughout the study.

Statistical analyses utilized commercially available computer packages, specifically, the Biomedical Computer Programs (BMDP) and the Statistical Package for the Social Sciences (SPSS). Two-way analyses of variance with repeated measures utilized BMDP2V. BMDP1R was used to perform multiple linear regression analyses. For pairwise t-tests and simple t-tests, SPSS-T-test was employed.

Results Qualitative An important aspect of this study was the careful and continued medical observation of all subjects.

The general clinical histories and physical examinations alone with subjective observations and qualitative laboratory tests throughout this study were accumulated in each subject's medical file. A careful

60

LUBBERS, CHAUAN AND BIANCHINE

inspection of each of these medical files presented a review of the general clinical health of each subject.

The careful clinical evaluation of every subject in Phases , II and III failed to reveal any clinically important impact upon the medical well-being of any subject as a result of disinfectant ingestion.

Further, there was no apparent grouping of the minor subjective symptoms and objective signs noted throughout the study; the "colds," "lymphadenopathy," "sore throats" and "flu" problems noted episodically appear to be randomly dispersed among the treatment groups. All subjects remained negative with respect to the Coombs tests and the sickle cell tests during the investigation. Hemoglobin electrophoresis results indicated that, in Phase II, a small number of subjects yielded abnormal hemoglobin distributions but these individuals were found to be randomly distributed in both the treatment groups and in the control group. Examination of electrocardiograms revealed no abnormalities.

Vital signs (blood pressure, pulse rate, respiration rate and body temperature) were measured on a regular basis to provide immediate feedback to the monitoring physician on the acute physiological response of study participants to treatment. The statistical analysis of the vital signs was limited to the calculation of arithmetic group means and standard deviations from the mean. The compiled vital signs were examined for evidence of consistent response to treatment. No such evidence was found.

The subjective evaluations of palatability indicated that few subjects found the test substances to have an objectionable taste at levels up to 24 mg/l.

Quantitative For the Phase I acute rising-dose tolerance study, a two-way analysis of variance with repeated measures was used to compare the treatment group values of each biochemical parameter to the corresponding values of the control group. The analysis of variance allowed distinctions to be made among the possible sources of variation. Differences between two groups that existed prior to treatment, parallel variations in quantitative chemical values due to

laboratory drift and authentic treatment-related changes in physiological parameters could be distinguished. Three probabilities were calculated for each case: the group main effect (G), the time main effect (R), and group-time interaction (RG). The treatment groups and the corresponding biochemical parameters for which a strong probability of treatment-related change was computed (that is, RG 0.05) are listed in the first column of Table 3.

To assist in determining the clinical importance of the statistically significant group time interactions, the group, mean and standard deviations -

from the mean were examined for the pretreatment baseline assay and each posttreatment assay for each of the treatment groups. In all instances, the group mean values remain well within the established normal ranges.

On the basis of the small magnitude of change within the normal range and the duration of the study, it was concluded that the trends identified by the analysis of variance are unlikely to be of clinical importance. The possibility that the trends might become clinically important with increased exposure cannot be excluded.

Alternative statistical techniques were employed for Phase II. An omnibus testing technique was used initially. To test the hypothesis that the response of one or more of the groups was different to that of the rest of the groups, an analysis of variance with repeated measures was performed in which values for all six treatment groups were included. For the parameters urea nitrogen and mean corpuscular hemoglobin, RG-values < 0.05 were obtained. Supplementary tests were performed. Analyses of variance with repeated measures in which the values of each treatment group were compared to the corresponding values of the control group were chosen. The use of the analysis of variance in this manner is flawed by the common control group.

However, the results of the analyses may be used with caution. The analysis of variance yielded statistically significant RG-values in the comparison of the group mean corpuscul ar hemoglobin values for the chlorite and the chlorate groups and of the group mean urea nitrogen values of chlorate and chlorine dioxide treatment groups to the corresponding control group values, as shown in Table 3.

No linear trends were detected by linear regression analysis of the chlorite group's mean corpuscular hemoglobin values, the chlorate group's urea nitrogen levels or the chlorite group's urea nitrogen values.

Mean corpuscular hemoglobin levels in the chlorate group yielded a probability of 0.01 upon linear regression analysis. The relative slope associated with the change during the 12-week treatment period was approximately 1% of the normal physiological range per week. We believe that no physiological importance may be attributed with confidence to the variation. However, it is impossible on the basis of this study to rule out the potential physiological significance of the trend. Further study is warranted.

The small number of subjects (three) in Phase III negated the value of many statistical procedures.

Linear regression analyses were chosen. The third column of Table 3 lists the biochemical parameters for which a high probability of change with respect to time was calculated. The p-values computed by

ORAL INTAKE OF CHLORINE DISINFECTANTS IN MAN

61

Table 3. Biochemical parameters and treatment groups in which statistical analyses indicated a high probability of change which could be attributed to ingestion of disinfectant.

Test Urea nitrogen (BUN)

Creatinine BUN/creatinine Ratio Uric acid Calcium Gamma glutamyl transferase Total bilirubin Albumin/globulin ratio Iron Methemoglobin T-4 (RIA) Free thyroxine index Mean corpuscular hemoglobin

Phase Ia

Chlorite

Phase l1b

Phase ITIC

Chlorate Chlorine dioxide

Chlorite Chlorine Chlorite Chlorine dioxide Chlorine Chlorine Chlorate Chlorite Chlorate Chlorate

Chlorite Chlorite Chlorite Chlorite Chlorate

Mean corpuscular hemoglobin concentration Chlorite Chlorine Lymphocytes aTwo-way analysis of variance yielded group-time interactions (RG values) S 0.05 in comparisons of treatment group values to those of the control group. bTwo-way analysis of variance yielded group-time interactions (RG-values) S 0.05 in both the omnibus and treatment group-control group comparisons. cLinear regression analysis indicated a strong probability of change with respect to time; p-values S 0.05.

the linear regression analysis were less than 0.05 for four biochemical parameters. To gauge the relative magnitude of change, the percent change of the normal range per week was computed. These statistical analyses indicate a good probability that, for A/G ratio, T-4 (RIA), free thyroxine, mean corpuscular hemoglobin concentration, and methemoglobin values, a change with respect to time occurs during the 12-week treatment period. However, in the absence of a concurrent control group and taking into consideration the small group size and the possibility of laboratory drift, one must exercise caution in dealing with the results. We can say with confidence only that trends were indicated. We cannot say that these trends were of physiological origin nor can we attribute physiological consequence to them.

Discussion Several researchers have addressed the physiological effects of oral ingestion of the oxidizing agents, chlorine dioxide, chlorite and chlorate. Musil et al. (9) associated oral chlorite ingestion with methemoglobin formation. In studies by Heffernan et al. (7,8), Abdel-Rahman et al. (5) and Couri et al.

(6), hemolytic anemia and suppressed glutathione levels were observed in animals treated with chlorite. The oral administration of chlorate to laboratory animals has been shown to induce oxidative destruction of hemoglobin and methemoglobin for-

mation (10, 11). The possibility of renal toxicity at high levels of chlorite ingestion was suggested by the increased kidney/body weight ratio reported by Heffernan et al. (7). Haller and Northgraves (12) and Fridlyand and Kagan (13) examined the chronic toxicity of orally consumed chlorine dioxide in rats; a slightly increased two-year mortality rate and a decreased rate of weight gain were observed. Oral administration of chlorite (14-16) to mice was shown to increase mean corpuscular volume, osmotic fragility, and glucose-6-phosphate dehydrogenase activity of erythrocytes; morphologic changes were reported. In the African Green monkey, chlorine dioxide adversely affected thyroid function; chlorite ingestion yielded transient changes in hemoglobin levels and red cell count (17). The maternal toxicity, embryonic toxicity and the teratogenic potential of concentrations of sodium chlorite was evaluated in rats (18).

Unfortunately, the information available on the impact of chlorine dioxide, chlorite, and chlorate ingestion in man is severely limited. Epidemiological studies (19,20) have failed to conclusively identify any significant exposure related effects. The clinical evaluation described in this report was an attempt to elucidate the effects of the chlorite, chlorine dioxide and chlorate in man under controlled clinical conditions.

During the course of the three-phase study, a massive volume of raw data was acquired. Routine urinalyses were performed and a meticulous exam-

62

ination of this body of information was made. No definitive finding ofdetrimental physiological impact was made in any of the three phases of this human investigation of the relative safety and tolerance of oral chlorine disinfectant ingestion. In several cases, statistically significant trends were associated with treatment; however, none of these trends were judged to have immediate physiological consequence.

One cannot rule out the possibility that, over a longer treatment period, these trends might indeed achieve proportions of clinical importance. However, within the limits of the study, the relative safety of oral ingestion of chlorine dioxide and its metabolites, chlorite and chlorate, was demonstrated by the absence of detrimental physiological response.

7.

8.

9.

10.

11.

12.

13.

REFERENCES 1. Lubbers, J. R., and Bianchine, J. R. The effects of the acute rising dose administration of chlorine dioxide, chlorate and chlorite to normal healthy adult male volunteers. J. Environ. Pathol. Toxicol. 5 (2, 3): 865-878 (1982).

2. Palin, A. T. Methods for the determination, in water of free and combined available chlorine, chlorine dioxide and chlorite, bromine, iodine and ozone, using diethyl-p-phenylene diamine (DPD). J. Inst. Water Engr. 21: 537-549 (1976).

3. Lubbers, J. R., Chauhan, S., Miller, J. K., and Bianchine, J. R. The effects of chronic administration of chlorine dioxide, chlorite and chlorate to normal healthy adult male volunteers. J. Environ. Pathol. Toxicol. 5 (2, 3): 879-888 (1982).

4. Lubbers, J. R., Chauhan, S., Miller, J. K. and Bianchine, J. R. The effects of chronic administration of chlorite to glucose-6-phosphate dehydrogenase deficient healthy adult male volunteers. J. Environ. Pathol. Toxicol. 5 (2, 3): 889-892 (1982).

5. Abdel-Rhaman, M. S., Couri, D., and Bull, R.J. Kinetics of C102 and effects of C102, and C102, and C103 in drinking water on blood glutathione and hemolysis in rat and chicken.

J. Environ. Path. Toxicol. 3(1,2): 431-449 (1979).

6. Couri, D., and Abdel-Rahman, M.S. Effect of chlorine dioxide and metabolites on glutathione dependent system in

14.

15.

16.

17.

18.

19.

20.

LUBBERS, CHAUAN AND BIANCHINE rat, mouse and chicken blood. J. Environ. Pathol. Toxicol.

3(1,2): 451-460 (1979).

Heffernan, W. P., Guion, C., and Bull, R. J. Oxidative damage to the erythrocyte induced by sodium chlorite in vitro. J. Environ. Pathol. Toxicol. 2(6): 1487-1499 (1979).

Heffernan, W. P., Guion, C., and Bull, R. J. Oxidative damage to the erythrocyte induced by sodium chlorite in vitro. J. Environ. Pathol. Toxicol. 2(6): 1501-1510 (1979).

Musil, J., Kontek, Z., Chalupa, J., and Schmidt, P. Toxicological aspects of chlorine dioxide application for the treatment of water containing phenol. Chem. Technol. Praze. 8: 327-345 (1964).

Richardson, A. P. Toxic potentialities of continued administration of chlorate for blood and tissues. J. Pharmacol. Exptl. Therap. 59: 101-103, (1937).

Jung, F., and Kuon, R. Zum inaktiven hemoglobin das Bluter. Naunyn-Schmiedebergs Arch. Exptl. Pathol. Pharmakol. 216: 103-111 (1951).

Haller, S. F., and Northgraves, W.W. Chlorine dioxide and safety. TAPPI 33: 199-202 (1955).

Fridyland, S. A., and Kagan, G. Z. Experimental validation of standards for residual chlorine dioxide in drinking water.

Hygiene Sanitation 36: 18-21 (1971).

Moore, G. S., and Calabrese, E. J. The effects of chlorine dioxide and sodium chlorite on erythrocytes of A-J and C-57L-J mice. J. Environ. Pathol. Toxicol. 4(2, 3): 513-524 (1980).

Moore, G. S. and Calabrese, E. J. G-6-PD-deficiency-a potential high-risk group to copper and chlorite ingestion. J. Environ. Pathol. Toxicol. 4(2, 3): 271-279 (1980).

Moore, G. S., Calabrese, E. J. and Ho, S. C. Groups at potentially high-risk from chlorine dioxide treated water. J. Environ. Pathol. Toxicol. 4(2, 3): 465-470 (1980).

Berez, J. P., DiBiasi, D. L., Jones, L., Murray, D., and Boston, J. Subchronic toxicity of alternate disinfectants and related compounds in the non-human primate. Environ.

Health Perspect. 46: 47-55 (1982).

Couri, D., Miller, C. H., Bull, R. J., Delphia, J. M., and Ammar, E. M. Assessment of maternal toxicity, embryotoxicity and teratogenic potential of sodium chlorite in SpragueDawley rats. Environ. Health Perspect. 46: 25-29 (1982).

Haring, B. J., and Zoetman, B. C. Corrosiveness of drinking water and cardiovascular diesase mortality. Bull. Environ.

Contam. Toxicol. 25: 658-662 (1981).

Michael, G. E., Miday, R. K., Bercz, J. P., Miller, R. G., Greathouse, D. G., Kraemer, D. F., and Lucas, J. B. Chlorine dioxide water disinfection: a prospective epidemiology. Arch. Environ. Health 36(1): 20-27 (1981).

29.10.2020

Behandlung mit Medikamenten, die noch nicht zugelassen sind

Behandlung mit Medikamenten, die noch nicht zugelassen sind Ehe ein Medikament auf den Markt kommt, müssen seine Wirksamkeit und Verträglichkeit in klinischen Studien ermittelt und seine technische Qualität geprüft werden. Nur Medikamente, deren Nutzen die Anwendungsrisiken überwiegt und die in einem zuverlässigen Verfahren hergestellt werden, erhalten nach vielmonatiger behördlicher Prüfung die Zulassung zur Vermarktung, können also auch bei Patienten außerhalb von Studien eingesetzt werden. Doch für Menschen mit schweren Erkrankungen, die mit allen zugelassenen Therapiemöglichkeiten „austherapiert“, aber keine Studienteilnehmer sind, werden sie damit manchmal zu spät verfügbar, um sie noch vor dem Tod oder Invalidität zu bewahren.

Unter bestimmten Umständen ist es deshalb zulässig, dass diese Patienten mit einem noch nicht zugelassenen Medikament (oder einem nur für andere Zwecke zugelassenen Medikament) behandelt werden. Hierfür gibt es Härtefall-Programme (auch Compassionate Use-Programme genannt) und die Möglichkeit des individuellen Heilversuchs.

Härtefall-Programme Härtefall-Programme (englisch Compassionate Use, "Anwendung aus Mitgefühl") müssen bei der zuständigen deutschen Arzneimittelbehörde gemeldet werden. Im Rahmen eines solchen Programms kann ein bestimmtes Medikament, das schon ein Stück weit erprobt, aber noch nicht zugelassen ist, unter genau aufgelisteten Umständen von einem Arzt angefordert werden, der versuchen möchte, einem entsprechenden Patienten in Not zu helfen.

Für welche Medikamente es derzeit diese Möglichkeit gibt, und für welche Patienten es genutzt werden kann, listen die Websites des Bundesinstituts für Arzneimittel und Medizinprodukte (BfArM) und des Paul Ehrlich Instituts (PEI) auf. Dabei stehen Angaben zu Medikamenten mit chemisch-synthetischen Wirkstoffen beim BfArM und solche zu gentechnischen Medikamenten, Blutprodukten und Impfstoffen beim PEI. Hier findet sich auch zu jedem Programm eine Kontaktadresse für die Anforderung des Medikaments. Viele Härtefallprogramme betreffen Medikamente gegen Krebserkrankungen.

Ein Arzt, der ein Medikament im Rahmen eines Härtefall-Programms einsetzt, muss Verdachtsfälle auf Nebenwirkungen an die für das Härtefall zuständige Person in der Firma weitergeben. Diese muss ihrerseits die Zulassungsbehörde darüber informieren. So können im Rahmen des Programms weitere Erkenntnisse über die Sicherheit des Medikaments gewonnen werden. https://www.vfa.de/print/de/patienten/artikel-patienten/behandlung-mit-medikamenten-die-noch-nicht-zugelassen-sind.html

1/3

29.10.2020

Behandlung mit Medikamenten, die noch nicht zugelassen sind

In Deutschland ist rechtlich klar geregelt, dass ein Arzneimittelhersteller, wenn er an einem Härtefallprogramm mitwirkt, das betreffende Medikament kostenfrei zur Verfügung stellen muss. Die gesetzlichen Krankenkassen zahlen für das Medikament nicht, nur für die Behandlung und gegebenenfalls für den Klinikaufenthalt. Härtefall-Programme gibt es aber auch in den anderen EU-Ländern (es gibt sogar einige wenige EU-weite Härtefall-Programme), und dort sind die Bestimmungen zur Kostenübernahme für das Medikament zum Teil andere.

Härtefall-Programme sind immer zeitlich befristet. „Auf Dauer“ angemeldete Härtefall-Programme gibt es nicht. Aber es ist möglich, die Dauer eines Härtefall-Programms im begründeten Bedarfsfall zu verlängern; ein Härtefall-Programm endet aber in jedem Fall mit dem Tag der Markteinführung des Medikaments.

Rechtlicher Rahmen für Härtefall-Programme Im Rahmen des 14. Änderungsgesetzes zum Arzneimittelgesetz (AMG) wurde der "Compassionate Use" in das deutsche Arzneimittelrecht aufgenommen, und die in § 80 vorgesehene Härtefall-Verordnung trat am 22. Juli 2010 in Kraft. Das Anzeigeverfahren für HärtefallProgramme ist auf nationaler Ebene in § 21 Abs. 2 Nr. 6 AMG und der Härtefall-Verordnung geregelt, auf europarechtlicher Ebene in der RL 2001/83/EG i.V.m. der VO 726/2004. Diese EU-Grundlagen waren auch der Anlass, Compassionate Use ins AMG aufzunehmen.

Was die europäische Ebene betrifft, finden sich auf der Website der EMA Informationen zum Compassionate Use in der EU.

Individuelle Heilversuche Bei einem individuellen Heilversuch handelt es sich um eine Anwendung eines nicht zugelassenen Medikaments im Einzelfall, über die der Arzt im Rahmen der Therapiefreiheit mit Zustimmung des Patienten allein und aus eigener Initiative entscheidet. Er ist nur angebracht, wenn alle anderen Behandlungsmöglichkeiten ausgeschöpft sind, und wenn der Arzt aufgrund von wissenschaftlichen Erkenntnissen einen Nutzen für den betreffenden Patienten vermutet. Der Arzt trägt dabei die Verantwortung für die Anwendung.

Beim individuellen Heilversuch steht also nicht die Gewinnung von neuen Forschungserkenntnissen im Vordergrund, sondern die Heilung des einzelnen Patienten, für den alle anderen Behandlungsmöglichkeiten bereits ausgeschöpft sind.

In Deutschland sind individuelle Heilversuche gesetzlich nicht geregelt. Sie sind nicht meldepflichtig, und es gibt kein Register dafür.

Dennoch ist anerkannt, dass von der ärztlichen Therapiefreiheit auch der individuelle Heilversuch umfasst ist und damit möglicherweise einhergehende tatbestandliche Verstöße gegen Normen des AMG durch die Notstandsregelung des § 34 StGB gerechtfertigt sein können. Klar ist hingegen, dass das betreffende Medikament nicht beworben werden darf und auch nicht bei BfArM oder PEI im Härtefall-Register gelistet ist. Insofern kann ein Arzt lediglich durch Teilnahme an Forschungskongressen, anhand der Fachliteratur oder durch Studienregister auf die Existenz des Mittels aufmerksam werden.

Erfolgt die Anwendung des Medikaments in einem individuellen Heilversuch, muss Folgendes sorgfältig dokumentiert werden: In der Patientenakte müssen unter anderem das Arzneimittel bzw. sein Wirkstoff, der Therapieplan, die Dosierung, das Auftreten unerwünschter Wirkungen und überhaupt der Verlauf der Behandlung ausführlich beschrieben werden. Für die Kostenübernahme gibt es keine eigenständigeRegelung. Das muss im Einzelfall zwischen den Beteiligten – Arzt, Patient, Firma und Krankenversicherung – geklärt werden.

Da individuelle Heilversuche nur in Ausnahmefällen und jeweils nur für einzelne Patienten durchgeführt werden dürfen, müssen die Mediziner und Forscher des Unternehmens jedes Mal neu entscheiden, ob sie dem Wunsch nach Bereitstellung ihres Medikaments für einen individuellen Heilversuch entsprechen können oder nicht. Und wenn sie eine Entscheidung dagegen treffen – in dem Wissen, dass dies für die Betroffenen schwer verständlich ist –, dann aus ihrer Verantwortung heraus.

Off-Label-Anwendung Von Härtefall-Programmen und individuellen Heilversuchen zu unterscheiden ist die Off-Label-Anwendung: Dabei wird bei einem Patienten ein Medikament angewendet, das zwar grundsätzlich eine Zulassung hat, aber nicht für den Fall dieses Patienten (denn dieser hat eine andere Krankheit; gehört einer anderen Altersgruppe an; etc.). „Label“ bezeichnet in diesem Fall den Zulassungstext.

Eine Kostenübernahme durch die Krankenkasse erfolgt im Wesentlichen nur in folgenden Fällen: Wenn eine positive Entscheidung des https://www.vfa.de/print/de/patienten/artikel-patienten/behandlung-mit-medikamenten-die-noch-nicht-zugelassen-sind.html

2/3

29.10.2020

Behandlung mit Medikamenten, die noch nicht zugelassen sind

Gemeinsamen Bundesausschusses (der Selbstverwaltung des deutschen Gesundheitswesens) vorliegt; wenn eine Kostenübernahmeerklärung nach § 2 Abs. 1a SGB V erteilt wird; wenn das Arzneimittel im Rahmen einer vom G-BA nicht widersprochenen klinischen Studie nach § 35c SGB V eingesetzt wird oder wenn die folgenden von der Rechtsprechung des Bundessozialgerichts definierten drei Kriterien vorliegen: 1. Notwendige Behandlung einer schwerwiegenden (lebensbedrohlichen oder die Lebensqualität auf Dauer nachhaltig beeinträchtigenden) Erkrankung; 2. Fehlen einer anderen Therapie und 3. aufgrund der Datenlage besteht begründete Aussicht, dass mit dem Arzneimittel ein Behandlungserfolg (kurativ oder palliativ) erzielt werden kann.

© vfa 2020 - https://www.vfa.de/

https://www.vfa.de/print/de/patienten/artikel-patienten/behandlung-mit-medikamenten-die-noch-nicht-zugelassen-sind.html

3/3

WMA Deklaration von Helsinki - Ethische Grundsätze für die medizinische Forschung am Menschen Verabschiedet von der 18. WMA-Generalversammlung, Juni 1964 Helsinki (Finnland) und revidiert durch die 29. WMA-Generalversammlung, Oktober 1975, Tokio (Japan), 35. WMA-Generalversammlung, Oktober 1983, Venedig (Italien), 41. WMA-Generalversammlung, September 1989, Hong Kong, 48. WMA-Generalversammlung, Oktober 1996, Somerset West (Republik Südafrika), 52. WMA-Generalversammlung, Oktober 2000, Edinburgh (Schottland), 53. WMA-Generalversammlung im Oktober 2002, Washington (Vereinigte Staaten) (ergänzt um einen klarstellenden Kommentar zu Ziffer 29), 55. WMA-Generalversammlung im Oktober 2004, Tokio (Japan), (ergänzt um einen klarstellenden Kommentar zu Ziffer 30), 59. WMA-Generalversammlung im Oktober 2008, Seoul (Korea) 64. WMA-Generalversammlung im Oktober 2013, Fortaleza (Brasilien)

Präambel 1.

Der Weltärztebund (WMA) hat mit der Deklaration von Helsinki eine Erklärung

ethischer Grundsätze für medizinische Forschung am Menschen, einschließlich der Forschung an identifizierbaren menschlichen Materialien und Daten, entwickelt.

Die Deklaration ist als Ganzes zu lesen, und ihre einzelnen Paragraphen sollen unter Berücksichtigung aller übrigen relevanten Paragraphen angewendet werden.

2.

Im Einklang mit dem Mandat des WMA wendet sich die Deklaration in erster Linie

an Ärzte. Der WMA regt andere an der medizinischen Forschung am Menschen Beteiligte an, diese Grundsätze zu übernehmen.

WMA

Seite 2

Allgemeine Grundsätze 3.

Die Genfer Deklaration des Weltärztebundes verpflichtet den Arzt mit den Worten

„Die Gesundheit meines Patienten soll oberstes Gebot meines Handelns sein“, und der Internationale Kodex für ärztliche Ethik legt fest: „Der Arzt soll bei der Ausübung seiner ärztlichen Tätigkeit im besten Interesse des Patienten handeln.“ 4.

Es ist die Pflicht des Arztes, die Gesundheit, das Wohlergehen und die Rechte der

Patienten zu fördern und zu erhalten, auch jener, die an der medizinischen Forschung beteiligt sind. Der Erfüllung dieser Pflicht dient der Arzt mit seinem Wissen und Gewissen.

5.

Medizinischer Fortschritt beruht auf Forschung, die letztlich auch Studien am

Menschen beinhalten muss.

6.

Vorrangiges Ziel der medizinischen Forschung am Menschen ist es, die Ursachen,

die Entwicklung und die Auswirkungen von Krankheiten zu verstehen und die präventiven, diagnostischen und therapeutischen Maßnahmen (Methoden, Verfahren und Behandlungen) zu verbessern. Selbst die nachweislich besten Maßnahmen müssen fortwährend durch Forschung auf ihre Sicherheit, Effektivität, Effizienz, Verfügbarkeit und Qualität geprüft werden.

7.

Medizinische Forschung unterliegt ethischen Standards, die die Achtung vor den

Menschen fördern und sicherstellen und ihre Gesundheit und Rechte schützen.

8.

Während vorrangiger Zweck der medizinischen Forschung ist, neues Wissen

hervorzubringen, darf dieses Ziel niemals Vorrang vor den Rechten und Interessen der einzelnen Versuchspersonen haben.

9.

Es ist die Pflicht des Arztes, der sich an medizinischer Forschung beteiligt, das

Leben, die Gesundheit, die Würde, die Integrität, das Selbstbestimmungsrecht, die Privatsphäre und die Vertraulichkeit persönlicher Informationen der Versuchsteilnehmer zu schützen. Die Verantwortung für den Schutz von Versuchspersonen muss stets der Arzt oder ein anderer Angehöriger eines Heilberufes tragen und nie die Versuchsperson selbst, auch dann nicht, wenn sie ihr Einverständnis gegeben hat.

WMA

10.

Seite 3

Ärzte müssen die ethischen, rechtlichen und behördlichen Normen und Standards

für Forschung am Menschen ihrer eigenen Länder sowie die maßgeblichen internationalen Normen und Standards berücksichtigen. Keine nationale oder internationale ethische, rechtliche oder behördliche Anforderung soll die in dieser Deklaration niedergelegten Bestimmungen zum Schutz von Versuchspersonen abschwächen oder aufheben.

11.

Medizinische Forschung sollte in einer Weise durchgeführt werden, die mögliche

Umweltschäden minimiert.

12.

Medizinische Forschung am Menschen darf nur von Personen durchgeführt

werden, die angemessen ethisch und wissenschaftlich ausgebildet, geübt und qualifiziert sind. Forschung an Patienten oder gesunden Freiwilligen erfordert die Überwachung durch einen kompetenten und angemessen qualifizierten Arzt oder anderen Angehörigen eines Heilberufes.

13.

Gruppen, die in der medizinischen Forschung unterrepräsentiert sind, sollten einen

angemessenen Zugang zur Teilnahme an der Forschung erhalten.

14.

Ärzte, die medizinische Forschung mit medizinischer Behandlung verbinden, sollten

ihre Patienten nur soweit in die Forschung einbeziehen, wie dies durch deren möglichen präventiven, diagnostischen oder therapeutischen Wert gerechtfertigt ist und der Arzt berechtigterweise annehmen kann, dass eine Beteiligung an dem Forschungsvorhaben die Gesundheit der Patienten, die als Versuchspersonen dienen, nicht nachteilig beeinflussen wird.

15.

Eine angemessene Entschädigung und Behandlung für Versuchspersonen, die

aufgrund ihrer Teilnahme an der Forschung geschädigt wurden, muss gewährleistet sein.

Risiken, Belastungen und Nutzen 16.

In der medizinischen Praxis und in der medizinischen Forschung sind die meisten

Maßnahmen mit Risiken und Belastungen verbunden.

WMA

Seite 4

Medizinische Forschung am Menschen darf nur durchgeführt werden, wenn die Bedeutung des Ziels die Risiken und Belastungen für die Versuchspersonen überwiegt.

17.

Jeder medizinischen Forschung am Menschen muss eine sorgfältige Abschätzung

der voraussehbaren Risiken und Belastungen für die an der Forschung beteiligten Einzelpersonen und Gruppen im Vergleich zu dem voraussichtlichen Nutzen für sie und andere Einzelpersonen oder Gruppen vorangehen, die von dem untersuchten Zustand betroffen sind.

Maßnahmen zur Risikominimierung müssen implementiert werden. Die Risiken müssen vom Forscher kontinuierlich überwacht, eingeschätzt und dokumentiert werden.

18.

Ärzte dürfen sich nicht an einem Forschungsvorhaben am Menschen beteiligen,

wenn sie nicht überzeugt sind, dass die mit der Studie verbundenen Risiken angemessen eingeschätzt worden sind und in zufriedenstellender Weise beherrscht werden können.

Sobald sich herausstellt, dass die Risiken den potentiellen Nutzen übersteigen oder wenn es einen schlüssigen Beweis für gesicherte Ergebnisse gibt, müssen Ärzte einschätzen, ob die Studie fortgesetzt, modifiziert oder unverzüglich beendet werden muss.

Vulnerable Gruppen und Einzelpersonen 19.

Einige Gruppen und Einzelpersonen sind besonders vulnerabel und können mit

größerer Wahrscheinlichkeit ungerecht behandelt oder zusätzlich geschädigt werden.

Alle vulnerablen Gruppen und Einzelpersonen sollten besonders bedachten Schutz erhalten.

20.

Medizinische Forschung mit einer vulnerablen Gruppe ist nur gerechtfertigt, wenn

das Forschungsvorhaben auf die gesundheitlichen Bedürfnisse oder Prioritäten dieser Gruppe reagiert und das Forschungsvorhaben nicht an einer nicht-vulnerablen Gruppe durchgeführt werden kann. Zusätzlich sollte diese Gruppe in der Lage sein, aus dem Wissen, den Anwendungen oder Maßnahmen Nutzen zu ziehen, die aus dem Forschungsvorhaben hervorgehen.

WMA

Seite 5

Wissenschaftliche Anforderungen und Forschungsprotokolle 21.

Medizinische Forschung am Menschen muss den allgemein anerkannten

wissenschaftlichen Grundsätzen entsprechen sowie auf einer gründlichen Kenntnis der wissenschaftlichen Literatur, anderen relevanten Informationsquellen, ausreichenden Laborversuchen und, sofern angemessen, auf Tierversuchen basieren. Auf das Wohl der Versuchstiere muss Rücksicht genommen werden.

22.

Die Planung und Durchführung einer jeden wissenschaftlichen Studie am

Menschen muss klar in einem Studienprotokoll beschrieben und gerechtfertigt werden.

Das Protokoll sollte eine Erklärung der einbezogenen ethischen Erwägungen enthalten und sollte deutlich machen, wie die Grundsätze dieser Deklaration berücksichtigt worden sind. Das Protokoll sollte Informationen über Finanzierung, Sponsoren, institutionelle Verbindungen, mögliche Interessenkonflikte, Anreize für Versuchspersonen und Informationen bezüglich Vorkehrungen für die Behandlung und/oder Entschädigung von Personen enthalten, die infolge ihrer Teilnahme an der wissenschaftlichen Studie einen Schaden davongetragen haben.

Bei klinischen Studien muss das Protokoll auch angemessene Vorkehrungen für Maßnahmen nach Abschluss der Studie beschreiben.

Forschungs-Ethikkommissionen 23.

Das Studienprotokoll ist vor Studienbeginn zur Erwägung, Stellungnahme, Beratung

und Zustimmung der zuständigen Forschungs-Ethikkommission vorzulegen. Diese Ethikkommission muss transparent in ihrer Arbeitsweise, unabhängig vom Forscher, dem Sponsor und von jeder anderen unzulässigen Beeinflussung, sowie angemessen qualifiziert sein. Sie muss den Gesetzen und Rechtsvorschriften des Landes oder der Länder, in dem oder denen die Forschung durchgeführt werden soll, sowie den maßgeblichen internationalen Normen und Standards Rechnung tragen, die jedoch den in dieser Deklaration festgelegten Schutz von Versuchspersonen nicht abschwächen oder aufheben dürfen.

WMA

Seite 6

Die Ethikkommission muss das Recht haben, laufende Studien zu beaufsichtigen. Der Forscher muss der Ethikkommission begleitende Informationen vorlegen, insbesondere Informationen über jede Art schwerwiegender unerwünschter Ereignisse. Eine Abänderung des Protokolls darf nicht ohne Erwägung und Zustimmung der Ethikkommission erfolgen. Nach Studienende müssen die Forscher der Kommission einen Abschlussbericht vorlegen, der eine Zusammenfassung der Ergebnisse und Schlussfolgerungen der Studie enthält.

Privatsphäre und Vertraulichkeit 24.

Es müssen alle Vorsichtsmaßnahmen getroffen werden, um die Privatsphäre der

Versuchspersonen und die Vertraulichkeit ihrer persönlichen Informationen zu wahren.

Informierte Einwilligung 25.

Die Teilnahme von einwilligungsfähigen Personen an der medizinischen Forschung

muss freiwillig sein. Auch wenn es angemessen sein kann, Familienangehörige oder führende Persönlichkeiten der jeweiligen Gemeinschaft hinzuzuziehen, darf keine einwilligungsfähige Person in ein Forschungsvorhaben aufgenommen werden, wenn sie nicht freiwillig zustimmt.

26.

Bei der medizinischen Forschung an einwilligungsfähigen Personen muss jede

potentielle Versuchsperson angemessen über die Ziele, Methoden, Geldquellen, eventuelle Interessenkonflikte, institutionelle Verbindungen des Forschers, den erwarteten Nutzen und die potentiellen Risiken der Studie, möglicherweise damit verbundenen Unannehmlichkeiten, vorgesehene Maßnahmen nach Abschluss einer Studie sowie alle anderen relevanten Aspekte der Studie informiert (aufgeklärt) werden. Die potentielle Versuchsperson muss über das Recht informiert (aufgeklärt) werden, die Teilnahme an der Studie zu verweigern oder eine einmal gegebene Einwilligung jederzeit zu widerrufen, ohne dass ihr irgendwelche Nachteile entstehen. Besondere Beachtung soll dem spezifischen Informationsbedarf der individuellen potentiellen Versuchspersonen sowie den für die Informationsvermittlung verwendeten Methoden geschenkt werden.

WMA

Seite 7

Nachdem er sich vergewissert hat, dass die potentielle Versuchsperson diese Informationen verstanden hat, hat der Arzt oder eine andere angemessen qualifizierte Person die freiwillige Informierte Einwilligung (Einwilligung nach Aufklärung - „informed

consent“) der Versuchsperson – vorzugsweise in schriftlicher Form – einzuholen. Falls die Einwilligung nicht in schriftlicher Form geäußert werden kann, muss die nichtschriftliche Einwilligung formell dokumentiert und bezeugt werden.

Allen Versuchspersonen medizinischer Forschung sollte die Möglichkeit gegeben werden, über den allgemeinen Ausgang und die allgemeinen Ergebnisse der Studie informiert zu werden.

27.

Beim Einholen der Informierten Einwilligung in die Teilnahme an einer

wissenschaftlichen Studie muss der Arzt besondere Vorsicht walten lassen, wenn die potentielle Versuchsperson in einem Abhängigkeitsverhältnis zum Arzt steht oder unter Zwang einwilligen könnte. In solchen Situationen muss die Informierte Einwilligung durch eine angemessen qualifizierte Person eingeholt werden, die in jeder Hinsicht außerhalb dieses Verhältnisses steht.

28.

Bei einer potentiellen Versuchsperson, die nicht einwilligungsfähig ist, muss der

Arzt die Informierte Einwilligung des rechtlichen Vertreters einholen. Diese Personen dürfen nicht in eine wissenschaftliche Studie einbezogen werden, die ihnen aller Wahrscheinlichkeit nach nicht nützen wird, sofern nicht beabsichtigt wird, mit der Studie die Gesundheit der Gruppe zu fördern, der die potentielle Versuchsperson angehört, die Forschung nicht mit Personen durchgeführt werden kann, die eine Informierte Einwilligung geben können, und die Forschung nur minimale Risiken und minimale Belastungen birgt.

29.

Ist eine potentielle Versuchsperson, die als nicht einwilligungsfähig eingestuft wird,

fähig, Entscheidungen über die Teilnahme an der Forschung zuzustimmen, muss der Arzt neben der Einwilligung des rechtlichen Vertreters auch die Zustimmung der potentiellen Versuchsperson einholen. Eine Ablehnung der potentiellen Versuchsperson soll respektiert werden.

30.

Forschung mit Personen, die körperlich oder geistig zu einer Einwilligung nicht fähig

sind, beispielsweise mit bewusstlosen Patienten, darf nur dann erfolgen, wenn der körperliche oder geistige Zustand, der das Einholen der Informierten Einwilligung

WMA

Seite 8

verhindert, ein erforderliches Merkmal für die beforschte Gruppe ist. Unter solchen Umständen muss der Arzt die Informierte Einwilligung des rechtlichen Vertreters einholen.

Ist ein solcher Vertreter nicht verfügbar und kann die Forschung nicht aufgeschoben werden, kann die Studie ohne Informierte Einwilligung und unter der Voraussetzung durchgeführt werden, dass die besonderen Gründe für den Einschluss von Versuchspersonen, die aufgrund ihres Zustands nicht zu einer Informierten Einwilligung fähig sind, im Studienprotokoll festgehalten worden sind und die Studie von einer Forschungs-Ethikkommission zustimmend bewertet worden ist. Die Einwilligung zur weiteren Teilnahme an der Forschung muss sobald wie möglich bei der Versuchsperson oder einem rechtlichen Vertreter eingeholt werden.

31.

Der Arzt muss den Patienten vollständig über die forschungsbezogenen Aspekte

der Behandlung informieren. Die Weigerung eines Patienten, an einer Studie teilzunehmen, oder der Entschluss des Patienten, aus der Studie auszuscheiden, darf niemals die Patienten-Arzt-Beziehung nachteilig beeinflussen.

32.

Bei medizinischer Forschung, bei der identifizierbare menschliche Materialien oder

Daten verwendet werden, wie zum Beispiel in Biobanken oder ähnlichen Depots enthaltenes Material oder Daten, müssen Ärzte für ihre Sammlung, Lagerung und/oder Wiederverwendung eine Informierte Einwilligung einholen. In Ausnahmesituationen kann es sich als unmöglich oder nicht praktikabel erweisen, eine Einwilligung für derartige Forschung zu erhalten. In solchen Situationen darf die Forschung erst nach Beurteilung und Zustimmung einer Forschungs-Ethikkommission durchgeführt werden.

Die Verwendung von Placebos 33.

Nutzen, Risiken, Belastungen und Wirksamkeit einer neuen Maßnahme müssen mit

denjenigen der nachweislich besten Maßnahme(n) verglichen werden, außer unter folgenden Umständen: 

Wenn keine nachgewiesene Maßnahme existiert, ist die Verwendung von Placebo oder das Unterlassen einer Maßnahme zulässig, oder

WMA 

Seite 9 wenn aus zwingenden und wissenschaftlich fundierten methodischen Gründen die Verwendung einer weniger wirksamen Maßnahme als die nachweislich beste, die Verwendung eines Placebos oder das Unterlassen einer Maßnahme, notwendig sind, um die Wirksamkeit oder Sicherheit einer Maßnahme festzustellen, und wenn die Patienten, die eine weniger wirksame Maßnahme als die nachweislich beste, ein Placebo oder keine Maßnahme erhalten, keinem zusätzlichen Risiko eines ernsten oder irreversiblen Schadens ausgesetzt werden, welches sich daraus ergibt, dass sie nicht die nachweislich beste Maßnahme erhalten haben.

Mit größter Sorgfalt muss ein Missbrauch dieser Option vermieden werden.

Maßnahmen nach Abschluss einer Studie 34.

Im Vorfeld einer klinischen Studie sollten Sponsoren, Forscher und Regierungen

der Einsatzländer Vorkehrungen für Maßnahmen nach Abschluss der Studie für alle Teilnehmer treffen, die noch eine Maßnahme benötigen, die in der Studie als nützlich erkannt wurde. Diese Information muss den Teilnehmern auch während des Aufklärungsund Einwilligungsprozesses mitgeteilt werden.

Registrierung von Forschung sowie Publikation und Verbreitung von Ergebnissen 35.

Jedes Forschungsvorhaben, an dem Versuchspersonen beteiligt sind, ist vor der

Rekrutierung der ersten Versuchsperson in einer öffentlich zugänglichen Datenbank zu registrieren.

36.

Forscher, Verfasser, Sponsoren, Herausgeber und Verleger haben im Hinblick auf

die Veröffentlichung und Verbreitung der Forschungsergebnisse ethische Verpflichtungen.

Forscher sind verpflichtet, die Ergebnisse ihrer Forschung am Menschen öffentlich verfügbar zu machen und sind im Hinblick auf die Vollständigkeit und Richtigkeit ihrer Berichte rechenschaftspflichtig. Alle Beteiligten sollen anerkannten Leitlinien für ethische Berichterstattung („ethical reporting“) folgen. Negative und nicht schlüssige Ergebnisse

WMA

Seite 10

müssen ebenso wie positive veröffentlicht oder in anderer Form öffentlich verfügbar gemacht werden. In der Publikation müssen Finanzierungsquellen, institutionelle Verbindungen und Interessenkonflikte dargelegt werden. Berichte über Forschung, die nicht mit den Grundsätzen dieser Deklaration übereinstimmt, sollten nicht zur Veröffentlichung angenommen werden.

Nicht nachgewiesene Maßnahmen in der klinischen Praxis 37.

Bei der Behandlung eines einzelnen Patienten, für die es keine nachgewiesenen

Maßnahmen gibt oder andere bekannte Maßnahmen unwirksam waren, kann der Arzt nach Einholung eines fachkundigen Ratschlags mit Informierter Einwilligung des Patienten oder eines rechtlichen Vertreters eine nicht nachgewiesene Maßnahme anwenden, wenn sie nach dem Urteil des Arztes hoffen lässt, das Leben zu retten, die Gesundheit wiederherzustellen oder Leiden zu lindern. Diese Maßnahme sollte anschließend Gegenstand von Forschung werden, die so konzipiert ist, dass ihre Sicherheit und Wirksamkeit bewertet werden können. In allen Fällen müssen neue Informationen aufgezeichnet und, sofern angemessen, öffentlich verfügbar gemacht werden. © 2013 Weltärztebund (World Medical Association, WMA), alle Rechte vorbehalten. Alle Urheberrechte der Deklaration von Helsinki liegen beim Weltärztebund. Vormalige Genehmigungen zur Veröffentlichung oder Vervielfältigung anderer WMA Positionen bis zum 1.

Januar 2014 beziehen sich nicht auf diese Version der Deklaration von Helsinki.

Bei weiteren Fragen, wenden Sie sich bitte an das WMA Sekretariat unter secretariat@wma.net.

© World Medical Association, Inc. - All rights reserved. © Weltärztebund, World Medical Association, WMA – alle Rechte vorbehalten

Ein Service des Bundesministeriums der Justiz und für Verbraucherschutz sowie des Bundesamts für Justiz ‒ www.gesetze-im-internet.de

Verordnung über das Inverkehrbringen von Arzneimitteln ohne Genehmigung oder ohne Zulassung in Härtefällen (ArzneimittelHärtefall-Verordnung - AMHV) AMHV Ausfertigungsdatum: 14.07.2010 Vollzitat: "Arzneimittel-Härtefall-Verordnung vom 14. Juli 2010 (BGBl. I S. 935)" Fußnote (+++ Textnachweis ab: 22.7.2010 +++)

Eingangsformel

Auf Grund des § 80 Satz 1 Nummer 3a in Verbindung mit Satz 3 und 4 und in Verbindung mit § 83 des Arzneimittelgesetzes, von denen § 83 zuletzt durch Artikel 1 Nummer 70 des Gesetzes vom 17. Juli 2009 (BGBl. I S. 1990) geändert worden ist, verordnet das Bundesministerium für Gesundheit: § 1 Anwendungsbereich (1) Diese Verordnung regelt das Verfahren für das Inverkehrbringen von Arzneimitteln ohne Genehmigung oder ohne Zulassung in Härtefällen unter den Voraussetzungen, die in § 21 Absatz 2 Nummer 6 des Arzneimittelgesetzes in Verbindung mit Artikel 83 der Verordnung (EG) Nr. 726/2004 des Europäischen Parlaments und des Rates vom 31. März 2004 zur Festlegung von Gemeinschaftsverfahren für die Genehmigung und Überwachung von Human- und Tierarzneimitteln und zur Errichtung einer Europäischen ArzneimittelAgentur (ABl. L 136 vom 30.4.2004, S. 1) genannt werden. Die Verordnung gilt für Fertigarzneimittel, die zur Anwendung im oder am Menschen bestimmt sind und weder im Geltungsbereich des Arzneimittelgesetzes noch in einem anderen Mitgliedstaat der Europäischen Union oder einem anderen Vertragsstaat des Abkommens über den Europäischen Wirtschaftsraum genehmigt oder zugelassen sind, aber der Genehmigungspflicht nach Artikel 3 Absatz 1 und 2 der Verordnung (EG) Nr. 726/2004 oder der Zulassungspflicht nach § 21 Absatz 1 des Arzneimittelgesetzes unterliegen.

(2) Diese Verordnung ist nicht anzuwenden für den individuellen Einsatz eines nicht genehmigten oder nicht zugelassenen Arzneimittels bei nur einer Patientin oder einem Patienten unter der unmittelbaren Verantwortung einer Ärztin oder eines Arztes. § 2 Begriffsbestimmungen (1) Ein Härtefallprogramm ist ein „compassionate-use“-Programm nach der Verordnung (EG) Nr. 726/2004.

Im Rahmen eines Härtefallprogramms können Arzneimittel ohne Genehmigung oder ohne Zulassung einer bestimmten Patientengruppe zur Verfügung gestellt werden, wenn ausreichende Hinweise auf die Wirksamkeit und Sicherheit des Arzneimittels vorliegen und für dieses eine klinische Prüfung durchgeführt wird oder ein Zulassungsantrag bei der Europäischen Arzneimittel-Agentur, der zuständigen Bundesoberbehörde oder einer für die Zulassung zuständigen Behörde eines Mitgliedstaates gestellt worden ist.

(2) Ein Härtefall im Sinne dieser Verordnung liegt vor, wenn eine Gruppe von Patientinnen oder Patienten, die an einer Erkrankung leiden, welche zu einer schweren Behinderung führen würde oder lebensbedrohend ist, nicht mit einem Arzneimittel zufriedenstellend behandelt werden kann, das zum Inverkehrbringen im Geltungsbereich des Arzneimittelgesetzes genehmigt oder zugelassen ist. § 3 Anzeige des Härtefallprogramms (1) Eine Person, die die Verantwortung für die Veranlassung, die Organisation und Finanzierung eines Härtefallprogramms übernimmt (verantwortliche Person), hat das Härtefallprogramm der zuständigen Bundesoberbehörde anzuzeigen. Hat die verantwortliche Person ihren Sitz nicht in einem Mitgliedstaat - Seite 1 von 5 -

Ein Service des Bundesministeriums der Justiz und für Verbraucherschutz sowie des Bundesamts für Justiz ‒ www.gesetze-im-internet.de

der Europäischen Union oder in einem anderen Vertragsstaat des Abkommens über den Europäischen Wirtschaftsraum, so benennt sie eine Vertreterin oder einen Vertreter mit Sitz in der Europäischen Union oder in einem anderen Vertragsstaat des Abkommens über den Europäischen Wirtschaftsraum.

(2) Für die Anzeige sind folgende Angaben und Unterlagen erforderlich: 1.

2.

3.

4.

5.

6.

7.

8.

9.

Name oder Firma sowie die Anschrift der verantwortlichen Person und, sofern vorhanden, des in der Europäischen Union oder in einem anderen Vertragsstaat des Abkommens über den Europäischen Wirtschaftsraum niedergelassenen Vertreters, Bezeichnung oder Code des Arzneimittels, Bezeichnung der Wirkstoffe nach Art und Menge, sonstige Bestandteile nach der Art, Darreichungsform, Art der Anwendung, Dosierung und Behandlungsschema,

Beschreibung der zu einer schweren Behinderung führenden oder lebensbedrohenden Erkrankung, an der die Patientinnen und Patienten leiden und für die das Arzneimittel bestimmt ist,

Kriterien für die Auswahl der Patientinnen und Patienten und Angabe der voraussichtlichen Patientenanzahl,

Begründung, dass diese Patientinnen und Patienten nicht zufriedenstellend mit einem zum Inverkehrbringen im Geltungsbereich des Arzneimittelgesetzes genehmigten oder zugelassenen Arzneimittel behandelt werden können,

Begründung dafür, dass die Patientinnen oder Patienten in eine laufende klinische Prüfung nicht einbezogen werden können,

Nachweise, dass das Arzneimittel die nach den anerkannten pharmazeutischen Regeln angemessene Qualität aufweist, sowie eine Bestätigung der sachkundigen Person nach § 14 des Arzneimittelgesetzes, dass das Arzneimittel nach den Grundsätzen und Leitlinien der Guten Herstellungspraxis für Arzneimittel hergestellt ist,

Belege und Begründung für die Annahme, dass das Arzneimittel sicher und wirksam bei der vorgesehenen Anwendung ist, in der Regel durch Vorlage der Ergebnisse konfirmatorischer klinischer Prüfungen,

Kriterien für das Aussetzen oder vorzeitige Beendigen des Härtefallprogramms,

10. Nähere Angaben

a) zur genehmigten klinischen Prüfung des Arzneimittels im vorgesehenen Anwendungsgebiet mit Angabe der EudraCT-Nummer oder b) zur klinischen Prüfung des Arzneimittels im vorgesehenen Anwendungsgebiet in einem Drittstaat und Nachweis, dass diese nach den international harmonisierten Standards der Guten Klinischen Praxis durchgeführt wird, oder

c) über den Genehmigungs- oder Zulassungsantrag, der für das Arzneimittel in dem vorgesehenen Anwendungsgebiet bei der Europäischen Arzneimittel-Agentur, der zuständigen Bundesoberbehörde oder einer für die Zulassung zuständigen Behörde eines Mitgliedstaates gestellt wurde,

11. bei Arzneimitteln, die aus einem gentechnisch veränderten Organismus oder aus einer Kombination von gentechnisch veränderten Organismen bestehen oder solche enthalten, Unterlagen gemäß Anhang II und III der Richtlinie 2001/18/EG des Europäischen Parlaments und des Rates vom 12. März 2001 über die absichtliche Freisetzung genetisch veränderter Organismen in die Umwelt und zur Aufhebung der Richtlinie 90/220/EWG des Rates (ABl. L 106 vom 17.4.2001, S. 1),

12. Begründung für eine Behandlung mit einem Arzneimittel, für das ein Antrag auf Genehmigung oder Zulassung des Arzneimittels versagt, zurückgenommen, widerrufen oder ruhend gestellt worden ist oder die Genehmigung einer klinischen Prüfung versagt, zurückgenommen, widerrufen oder ruhend gestellt oder unter Nebenbedingungen erteilt worden ist, unter Angabe der Entscheidungsgründe,

13. die in der klinischen Prüfung der Prüferin oder dem Prüfer zur Verfügung gestellte aktuelle Prüferinformation oder der im Antrag auf Zulassung oder Genehmigung vorgesehene Entwurf einer Zusammenfassung der Produktmerkmale des Arzneimittels,

14. Informationen und Unterlagen, die die Patientinnen und Patienten erhalten, in deutscher Sprache sowie eine Darstellung des Verfahrens der Einwilligung nach Aufklärung durch eine teilnehmende Ärztin oder einen teilnehmenden Arzt,

15. Anforderungen an die ärztlichen Einrichtungen und an die Qualifikation der teilnehmenden Ärztinnen und Ärzte,

- Seite 2 von 5 -

Ein Service des Bundesministeriums der Justiz und für Verbraucherschutz sowie des Bundesamts für Justiz ‒ www.gesetze-im-internet.de

16. Beschreibung der vorgesehenen Maßnahmen zur Gewährleistung einer sicheren Aufbewahrung, Anwendung sowie des Verbleibs der zur Verfügung gestellten Arzneimittel, 17. Angaben, soweit zutreffend, zu bereits laufenden Härtefallprogrammen in Mitgliedstaaten der Europäischen Union oder anderen Vertragsstaaten des Abkommens über den Europäischen Wirtschaftsraum und, soweit vorhanden, Gutachten des Ausschusses für Humanarzneimittel nach Artikel 83 Absatz 4 der Verordnung (EG) Nr. 726/2004,

18. Einverständniserklärung dafür, die Information über die Grundzüge des angezeigten Härtefallprogramms an die Öffentlichkeit weiterzugeben.

(3) Die Anzeige und die erforderlichen Unterlagen nach Absatz 2 bedürfen der Schriftform und der elektronischen Form. Die Unterlagen können in deutscher oder englischer Sprache eingereicht werden; davon ausgenommen sind die Unterlagen nach Absatz 2 Nummer 14.

(4) Die zuständige Bundesoberbehörde veröffentlicht Hinweise zur Einreichung der Angaben und Unterlagen auf ihrer Website. § 4 Beginn des Härtefallprogramms, Widerspruch (1) Die zuständige Bundesoberbehörde bestätigt der verantwortlichen Person innerhalb von zwei Wochen den Eingang der ordnungsgemäßen Anzeige unter Angabe des Eingangsdatums (bestätigte Anzeige). Wenn Unterlagen zur Anzeige fehlen oder die Anzeige aus sonstigen Gründen nicht ordnungsgemäß ist, fordert die zuständige Bundesoberbehörde die verantwortliche Person ebenfalls innerhalb von zwei Wochen auf, diese Formmängel zu beheben. Die zuständige Bundesoberbehörde bestätigt der verantwortlichen Person innerhalb von zwei Wochen nach Eingang der nachgereichten Unterlagen den Eingang der ordnungsgemäßen Anzeige (bestätigte Anzeige), sofern die Anzeige nun vollständig und ordnungsgemäß ist.

(2) Mit der Durchführung des Härtefallprogramms kann begonnen werden, sobald die bestätigte Anzeige zugegangen ist und die zuständige Bundesoberbehörde nicht widersprochen hat.

(3) Die zuständige Bundesoberbehörde kann widersprechen, wenn die Voraussetzungen für die Durchführung des Härtefallprogramms nicht vorliegen oder Anhaltspunkte dafür vorliegen, dass die gemachten Angaben unrichtig sind oder dass keine sichere Anwendung des Arzneimittels gewährleistet ist. Die zuständige Bundesoberbehörde kann auch nachträglich widersprechen, wenn die in Satz 1 genannten Gründe nach dem Beginn des Härtefallprogramms eintreten. Hat die zuständige Bundesoberbehörde widersprochen, darf das Härtefallprogramm nicht begonnen oder weitergeführt werden.

(4) Bezieht sich die Anzeige auf ein Arzneimittel für neuartige Therapien oder auf ein Arzneimittel, das von der zuständigen Bundesoberbehörde noch in keinem Genehmigungsverfahren einer klinischen Prüfung bewertet worden ist, gilt abweichend von Absatz 1 eine angemessene Frist, die 60 Tage nicht überschreiten soll.

(5) Bei Arzneimitteln, die aus einem gentechnisch veränderten Organismus oder aus einer Kombination von gentechnisch veränderten Organismen bestehen oder solche enthalten, umfasst die Genehmigung der klinischen Prüfung durch die zuständige Bundesoberbehörde auch die Genehmigung der Freisetzung dieser gentechnisch veränderten Organismen im Rahmen des Härtefallprogramms. Dies gilt nur, soweit keine Änderungen gegenüber der genehmigten klinischen Prüfung vorgenommen werden, die geeignet sind, die Risikobewertung für die Gesundheit Dritter und für die Umwelt zu verändern. Bei Änderungen, die geeignet sind, die Risikobewertung für die Gesundheit Dritter und für die Umwelt zu verändern, ist von der zuständigen Bundesoberbehörde im Benehmen mit dem Bundesamt für Verbraucherschutz und Lebensmittelsicherheit innerhalb einer angemessenen Frist zu entscheiden. Abweichend von Absatz 2 und 4 darf ein Härtefallprogramm mit Arzneimitteln, für die eine Risikobewertung nach Satz 2 erforderlich ist, erst mit Zugang der hierfür erforderlichen Genehmigung begonnen werden. § 5 Dauer des Härtefallprogramms, erneute Anzeige (1) Das Härtefallprogramm endet unbeschadet von § 4 Absatz 3 mit dem Abbruch durch die verantwortliche Person oder mit der tatsächlichen Verfügbarkeit des Arzneimittels auf dem Markt, spätestens jedoch ein Jahr nach Zugang der bestätigten Anzeige oder der nach § 4 Absatz 5 erforderlichen Genehmigung.

(2) Eine erneute Anzeige ist zulässig. Dabei kann auf bereits vorgelegte Unterlagen Bezug genommen werden, soweit sich keine Änderungen hierzu ergeben haben.

- Seite 3 von 5 -

Ein Service des Bundesministeriums der Justiz und für Verbraucherschutz sowie des Bundesamts für Justiz ‒ www.gesetze-im-internet.de

§ 6 Mitteilungspflichten der verantwortlichen Person (1) Die verantwortliche Person nach § 3 Absatz 1 hat 1. jeden Verdachtsfall einer schwerwiegenden Nebenwirkung, der ihr von den teilnehmenden Ärztinnen und Ärzten mitgeteilt oder ihr auf anderem Weg zur Kenntnis gebracht worden ist, zu dokumentieren und der zuständigen Bundesoberbehörde unverzüglich, spätestens jedoch innerhalb von 15 Tagen nach Bekanntwerden, elektronisch mitzuteilen, 2. jede Änderung der Angaben nach § 3 der zuständigen Bundesoberbehörde unverzüglich anzuzeigen und die entsprechenden Unterlagen beizufügen,

3. der zuständigen Bundesoberbehörde unverzüglich den Abbruch des Härtefallprogramms unter Angabe der Gründe mitzuteilen,

4. nach Abschluss des Härtefallprogramms der zuständigen Bundesoberbehörde einen Sicherheitsbericht vorzulegen, der insbesondere alle schwerwiegenden Nebenwirkungen und alle nicht schwerwiegenden unerwarteten Nebenwirkungen erfasst und bewertet,

5. der zuständigen Bundesoberbehörde neue Gutachten nach Artikel 83 Absatz 4 der Verordnung (EG) Nr. 726/2004 und Erkenntnisse aus anderen Härtefallprogrammen, die in einem anderen Mitgliedstaat der Europäischen Union oder in einem anderen Vertragsstaat des Abkommens über den Europäischen Wirtschaftsraum durchgeführt werden, unverzüglich vorzulegen oder mitzuteilen.

(2) Änderungen, die sich auf das Anwendungsgebiet, die Stärke oder die Darreichungsform des Arzneimittels im Rahmen des Härtefallprogramms beziehen sowie Änderungen, die geeignet sind, sich auf die Sicherheit der Patientinnen und Patienten auszuwirken, dürfen erst mit Zugang einer erneuten bestätigten Anzeige nach § 4 vollzogen werden.

(3) Die verantwortliche Person hat dafür Sorge zu tragen, dass Arzneimittelrisiken der zuständigen Bundesoberbehörde unverzüglich mitgeteilt werden. Sie hat die zur Risikoabwehr erforderlichen Maßnahmen unverzüglich zu ergreifen. § 7 Verantwortungsbereich der verantwortlichen Person Die verantwortliche Person nach § 3 Absatz 1 hat sicherzustellen, dass 1. die Umsetzung des Härtefallprogramms ordnungsgemäß erfolgt, 2. alle Bedingungen und Einschränkungen hinsichtlich der sicheren und wirksamen Anwendung des Arzneimittels erfüllt werden und den beteiligten Personen die hierfür erforderlichen Informationen gegeben werden,

3. das Arzneimittel nur in Verkehr gelangt, wenn auf den Behältnissen und soweit verwendet auf den äußeren Umhüllungen mindestens folgende Angaben enthalten sind:

a) Bezeichnung oder Code des Arzneimittels, b) Name und Anschrift der verantwortlichen Person,

c) Chargenbezeichnung,

d) Art der Anwendung,

e) Wirkstoffbezeichnung,

f) Verfalldatum,

g) soweit erforderlich, Aufbewahrungs- und Lagerungshinweise,

h) Hinweis, dass das Arzneimittel ohne Genehmigung oder Zulassung im Rahmen eines Härtefallprogramms zur Verfügung gestellt wird,

4. die Herstellung, Einfuhr und Freigabe nach den in der Arzneimittel- und Wirkstoffherstellungsverordnung vorgesehenen Regelungen für Prüfpräparate erfolgt,

5. die wesentlichen Unterlagen des Härtefallprogramms nach dessen Beendigung oder Abbruch mindestens zehn Jahre aufbewahrt werden; andere Vorschriften zur Aufbewahrung von medizinischen Unterlagen bleiben unberührt.

- Seite 4 von 5 -

Ein Service des Bundesministeriums der Justiz und für Verbraucherschutz sowie des Bundesamts für Justiz ‒ www.gesetze-im-internet.de

§ 8 Informationspflichten der zuständigen Bundesoberbehörde (1) Bei Arzneimitteln, die aus einem gentechnisch veränderten Organismus oder aus einer Kombination von gentechnisch veränderten Organismen bestehen oder solche enthalten, informiert die zuständige Bundesoberbehörde das Bundesamt für Verbraucherschutz und Lebensmittelsicherheit über den Eingang der Anzeige.

(2) Die zuständige Bundesoberbehörde informiert die Europäische Arzneimittel-Agentur über ihr angezeigte Härtefallprogramme.

(3) Die zuständige Bundesoberbehörde stellt sicher, dass jede ihr angezeigte vermutete schwerwiegende Nebenwirkung erfasst und der Europäischen Arzneimittel-Agentur unverzüglich, spätestens jedoch innerhalb von 15 Tagen nach Erhalt der Information mitgeteilt wird.

(4) Die zuständige Bundesoberbehörde stellt der Öffentlichkeit Informationen über ihr angezeigte Härtefallprogramme zur Verfügung. § 9 Übergangsregelung Härtefallprogramme, die bei Inkrafttreten der Verordnung durchgeführt werden, können unabhängig von den Vorgaben dieser Verordnung weitergeführt werden. § 10 Inkrafttreten Diese Verordnung tritt am Tag nach der Verkündung in Kraft.

- Seite 5 von 5 -

(Deutsches Deckblatt)

EXPERIMENTELLE FORSCHUNGSARBEITEN DER ECUADORIANISCHEN VEREINIGUNG VON FACHÄRZTEN FÜR INTEGRATIVE MEDIZIN.

CHLORDIOXID EINE WIRKSAME THERAPIE ZUR BEHANDLUNG VON SARS-COV2 (COVID19) PRÄSENTIERT VON AEMEMI Guayaquil, Mai 2020 https://www.aememiecuador.org/

Ärzte von AEMEMI (Ecuador) stellen wässrige Chlordioxidlösung vor.

Herstellung von Chlordioxidlösung in Krankenhausapotheke.

Verkauf von Chlordioxidlösung in einer südamerikanischen Apotheke.

Straßenverkauf von CDS (Chlorine Dioxide Solution), dt. CDL=Chlordioxidlösung.