Documented use of chlorine dioxide (ClO₂) in academic literature dates back to 1946¹ when ClO₂ began to be used for paper bleaching and to purify water because of its oxidative power to eliminate pollutants and remove the unpleasant taste and odor from water.² In the 60s, several studies began to explore the kinetics, mechanisms, and effectiveness of ClO₂ as a disinfectant against bacteria, viruses, and protozoa.^3,4

Furthermore, hypotheses identifying ClO₂ as a possible therapeutic agent based on its oxidative properties began to emerge, and studies on the acute and chronic toxicology of ClO₂ and its metabolites in animals began to be published.⁵ Since then, several patents concerning the use of ClO₂ and its derivatives as a treatment for several diseases (including HIV) were published without solid evidence support.⁶

In 2006, a book claimed that a product made of a sodium chlorite solution in combination with citric or hydrochloric acid (known as a "Miracle Mineral Solution" or MMS) was able to cure malaria and other diseases.⁷ These claims on MMS were later amplified on the internet, particularly as a treatment for cancer and autism.⁸ Nevertheless, the increase in reports of adverse events linked to the consumption of MMS —including vomiting and diarrhea— resulted in warnings against its use issued by the Food and Drug Administration (FDA) in 2010 and by the regulatory agencies of other countries.⁹

In 2020, with the surge of the COVID-19 pandemic, the use of products made of ClO₂ and related compounds as a treatment for the disease gained popularity on social media platforms. A group of supporters, such as the World Health and Life Coalition (COMUSAV, allegedly integrated by health professionals, mainly from Spain and Latin American countries), argued in favor of its innocuity and efficacy, lobbying governments and promoting "treatment protocols" for the use of a chlorine dioxide solution (CDS), gaining thousands of followers.¹⁰

Given this renewed interest in ClO₂, some authors have collected information regarding the available evidence of this compound as a human treatment. In their 2020 systematic review, Burela et al.¹¹ did not find publications evaluating ClO₂ and its derivatives as prophylactic or curative agents for COVID-19 or other coronavirus infections. Authors reported not having found sufficient clinical evidence to support the effectiveness of ClO₂ against COVID-19; however, other research groups kept arguing the opposite.¹²

Due to the conflicting evidence, in this scoping review, we systematically identified, characterized, and synthesized the available evidence (animal and human studies) associated with the toxicity of ClO₂, their related compounds, and their efficacy as a treatment for diseases. This study aims to provide comprehensive answers for the scientific public and lay people regarding which information can be relied upon on the issue of ClO₂ consumption at the moment.

Literature Source and Search Strategy

A scoping systematic review of academic literature following an adapted version of the PRISMA guidelines was developed to integrate academic literature on chlorine dioxide's efficacy and toxicity outcomes in animal and human studies.

The systematized search collected English, Spanish, French, Portuguese, and German records from MEDLINE (PubMed) and EMBASE from the databases' origins to October 20, 2020. The search strategy included MeSH terms and keywords focusing on chlorine dioxide's therapeutic use and toxicity (Suppl. Table 1).

Screening

Peered-review screening (IR, IL, LM) was subsequently performed by title, abstract, and full-text.

Inclusion and Exclusion Criteria

This study included academic articles (indexed in journals, including letters to the editor) but not grey literature (press publications and reports lacking peer review). Systematic reviews were screened for single pertinent articles, which were included manually.

Studies were only included if ClO₂ or its precursors (MMS in particular) were part of the studied substances; byproducts were only considered when resulting from the exposure or administration of the first. There was no predefined study design limitation for the inclusion of studies regarding toxicity and efficacy outcomes (no disease target was specified either).

For animal studies, only the systemic route of administration was considered (it was deemed the closest to oral and parenteral use, which was our outcome of interest in humans). For human studies, the administration route included systemic (oral and parenteral), topical, and buccopharyngeal administration.

Publications regarding uses for water bleaching, surface cleaning (disinfectant properties), or purifying properties of ClO₂ were excluded. In vitro, plant, and insect studies were not part of the scope of the study. Protocols and articles with unavailable full-text were also excluded.

Extraction and Data Synthesis

Four researchers extracted relevant data on the publication and study characteristics.

The studied variables for animal studies included the type and number of animals, study type, administration route, tested substances, exposure and dosage, control group, measurements, and outcomes of interest.

Human case reports described individuals involved, exposure context and formulation, toxic or adverse effects, treatment, monitoring, and mechanism behind toxicity. Clinical studies included information on the study design, participants, intervention (formulation, route of administration, and dosage), results, and conclusions.

Methodological Quality and Risk of Bias

Each of the selected animal studies was evaluated for potential sources of selection, performance, detection, attrition, and reporting bias (Suppl. Table 2) using elements adapted from the CAMARADES¹³ and SYRCLE¹⁴ risk of bias tools (A. Animal allocation & randomization, B. Blinding, C. Population size, D. Outcome reporting, complete follow-up, and E. Compliance with animal welfare requirements).

Two critical appraisers evaluated clinical studies using the Joanna Briggs Institute (JBI) risk bias evaluation tools¹⁵ for case reports, case-controls, case series, quasi-experiments, and randomized controlled trials (RCT). JBI appraisal final scoring can be found in Supplementary Tables 3 (A-E).

A total of 752 articles were identified through the systematized search. After duplicate removal and screening (title, abstract, and full text), a total of 34 studies were included: 15 in animals and 19 in humans (Figure 1).

Animal Studies

A total of 15 in vivo studies in animals published between 1980 and 2020 were identified (Table 1). Chlorine dioxide (n=10) and sodium chlorite (NaClO₂) (n=9) were more frequently tested in animal studies, followed by other chloride species.

Most studies were performed in rats (n=8) and mice (n=5); however, there were also other ones on monkeys (n=2) and chicken embryos (n=1). The majority of animal studies (14 out of 15) aimed to investigate the systemic toxicity of ClO₂.

Seven studies assessed reproductive and developmental toxicity; six studies investigated toxic effects on thyroid function; four studies evaluated hematologic biomarkers of toxicity, and one studied genotoxicity (n=2), nephrotoxicity (n=2), and immunotoxicity (n=1). Harrington et al.²⁷ ran the most comprehensive study (1995) where practically all organs, systems, and tissues were examined after testing oral exposure to NaClO₂ in a rat model.

Regarding ClO₂, only Moore et al. 1982¹⁸ found no effects on the reproduction or development of mice, while other studies found a wide range of abnormalities at different concentrations (Table 1). Sodium chlorite also demonstrated detrimental effects in mice by decreasing the conception rate and affecting pups' weight and growth rate.

Chlorine dioxide affected the thyroid function at different doses in rats and monkeys in studies published in the 80s. Nevertheless, studies in rats and mice in the early 90s and 2000s did not replicate these findings for ClO₂ or NaClO₂.

Hematological toxic effects were not observed in monkeys, mice, or rats exposed to ClO₂. Nonetheless, other studies in mice and monkeys reported that NaClO₂ did derive in hematological alterations. Only Gill et al.²⁸ formally reported a "no observable effect level" (NOEL) of NaClO₂ regarding the effects on reproduction and thyroid toxicity biomarkers and a "no observed adverse effect level" (NOAEL) for hematological and neurotoxicity.

Chlorine dioxide and NaClO₂ were found to be potential genotoxins; however, they were not identified as a cause of nephrotoxicity in mice or rats. In addition, slight immunotoxic effects were observed after exposure to NaClO₂, and only OCl-, one of the byproducts of chlorine at normal drinking water pH, showed to be a potential mutagenic chemical. No studies investigated immunotoxicity caused by ClO₂.

Solely one pre-clinical efficacy study was found and included in our scoping review. Zambrano-Estrada et al.³⁰ infected 30 chick embryos with avian coronavirus. After treating them with ClO_2, they observed lower viral titers, decreased mortality, and significantly less severe lesions without evidence of ClO₂ toxicity.

After assessing the selected study-quality items, most studies showed satisfactory quality (Suppl Table 2). Population size and outcome reporting were described by most, but not all of the studies; others did not inform drop-outs, deviations from the protocol, or how they were handled. Regarding the risk of bias, 7 out of 15 studies did not specify animal allocation and randomization. Moreover, the blinding of study personnel and outcome assessors were not stated in any studies.

Case Reports

The review identified 13 case reports in humans (Table 2). Most of them (85%) were published between 2012 and 2020, involving seven men, four women, and three children. Four reports were related to a ClO₂ product exposure, nine referred to a NaClO₂ exposure, and one referred to both. At least six reports identified the ingestion of a commercially-developed product. Oral exposure was the most frequent one (77%); other exposures included inhalation, topical exposure, and oral exposure in combination with intravenous administration.

Most of the exposures to these substances were intentional (69.2%). In some cases, different concentrations, presentations, and dilutions of NaClO₂ and ClO₂ (Table 2) were used/consumed to treat diseases such as prostate cancer, a cutaneous fungal infection, gastroenteritis in a patient with idiopathic thrombocytopenic purpura, hypertension with concomitant COVID-19, or for unspecified reasons.

Specifically, in the context of COVID-19, there was a fatal case due to the intentional ingestion of complementary therapy (ClO₂ solution with intravenous saline solution and NaClO₂). Likewise, during the pandemic, two 8- and 9-year-old siblings accidentally ingested an undiluted sodium chlorite solution; the youngest had mild symptoms, but the oldest suffered severe poisoning that kept him hospitalized for several weeks until he recovered.

Adverse events were reported in five out of seven voluntary consumption cases; three of seven experienced toxic effects due to an erroneous dosage consumption.

Additionally, there were six accidental exposures and two suicide attempts. Only in 5 out of 13 cases was possible to know or estimate the amount of ClO₂ or NaClO₂ the patients consumed. In such cases, it was evident that the doses were much higher than permissible or recommended by the manufacturers of the products.

Overall, all case reports showed good methodological quality after critical appraisal using the corresponding JBI checklist (Supp. Table 3-A); almost two-thirds (62%) of them reached the highest possible score.

Clinical Studies

The review identified six clinical studies: one case series, two case controls, one quasi-experimental study, and two RCTs (Table 3).

Four studies employed topical administration of ClO_2, and only two referred to systemic administration.

Regarding topical treatment, the case series consisted of a descriptive study on female patients with keratosis pilaris who were given a ClO₂-based commercial foam cleanser. This study scored very low in terms of quality and reliability of the validity of its results (Suppl. Table 3). No control group was included, and the total duration of treatment for each patient was not clearly explained.

Likewise, unstandardized reports of variability in size and resolution time of the corporal papules were employed as confirmation of some patients who showed improvements.

Furthermore, inclusion/exclusion criteria, patient demographics, validated methods for diagnosing the condition and assessing outcomes, and information about the consecutive and complete inclusion of participants were missing or not clearly stated.

Regarding topical treatment, two RCTs conducted by the same research team and reporting outcomes for the same group of 15 male volunteers aimed to assess the efficacy of rinsing with a mouthwash based on ClO₂ (single dose) in order to inhibit morning oral malodor (halitosis). The 7-day trial study also measured the effect of mouthwash on plaque accumulation and in other biomarkers of periodontal diseases; both showed significant improvements.

The two RCTs obtained the highest score in terms of methodological quality and analysis of the risk of bias according to the corresponding JBI checklist.

Paraskevas et al. 2008⁴⁵ also evaluated a ClO₂ mouth rinse product in the form of a case-control study with 77 subjects. They concluded that it could reduce plaque accumulation; however, it was less effective than a chlorhexidine-based control product.

Lubbers et al. 1982⁴⁴ conducted a case-control study of acute and subchronic toxicity in a total of 123 male volunteers to assess tolerability to ClO₂ and four other disinfectants diluted in water for oral ingestion.

Several biochemical parameters showed changes possibly related to the ingestion of one of the disinfectants in the different phases of the study. Still, in general terms, no physiologically relevant alterations were observed in any of the study phases. The authors emphasized the need for further studies assessing extended treatment periods and broader group sizes.

The two case-control studies included in our review also obtained the highest score after evaluating them with the corresponding JBI checklist.

Finally, the most recent quasi-experimental efficacy study by Insignares-Carrione et al.¹² sought to demonstrate the effectiveness of oral ingestion of ClO₂ in the complementary management of 40 patients with COVID-19; twenty of them decided to ingest the self-prepared experimental treatment in comparison with other 20 control patients who disagreed.

The study groups had different follow-up periods and displayed unclear or unspecified inclusion/exclusion criteria.

Specific outcomes were assessed through laboratory tests (Table 3) and subjective scales such as the Likert and visual analog scales.

Patient oxygenation levels were missing despite the authors stating that they did measure them.

The authors concluded that ClO₂ effectively managed COVID-19 without relevant adverse effects and that future double-blind studies were required. According to the JBI checklist for quasi-experimental studies, this study had a moderate methodological design quality and risk of bias.

A total of 34 toxicological and clinical efficacy studies on the use of ClO₂ and related compounds as treatment in animals and humans were identified. Almost half of the studies (47%) were more than 20 years old. Most human studies were toxicity case reports (68%), and only 6 were clinical studies. Regarding methodological quality and risk of bias, 56% (19 out of 34 studies) of the total animal and human studies displayed good quality (upper quintile). In contrast, the others did not comply with the quality elements considered necessary in each type of study.

Toxicological Evidence on Animals Should Be Noted

Chlorine dioxide and NaClO₂ were the most studied chemicals in animals, presenting relevant findings on toxicity. However, since 1991 there have been no new publications concerning these studies.

Given the progress in technology and scientific development, better pre-clinical evidence (including non-rodent mammalian models) regarding ClO₂ toxicity will undoubtedly be needed to update procedures, biomarkers employed to investigate various forms of toxicity, different routes of administration, pharmacokinetic profiles, and parameters. The latter, such as the NOAEL values obtained in pre-clinical studies, should be divided by uncertainty factors to scale the safety margins from the "most sensitive" of the tested species to human dose levels.

Such escalation is fundamental to either developing clinical efficacy studies (if safe for human consumption) or establishing reference consumption doses (in case of accidental ingestion).⁴⁹ This should not be done conversely, without scaling to design or justify dosage regimens directly in humans, such as the presented cases in which ClO₂ promoters justify its use in humans (specifically for COVID-19 treatment) by arguing that a NOAEL (after oral ingestion) reported in a reproduction toxicity study in rats can be extrapolated to a "safe" human consumption dose in adults.^50,51

Additionally, the authors of this review believe that information on routes of administration in animals is still incomplete, as only a few studies of oral and intravenous administration were identified. Besides, knowledge of potential target organs and systems is needed, which can be complemented with a more comprehensive panel of animal tests for this substance (in different species), such as carcinogenicity studies, specific genetic toxicology assays, and immunotoxicity studies, which were absent in the reviewed literature.

Our review also identified specific gaps in outcome reporting in the included animal studies, which decreases the strength of the generated evidence and the interpretations that can derive from the experimental outcomes.

We identified that randomization, allocation concealment, and blinded outcome assessments to eliminate potential bias in individual experiments⁵² were rarely reported in animal studies.

The absence of these elements can be partially explained by the fact that guidelines for quality reporting are relatively recent in animal experimentation. Most likely, some of these publications cannot be taken as solid evidence until these issues are fully taken care of.⁵³

Only one modern animal study aimed to report the efficacy outcomes of ClO₂ as a treatment for COVID-19³⁰, and successfully complied with quality reporting elements. Nevertheless, it is still only available as a pre-print, potentially indicating that it has not been peer-reviewed.

In addition, out of the time frame of this review, a recent study in mice, intended as a safety evaluation of a commercial product liberating aqueous ClO₂ and used to treat halitosis, concluded that if ingested, this product could cause multiorgan failure (the quality of this study, however, could not be evaluated).⁵⁴

In this review, animal studies demonstrated increasingly higher critical appraisal scores as the years of publication approached the present, potentially meaning they had a more careful experimental design. Nevertheless, this does not always apply, as we found a newly published low-quality narrative review citing in vitro, animal, and human studies to justify the nasal irrigation of ClO₂ for the prevention of COVID-19.⁵⁵ This publication did not account for any of the JBI critical appraisal checklist points.

Animal studies represent the critical step before translation to clinical studies; therefore, researchers lacking complete and reliable data reports on animals (mainly basing themselves on data from older studies) should refrain from continuing with human trials.⁵⁶

How Much Is Enough, and Under What Circumstances?

Chlorine dioxide may not be as toxic to humans as other substances. Still, as seen in this review, there is always a possibility of accidentally reaching a threshold of ClO₂ toxicity (overdose) by deviating from the product preparation indications, such as not diluting ClO₂ in water.

Additionally, cumulative exposure to ClO₂ through disinfected water consumption should be reviewed. It is also possible that preconditions may play a role in exacerbating potential toxic effects after the ingestion of miracle cures for illnesses despite not exceeding the toxicity thresholds calculated by the manufacturers.

Particular attention should be paid to warning labels since products based on ClO₂ or NaClO₂ are used as disinfectants or acquired for therapeutic purposes. These represent a latent risk of accidental ingestion for children, which could derive in severe or even lethal consequences.

Clinical efficacy studies for any substance should always emphasize the need to collect and analyze toxicity outcomes, as some authors have noted that this data is sometimes overlooked in studies in progress.⁵⁷

Our review also includes evidence for the management of exposure/intoxication due to ClO₂ in different circumstances, which may be of use to guide clinical decision-making in similar situations.

This information can be beneficial during the COVID-19 pandemic due to the booming demand for prophylactic or curative products based on ClO₂ and other chlorine derivatives, sometimes resulting in adverse events.⁵⁸ Of note, other human toxicity reports added to the piling evidence amid the COVID-19 pandemic.^59-62

An Effective Treatment?

In general, three clinical studies displayed high methodological quality scores and were focused on investigating the topical use of ClO₂ for oral health management.^45-47 However, only two studies dealt with the systemic use of ClO₂; this also excluded high-quality clinical safety study (according to the JBI tool) by Lubbers et al., 1982.⁴⁴ The latter should be replicated and expanded, focusing on ClO₂; likewise, it should measure a broader panel of biomarkers using different doses and more extensive exposure periods.

Recent reviews and meta-analyses of RCTs concerning the daily use of ClO₂ to effectively treat halitosis have been published. They conclude that this remedy is effective and does not present adverse effects⁶³, but they are also cautious, suggesting longer follow-up times in similar experimental designs.⁶⁴

Finally, the quasi-experimental clinical study by Insignares-Carrione et al.¹², seeking to demonstrate the effectiveness of ClO₂ in managing COVID-19 patients, displayed numerous methodological and overall quality deficiencies.

It is worth mentioning that the sample of participants was small and that since COVID-19 can be a self-limiting disease in non-severe cases, any assertion of improvement due to ingested substances must be taken with caution.

Likewise, the study does not objectively specify the severity of the COVID-19 disease experienced by participants (neither in the case group nor in the controls), which introduces a high probability of bias. The fact that participants prepared their ClO₂ solution at home might have introduced variability in exposure conditions.

Despite denying any conflicts of interest, the authors, at the end of the article, thank Andreas Kalcker, the leading promoter of CDS consumption, with whom they have published other works directly.⁶⁵

Out of the time scope of our review, but worth mentioning in 2021, Aparicio et al.⁵¹ published a retrospective observational study in which ClO₂ is administered as prophylaxis treatment to family members of individuals with COVID-19 (confirmed by PCR).

Exposed family members with no COVID-19 symptoms were considered a success; however, this statement ignores the asymptomatic presentation of the disease, which the appropriate laboratory tests can only identify. It is noteworthy that this study did not undergo an Institutional Review Board (IRB) process review, and the first author's clinic (most probably supplying the ClO₂ preparation) was closed by health authorities due to non-compliance with sanitary requirements.⁶⁶

Implications for Public Health

As confirmed in this study, evidence concerning ClO₂ is inconclusive. Advocates of this substance have defended its use, arguing the existence of registered clinical trials which have not reported results yet and lack a national regulatory authorization of execution.⁶⁷ As a matter of fact, a recent exploration of the website https://clinicaltrials.gov yielded 18 clinical trials using ClO₂ as an intervention, most of them addressing mouth conditions and 3 concerning COVID-19; none of the latter reported results.

Additionally, it is worrisome that decision-makers have delivered this substance to their people, and, in some countries, the Senate has authorized its production.^68,69 Supporters have even filed applications in order to consider the Helsinki declaration for the compassionate use of this substance for COVID-19 treatment in the absence of other solutions, despite its discussed toxicity and lack of evidence on effectiveness.⁷⁰

Public health should call on the precautionary principle to prevent toxicity cases derived from the use of this substance as a treatment as long as there is no conclusive evidence. Most importantly, tight advertising, sales, and consumption regulations should be enforced until further evidence is published.

Limitations

Despite careful search strategy design, the present scoping review may have missed academic articles due to search engines' inherent restrictions and online unavailability, while case reports are usually published in grey literature such as national databases⁷¹ and the press. Finally, excluded in vitro studies could help understand the kinetics of ClO₂.