Resumen
En este estudio, se produjo una solución de dióxido de cloro (UC-1) compuesta de dióxido de cloro mediante un método electrolítico y posteriormente se purificó mediante una membrana. Se determinó que el UC-1 contenía 2000 ppm de dióxido de cloro gaseoso en el agua. Se evaluó la eficacia y la seguridad del UC-1. La actividad antimicrobiana fue de más del 98,2% de reducción cuando las concentraciones de UC-1 fueron de 5 y 20 ppm para las bacterias y los hongos, respectivamente. Las concentraciones inhibidoras máximas a la mitad (IC50) del H1N1, el virus de la gripe B/TW/71718/04, y el EV71 fueron 84,65 ± 0,64, 95,91 ± 11,61, y 46,39 ± 1,97 ppm, respectivamente. Una prueba de bromuro de 3-(4,5-Dimetiltiazol-2-il)-2,5-difeniltetrazolio (MTT) reveló que la viabilidad celular de las células de fibroblasto pulmonar de ratón L929 era del 93,7% a una concentración de 200 ppm de UC-1 que supera la prevista en el uso rutinario. Además, 50 ppm de UC-1 no mostraron ningún síntoma significativo en una prueba de irritación ocular de conejo. En una prueba de toxicidad por inhalación, el tratamiento con 20 ppm de UC-1 durante 24 h no mostró ninguna anormalidad ni mortalidad en los síntomas clínicos y el funcionamiento normal del pulmón y otros órganos. Una concentración de ClO2 de hasta 40 ppm en el agua potable no mostró ninguna toxicidad en una prueba de toxicidad oral subcrónica. Aquí, el UC-1 mostró una actividad de desinfección favorable y una tendencia de perfil de seguridad más alta que en informes anteriores.
Palabras clave: dióxido de cloro (PubChem CID: 24870), eficacia antimicrobiana, ensayo antiviral, toxicidad por inhalación, toxicidad oral subcrónica
Fuente: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5369164/
Artículo original
Efficacy and Safety Evaluation of a Chlorine Dioxide Solution
Miklas Scholz, Academic Editor
This article has been cited by other articles in PMC.
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 [1,2,3,4,5].
Studies have investigated the application of chlorine dioxide in
numerous fields such as water or wastewater treatment, environment and
food disinfection, and medicine [6,7,8,9,10,11,12,13]. 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 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% H2SO4 and 15% NaClO2 or reaction byproducts such as Cl2 and chloroxy anion. For example, 10% H2SO4 and 15% NaClO2 contain 90% and 85% unknown impurities, respectively. The chlorine dioxide product obtained from a mixture of 10% H2SO4 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.
Flowchart of chlorine dioxide solution production.
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 than in the case of other ClO2 generators because the electrolyte applied by us does not contain any NaClO2.
The UC-1 solution was produced by using only 25% NaCl solution, with no
other additive, which is an obvious advantage. At the same time,
despite the higher total chlorine content (which is present in the
diluted UC-1 solutions mostly as HOCl), no detectable adverse effects
were observed on the test animals or animal tissues.
2.2. Antimicrobial Efficacy Test
The test was performed following U.S. Pharmacopeia 34 NF29 Microbiological Tests/<51> [17]. Antimicrobial Effectiveness Testing. The test organisms were as follows: Escherichia coli (BCRC 11634/ATCC 8739), Staphylococcus aureus (BCRC 10451/ATCC 6538P), Pseudomonas aeruginosa (BCRC 11633/ATCC 9027), S. aureus subsp. aureus (BCRC 15211/ATCC 33591), Bacillus subtilis subspecies. (BCRC 10447/ATCC 6633), Listeria monocytogenes (BCRC 14848/ATCC 19114), Acinetobacter baumannii (BCRC 10591/ATCC 19606), Salmonella enterica subspecies. (BCRC 12947/ATCC 13311), Klebsiella pneumoniae (BCRC 16082/ATCC 4352), Penicillium funiculosum (BCRC 30438/ATCC 11797), and Candida albicans (BCRC 21538/ATCC10231).
2.3. Antiviral Assay
Viruses
were amplified in MDCK/RD cells. MDCK/RD cells were cultured in 10%
fetal bovine serum Dulbecco’s modified Eagle’s medium (FBS DMEM). When
the cells reached 90% confluence, they were washed with
phosphate-buffered saline (PBS) and infected at a multiplicity of
infection of 0.01. Following the infection, 0% FBS DMEM was added, and
the cells were incubated at 35 °C in a 5% CO2 incubator for 48 h.
A 1-mL cell suspension (6 × 105
cells) was loaded into each well of a 6-well plate, which was incubated
at 37 °C for 18–24 h. PBS was used to dilute UC-1 to final
concentrations of 0, 25, 50, 100, and 200 ppm in wells reacted with
cells and viruses for 2 min at 37 °C. Following the reaction, the total
reaction mixture was diluted to 10−8. Subsequently, the 10−8
dilution mixture was incubated at 37 °C for 48–64 h. The cells were
fixed with 10% formalin for 1 h and stained with 0.1% crystal violet for
5 min. The virus-formed plaque number was counted and compared between
the test and control groups. The antiviral activity is shown as the
percentage of virus control = plaques in the test group/plaques in the
control group × 100. The virus control is defined as infected virus with
cells without the testing agent and is considered as 100%.
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.
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.
3.1. Antimicrobial Efficacy Test
The
in vitro antimicrobial activity of UC-1 was examined. The in vitro
antimicrobial 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.
Table 1
Antimicrobial efficacy of UC-1.
| Organisms | Original Inoculum (CFU/mL) | Counts of UC-1 at Contact Time a (CFU/mL) | Percent Reductions (R) b |
|---|---|---|---|
| Escherichia coli c,* | 2.55 × 105 | <1 | >99.9 |
| Staphylococcus aureus c,* | 3.15 × 105 | <1 | >99.9 |
| Pseudomonas aeruginosa c,* | 2.55 × 105 | <1 | >99.9 |
| Staphylococcus aureus subsp. Aureus c,* | 2.60 × 105 | <1 | >99.9 |
| Bacillus subtilis subspecies c,* | 3.75 × 105 | 1.35 × 103 | 99.6 |
| Listeria monocytogenes c,* | 8.20 × 105 | <1 | >99.9 |
| Acinetobacter baumannii c,* | 5.40 × 105 | <1 | >99.9 |
| Salmonella enterica subspecies c,* | 4.80 × 105 | <1 | >99.9 |
| Klebsiella pneumoniae c,* | 9.30 × 105 | <1 | >99.9 |
| Penicillium funiculosum ,cΔ | 3.70 × 105 | 6.70 × 103 | 98.2 |
| Candida albicans c,Δ | 3.20 × 105 | <1 | >99.9 |
a The contact time was 10 min. b Percent reductions of <1% represent no significant bacteriostasis or fungistasis. c UC-1 concentrations were 5 and 20 ppm for bacteria and fungi, respectively. * presented as bacteria; Δ presented as fungi. CFU: colony-forming unit.
3.2. Antiviral Assay
The antiviral activity of 0, 25, 50, 100, and 200 ppm UC-1 after 2 min of reaction is shown in Figure 2.
For H1N1 and influenza virus B/TW/71718/04, 200 ppm UC-1 had the most
significant effect in inhibiting viral plaque formation. The half
maximal inhibitory concentration (IC50) of H1N1 was 84.65 ±
0.64 ppm and that of influenza virus B/TW/71718/04 was 95.91 ± 11.61
ppm. For EV71, 50 ppm UC-1 showed significant inhibition activity, with
an IC50 of 46.39 ± 1.97 ppm at 2 min. The results showing statistical significance (p < 0.05) are presented. Bars are plotted as means ± SD.
Antiviral
efficacy against influenza virus A/WSN/33, influenza virus
B/TW/71718/04, and enterovirus 71. Bars are plotted as means ± standard
deviation (SD). Means with the same letter did not differ significantly
at p < 0.05 according to the ANOVA (Analysis of Variance) F-test and Duncan’s new multiple range test.
3.3. In Vitro Cytotoxicity Test (MTT Assay)
The
cytotoxic effect of 0, 200, 400, 600, and 800 ppm UC-1 against L929
lung fibroblast cells was analyzed. The cell viability was 74.0%–100.0%
at UC-1 concentrations below 600 ppm. L929 cell viability was reduced to
40.3% at 800 ppm UC-1 (Figure 3).
Cytotoxic effects of various UC-1 concentrations on L929 cells.
3.4. Ocular Irritation Test
The
cornea, iris, and conjunctivae were evaluated in a rabbit ocular
irritation test. The 50 ppm 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 ocular irritation in rabbits.
Table 2
Grades in the clinical observation of individual rabbits for the ocular irritation test.
| Applied Regions | Test Component | Animal No. | Items for Grading | Clinical Observation (Time point/h) | |||
|---|---|---|---|---|---|---|---|
| 1 | 24 | 48 | 72 | ||||
| Left Eye | Test (50 ppm UC-1) | RB-160114-01 | Cornea | 0 | 0 | 0 | 0 |
| Iris | 0 | 0 | 0 | 0 | |||
| Conjunctivae | 0 | 1 | 0 | 0 | |||
| RB-160114-03 | Cornea | 0 | 0 | 0 | 0 | ||
| Iris | 0 | 0 | 0 | 0 | |||
| Conjunctivae | 0 | 0 | 0 | 0 | |||
| RB-160114-06 | Cornea | 0 | 0 | 0 | 0 | ||
| Iris | 0 | 0 | 0 | 0 | |||
| Conjunctivae | 0 | 0 | 0 | 0 | |||
| Right Eye | Control (0.9% Saline) | RB-160114-01 | Cornea | 0 | 0 | 0 | 0 |
| Iris | 0 | 0 | 0 | 0 | |||
| Conjunctivae | 0 | 0 | 0 | 0 | |||
| RB-160114-03 | Cornea | 0 | 0 | 0 | 0 | ||
| Iris | 0 | 0 | 0 | 0 | |||
| Conjunctivae | 0 | 0 | 0 | 0 | |||
| RB-160114-06 | Cornea | 0 | 0 | 0 | 0 | ||
| Iris | 0 | 0 | 0 | 0 | |||
| Conjunctivae | 0 | 0 | 0 | 0 | |||
3.5. Inhalation Toxicity Test
In
an inhalation toxicity test, we used 0, 10, and 20 ppm UC-1, which was
administered as mist by using a humidifier in an airtight box containing
five mice. The test showed no abnormality and 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.
Hematoxylin
and eosin staining of mouse lung sections in the inhalation toxicity
test. The Scale bar labeled in this figure was 100 μm.
Table 3
Evaluation of clinical symptoms in the inhalation toxicity test.
| Test Component | Animal No. | Abnormality Rate (%) | Mortality (%) |
|---|---|---|---|
| Control: PBS | 5 | 0 | 0 |
| 10 ppm UC-1 | 5 | 0 | 0 |
| 20 ppm UC-1 | 5 | 0 | 0 |
PBS: phosphate-buffered saline.
Table 4
Evaluation of organ weight for the inhalation toxicity test.
| Test Component | Heart (g) | Liver (g) | Spleen (g) | Kidney (g) |
|---|---|---|---|---|
| Control: PBS | 0.3 ± 0.1 | 0.7 ± 0.2 | 0.5 ± 0.1 | 0.6 ± 0.1 |
| 10 ppm UC-1 | 0.4 ± 0.1 | 0.7 ± 0.2 | 0.5 ± 0.2 | 0.6 ± 0.2 |
| 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).
3.6. Subchronic Oral Toxicity Test
In
the subchronic oral toxicity test, 0, 5, 10, 20, and 40 ppm UC-1 was
prepared to feed the mice. Clinical observations of the mice showed no
abnormality and no mortality after 90 days for the control and test
groups (Table 5). The mouse weight was not influenced (Figure 5). Moreover, necropsy and gross examination did not show any pathological symptoms (Figure 6).
The weights of the heart, liver, spleen, and kidney of the test groups
did not differ significantly compared with those in the control group (Table 6). Therefore, administration of up to 40 ppm UC-1 to mice for 90 days is nontoxic.
Mouse weight trend chart in the subchronic oral toxicity test. CTL: control.
Observation of mouse lungs and organs in the subchronic oral toxicity test.
Table 5
Evaluation of clinical symptoms in the subchronic oral toxicity test.
| Test Component | Animal No. | Abnormality Rate (%) | Mortality (%) |
|---|---|---|---|
| Control: PBS | 5 | 0 | 0 |
| 5 ppm UC-1 | 5 | 0 | 0 |
| 10 ppm UC-1 | 5 | 0 | 0 |
| 20 ppm UC-1 | 5 | 0 | 0 |
| 40 ppm UC-1 | 5 | 0 | 0 |
Table 6
Evaluation of organ weight in the subchronic oral toxicity test.
| 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 |
| 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 |
| 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).
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 [6,9,18,19,20,21]. In these studies, chlorine dioxide was produced using various methods (e.g., 2% NaClO2 with H3PO4, 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.
References
1. 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. doi: 10.4265/bio.15.45. [PubMed] [CrossRef] [Google Scholar]
2. Ogata N., Shibata T. Protective effect of low-concentration chlorine dioxide gas against influenza a virus infection. J. Gen. Virol. 2008;89:60–67. doi: 10.1099/vir.0.83393-0. [PubMed] [CrossRef] [Google Scholar]
3. 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. doi: 10.4265/bio.14.147. [PubMed] [CrossRef] [Google Scholar]
4. Tanner R.S. Comparative testing and evaluation of hard-surface disinfectants. J. Ind. Microbiol. 1989;4:145–154. doi: 10.1007/BF01569799. [CrossRef] [Google Scholar]
5. 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. doi: 10.1016/S0043-1354(96)00276-X. [CrossRef] [Google Scholar]
6. 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. doi: 10.1021/jf981397h. [PubMed] [CrossRef] [Google Scholar]
7. 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. doi: 10.1021/es801930c. [PubMed] [CrossRef] [Google Scholar]
8. 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 doi: 10.1155/2014/619038. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
9. 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. doi: 10.1016/j.ijfoodmicro.2015.06.009. [PubMed] [CrossRef] [Google Scholar]
10. 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. doi: 10.1021/acs.jafc.5b04153. [PubMed] [CrossRef] [Google Scholar]
11. 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. doi: 10.1016/j.serj.2016.04.009. [CrossRef] [Google Scholar]
12. 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. doi: 10.1016/j.jhin.2016.01.005. [PubMed] [CrossRef] [Google Scholar]
13. 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. doi: 10.1016/j.jobcr.2015.08.008. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
14. U.S. Environmental Protection Agency . Microbial and Disinfection Byproduct Rules Simultaneous Compliance Guidance Manual. U.S. Environmental Protection; Washington, DC, USA: 1999. [Google Scholar]
15. Couri D., Abdel-Rahman M.S., Bull R.J. Toxicological effects of chlorine dioxide, chlorite and chlorate. Environ. Health Perspect. 1982;46:13–17. doi: 10.1289/ehp.824613. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
16. 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. [Google Scholar]
17. U.S. Pharmacopeia . 34 NF29 Microbiological Tests/<51> Antimicrobial Effectiveness Testing. U.S. Pharmacopeial Convention; Rockville, MD, USA: [Google Scholar]
18. Trinetta
V., Morgan M., Linton R., Dermirci A., Ngadi M.O. Chlorine dioxide for
microbial decontamination of food. In: Demirci A., Ngadi M.O., editors.
Microbial Decontamination in the Food Industry. Woodhead Publishing; Cambridge, UK: 2012. pp. 533–562. [Google Scholar]
19. 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. doi: 10.1016/j.watres.2014.01.038. [PubMed] [CrossRef] [Google Scholar]
20. 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. doi: 10.1016/j.lwt.2014.09.031. [CrossRef] [Google Scholar]
21. 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. doi: 10.1016/j.fpsl.2016.05.003. [CrossRef] [Google Scholar]
22. Paulet G.D., Desbrousses S. On the action of ClO2 at low concentrations on laboratory animals. Arch. Mal. Prof. 1970;31:97–106. [PubMed] [Google Scholar]
23. Paulet G.D., Desbrousses S. Actions of discontinuous exposure to chlorine dioxide (ClO2) on the rat. Arch. Mal. Prof. 1974;35:797–804. [Google Scholar]
24. 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. [Google Scholar]
25. 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. doi: 10.1289/ehp.824647. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Articles from International Journal of Environmental Research and Public Health are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)
60 comentarios:
Excelente
Excelente.. esto es fabuloso
Genial esto es maravilloso
Que bueno. Se debe compartir el estudio.
Excelente!!
Excelente!!
Es lo mejor que ha pasado en este tiempo de pandemia deberian difundirlo yo lo comparti en mis redes
Muy importante este resultado para continuar con el combate al Covi 19
Gracias
El estudio fue publicado en marzo de 2017 y desarrolado centro nacional de investigación animal de China. No fue pensado para humanos, ni se constató su acción contra el coronavirus!!!
En ningun caso se habla de uso en humanos!!
Efectivamente no fue en humanos pero no hubo efectos nocivos en animales y en estos tiempos que no hay nada para combatir a este virus y con los estudios y resultados. De más de 13 años al menos por en Biofisico Andreas Kalcker yo ya me lo edtoy tomando y a todos mis seres queridos se los he compartido y ha sido testigo de por lo menos 5 pacientes que se han curado usandolo y tenían saturacion de menos de 70 de O2. Pero se ve la negligencia por parte de los grandes que podrían hacer el cambio y el fin de la pandemia.
Gracias Espero se utilice pronto en humanos.y salven VIDAS. Es efectivo en muchos problemas de salud. En mi familia lo usamos todos. Somos siete .desde hace tres meses.GRACIAS lo comparto
Excelente trabajo de investigacion. Gracias.
En conclusión, el estudio es para que estés seguro que al desinfectar superficies, alimentos y dientes, no te estarás haciendo daño en concentraciones de UC-1 de 5 y 20 ppm. NO ES PARA QUE TE CURE ALGUNA PATOLOGÍA.
Y dónde se compra
Cómo lo consigo?
¡Identifícate!
¡ Un servicio, por favor; en nombre de todas y todos los muertos innecesarios, por eror, omisión ó adrede; DIFUNDIR ! .
¡ DEFENDAMOS SIEMPRE LA VIDA EN EL PLANETA ! .
Excelente
Para seguir enriqueciendo sus conocimientos sobre el dioxido de cloro, les adjunto otro link que va en la misma dirección que este artículo: https://akjournals.com/view/journals/2060/107/1/article-p1.xml.
la primera persona qu descubrio la aplicacion de este quimico (qu llamaron MMS) es el ingeniero quimico dr Jim Humble, hace muuchis anios atras. Entonces, Andreas Kalcker uso el producto y conocio a Jim. Luego lo estudio y mejoro el proceso de preparado =CDS. Los protocolos de Jim Humble se mantienen, adaptados a la nueva forma de prepararlos
desde que comenzó la plandemia el cientifico Andreas Kalcker que lo estudia hace trece años y coonoce los protocolos de administracion lo viene diciendo y sigue siendo censurado y perseguido no solo el sino todos los que venimos tratando a traves del teclado de ayudarlo en su lucha contra los molinos de vento Es por intereses multimillonarios de los laboratorios que no quieren que la verdad se haga masiva entonces confunden a la gente a través de sus miedos de comunicación Ya uqe esta solución acabará con infinidad de enfermedades crónicas que se mantienen en esa condición no por falta de tecnologias aplicables para erradicarlo sino c por conveniencias de gente inescrupulosa que lucra con el sufrimiento y hasta con la muerte. Espero que ahora entiendan la diferencia entre dioxido de cloro y legia o lavandina .
Pero los humanos que lo tomamos y nos hemos recuperado de diferentes patologias estamos aqui,y somos pruebas de que si funciona y ademas maravilkosamente.
Testimonios en video, chat para para consultar con otros usuarios por telegram,como hacer el dióxido de cloro en casa, y donde comprarlo visita www.cdsmexico.org
Puea mas m favor !!!. Si desde 2017 es efectivo, porqué no le permitieron a ese Dr escalarlo a pruebas en humabos? Se hubiera tenido muuuucho tiempo para indagar y concluir si es efectivo o no. No sea Necio ! Isted trata de comparar peras con manzanas aplicando protocolos que se siguen en investigaciones y tiempos normales. Hoy por hoy ESTAMOS EN GUERRA !!! Y se deberia apoyar estos estudios que por la urgencia, ya hay muchos humanos que gracias a Dios han sido beneficiados. Aaahhh, claro, como no lo dice Soros, Hqrvard, Oxford o cualquier otro interes corporativo que presuntamente usted sigue, pues entonces se "rechaza". Poderoso caballero es Don Dinero. "El interea tiene pies, y cua do es dineri, hasta oarece cienpies !!. Vamos todos cintra kos corporativos abusivos , el mundo se debe levantar YA contra estos insensatis que se venden por un plato de lentejas !!! El mundo unido, jamás será vencido !!!
Lo siento amigo el contenido ya no existe, como todo lo que tiene que ver con la difusion del dioxido de cloro
Es una barbaridad difundir artículos científicos sobre producción, seguridad y uso “un vitro” del dióxido de cloro, para desinfectar agua y materiales, como queriendo justificar su uso para combatir enfermedades causadas por bacterias y virus “un vivo”.... Veo la mano crimínal de quienes medran del Covid sin otro propósito de lucrar a costa de la ingenuidad y el miedo a la pandemia ... Por favor lean con cuidado esta información, que como ella, es muy abundante, pero que no trata de manera alguna de emplear este producto como medicamento.....
1ro) este estudio es publicado en un blog independiente.
2do) todo éste estudio esta hecho en ratas... lo leíste, o dilo leyeron el título?
3ro) el estudio fue en china
La desinformación es un arma muy grave.
Es una batalla de de dos bandos encontrados. No es nuevo el producto y ha tenido mucho éxito en muchas enfermedades y ahora con covid.conozco casos de gente con covid que se han recuperado, vean los resultados del centro médico de Jurica , lean el libro de salud prohibida. etc. Yo lo tomo y me he hecho el check up médico y no hay afectación en órganos como se dice. Les comparto por si quieren un testimonio más
TOXICIDAD del dióxido de cloro demostrada en un estudio publicado en marzo del 2017, por científicos de Taiwán. (Int J Environ Res Public Health. 2017 Mar; 14(3): 329.). Preocupados por efectos tóxicos del dióxido de cloro, usado en procesos relacionados con plantas de tratamiento de aguas negras, protección de alimentos, y otros, investigadores chinos (Taiwán) demostraron cuán importante es la elaboración de esa solución desinfectante, usada para destruir bacterias, hongos, virus y otros patógenos, en procesos industriales. Dicen en parte del estudio: "El uso doméstico e industrial del dióxido de cloro debe evaluarse de acuerdo con la pureza del producto, para lo cual el método de preparación es un paso esencial. Los materiales de partida de baja pureza (p. Ej., 5% de HCl y 10% de NaClO2) tienen un alto contenido de impurezas". (Como el que se vende en Costa Rica, agregamos). Y agregan, "Si el producto de estas reacciones no se purifica más, muestra altos niveles de impurezas, por lo que son útiles solo para el tratamiento de aguas residuales pero no adecuados para el contacto con humanos o alimentos debido a las impurezas dañinas". O sea, el dióxido de cloro producido sin cumplir con controles de calidad rigurosos, pueden ser muy peligrosos para las personas que lo manipulan. Para sus experimentos, los científicos usaron "una solución de dióxido de cloro utilizando un método electrolítico que fue posteriormente purificada utilizando una membrana, determinándose que la solución contenía 2000 ppm (partes por millón) de dióxido de cloro gaseoso en agua", de gran pureza. En sus ensayos con animales, indican que "Los ratones fueron alimentados con agua potable que contenía hasta 40 ppm de dióxido de cloro durante 90 días; no detectándose toxicidad en la prueba de toxicidad oral subcrónica, a diferencia de lo reportado por Daniel et al, que encontraron toxicidad oral expuesta del dióxido de cloro en el agua potable administrada a ratas durante 90 días; utilizando varias concentraciones de dióxido de cloro, donde el peso del bazo y el hígado disminuyó significativamente a 25 y 50 mg / L, respectivamente, y mostraron lesiones nasales causadas por 25 mg / L de vapores de dióxido de cloro en el agua potable. L. Bercz et al realizaron una prueba similar en monos verdes africanos (Cercopithecus aethiops) utilizando 0, 30, 100 y 200 mg / L de dióxido de cloro durante 4–6 semanas, donde 200 mg / L de dióxido de cloro causó eritema y ulceración de la mucosa oral después de 1 semana, y 100 mg / L de dióxido de cloro redujo los niveles séricos de tiroxina después de 6 semanas". Resultados que demuestran que *para los procesos señalados (que ¡no tienen nada que ver con las "calidades medicinales" que algunos alegan sin aportar ni un solo estudio que avale sus afirmaciones!) es importante utilizar soluciones de dióxido de cloro, obtenidas mediante procesos como el descrito, mediante método electrolítico, filtrado ultrafino y adición de agua de ósmosis inversa. Ello porque, como reseñan los investigadores, "El producto Cl2 puede reaccionar con la materia orgánica para formar trihalometano, que es un carcinógeno, y los aniones cloroxi, como ClO2- o ClO3−, pueden ser perjudiciales para la salud humana". El estudio reafirma lo conocido, o sea que es una sustancia desinfectante muy usada, por ejemplo en recintos hospitalarios y para el tratamiento de aguas negras y desinfección alimentos, pero que aún para ello, se debe tener especial cuidado con las concentraciones usadas y el origen del producto vendido comercialmente, pues su alta toxicidad puede afectar la salud de los que se exponen a sus gases y soluciones. (FREDDY PACHECO LEÓN, PhD). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5369164/
¿Cómo podría conocer ese falso doctor que el tóxico dióxido de cloro podía acabar con el SARS-Cov-2... ANTES de que éste provocará la pandemia?
Pero el estudio hecho hace más de tres años en Taiwán, no tiene NINGUNA relación con la pandemia.
Para empezar,no es un falso doctor,es un científico acreditado y el único estudioso de los efectos del CDS y sus protocolos...y sabía desde que "apareció"el famoso virus que lo podía combatir con el CDS...no seas torpe unknown...
No entiendo de donde sacan que esto va a solucionar el Covid-19. En ninguna parte del artículo se menciona siquiera el virus; esto es un sesgo de confirmación. El estudio trabaja con ratas y la discusión no declara ningún vínculo con el nuevo Coronavirus ¿si saben interpretar artículos científicos o solo quieren reforzar sus creencias conspirativas?
Pues yo soy Humano y no le desearía el mal a nadie con tanto desprestio al CDS, yo lo tomo y lo seguiré tomando toda la vida, seguiré siendo un ejemplo vivo de salud..
Viva Andreas.
Pronto la Plandemía se caerá y todos sus cómplices al igual que todos los desinformantes ..
Ojalá las personas del mundo entero lo tomen,y sabrían los beneficios del CDS
creo que la intención no es para saber cuándo se hizo sino para corroborar lo que tantos niegan o ignoran. Cuál es tu objeción? no se entiende bien
Si, lo ideal es que siga muriendo gente, lo ideal es que más personas mueran mientras nosotros nos sentamos esperando que alguna farmacéutica multimillonaria haga una vacuna, lo ideal es qué más y más personas lloren por sus familiares por Que los gobiernos no saben qué hacer. .... somos miles de personas tomando el CDS, somos miles los sanos. Son miles los testimonios y aún así, se niegan a aceptarlo Por neglicencia, orgullo, o brutalidad !, aún no entiendo por qué no investigan o estudian un poco más.
Que buena información y aplicarla con seriedad
Lo ovio es que a los laboratorios no les conviene, a mi me ha caido bien, ademas no sufro de stress y miedo.
https://youtu.be/aWa4CZdIROM
No se si ea I o l
testimonio de sanación: me esta enderezando los dedos de la mano que tenia con artritis, me quitó una mancha de vitiligo en la muñeca, me quitó terigios en ambos ojos, me quitó caspa del pelo, me quito una molestia en el oido izquierdo, me quitó TODOS LOS PARASITOS que despues de mas de 30 años, no habia medicamento que me pudiera sacar, duermo toda la noche, descanso con un sueño reparador, me levanto con alegría, energía y recuerdo todo lo que tengo que hacer VIVO FELIZ, gracias Andreas por compartir esta fórmula....comparto con mis amistades que se han curado de diabetes, colon irritado, de alergias, de dolores de cabeza, de problemas de erección, de vicio como el cigarrillo, de una intoxicación por un robo con escopolamina, se recuperó en 2 dias. Y muchos testimonios mas del OXIGENO QUE BRINDA AL ORGANISMO y de la purgada de parásitos tan efectiva.
Extraordiaria publicación, cambia todo y confirma el origen vil de todas las locuras que hemos tenido que padecer gracias a los enfermos con poder y los gobiernos corruptos que les dan lugar!!! Una genialidad este blog :)
ya censuraron el video, no les conviene a las farmacéuticas les arruinan el negocio
Estás engañando a la gente y lo sabés, no es una publicacion del gobierno de EUA. Quien tenga dudas lo espero en twitter @jorgenomefui y le explico qué es, de dónde viene, y qué significa este paper
Calla llorón ,vacúnate nomas con la vacuna genocida, borrego estúpido , hay quienes conocemos años esta solución de la fórmula de Andreas Kalcher ,no nos vas a venir con tus payasadas, los registros de toxicidad existen y los protocolos que se usan para curar CON CDS no sobrepasan ni al mínimo los niveles de toxicidad . El CDS a 3000ppm igual al 0.3 % concentración es apto para todos los seres humanos!
Quieren matar al Dr. Eduardo Brandes de Argentina por pedir los ensayos clínicos con el CDS, no lo van a lograr, somos miles de testigos si le pasa algo al Dr.
Quieren matar al Dr. Eduardo Brandes de Argentina por pedir los ensayos clínicos con el CDS, no lo van a lograr, somos miles de testigos si le pasa algo al Dr. Tenemos pruebas!!
Si es dañino entonces por qué la FDA no lo retira de las bolsas de transfusión sanguínea que ahí el Dióxido de cloro va a la vía sanguínea diractamente
Busca en you tube a Andreas kalcker.
Señor Welton hay muchos testimonios a favor del CDS, ya esta CURANDO a miles de personas por todo el mundo buesque doctores por la verdad o comusav, ellos son doctores que ya están usando el dioxido de cloro, digo miles de doctores NO pueden equivocarse. Busque le talvez un día lo necesite🙏🇺🇸
Holae por favor alguien sabe si tiene efectos positivos sobre el cancer de próstata? Y si alguien tiene una dirección del doctor alemán para escribirle, gracias.
es un falso cientifico, no acreditado y no estudioso dado que no ha publicado ningun estudio al respecto, es mas sus "estudios" sobre el cds y la malaria fueron desmentidos y esta acusado en alemania de fraude y estafa,decia que curaba el sida, mentira, el cancer, mentira, el parkinson, mentira, el autismo, mentira,y resulta que ahora tambien cura el covid? ahora si es verdad?
El que tu no sepas de Andreas Kalcker no quiete decir que no sea cientifico, y si tu no confias en el CDS pues no lo tomes.
Sólo tienes que ir a la fuente, los vídeos explicativos de Andreas Kalcker para entender, si es que es posible que lo entiendas. Yo lo uso desde hace años y no es tóxico. Repito, no es tóxico. Por cierto, No Es Tóxico.
No seas ingenuo. Miles de médicos ya lo están usando, miles de personas ya lo usan, yo mismo también. Quítate la venda de los ojos. Despierta!!!
No hay peor ciego que aquel que no quiere ver.
Y dale, investiga un poco más y úsalo como hacían los pioneros científicos que lo probaba con ellos mismos. Yo lo uso hace años y No ES TÓXICO. Hay muchos estudios que lo demuestran. Esfuérzate un poco e investiga.
Andreas Kalcker si termino mi carrera de meicina y no sigo los protocolos establecidos (por que no los hay) para curar una enfermedad, y logro curar la enfermedad, la comunidad medica me desacredita por no haber seguido los protocolos, aunque haya curado la enfermedad. Eso es lo que le paso a Andreas Kalcker. Pero eso no significa que no haya estudiado y graduado de la carrera. Eso no quiere decir que, repentinamente ya no tenga el conocimiento adquirido durante la carrera y durante sus años de investigacion.
Ademas, Kalcker si hizo las investigaciones que dice haber hecho, pero la industria farmaceutica no le conviene que algo tan simple, no patentable y tan barato, sea tan eficaz para tantos padecimientos considerados "incurables". Si el CLO2 no cura el padecimiento que dice curar, mejora muchisimo la calidad de vida del paciente. La industria farmaceutica hara hasta lo imposible por desacreditar, incluso llevar a la carcel o asesinar, eliminar a quien sea necesario para continuar obteniendo la maxima ganancia de la persona enferma, para ser la unica esperanza de mejoria de la persona enferma, manteniendo al enfermo en la ignorancia, otorgando placebos y prolongando la enfermedad lo mas posible.
Ademas, puedes tambien leer las otras investigaciones hechas en otros paises, como Japon y EEUU. No necesitas ser erudito para sacar conclusiones muy interesantes acerca de los beneficios del CLO2. Hay que escarbar un poco mas a fondo y comienzas a encontrar mucho mas de lo que dicen unos pocos.
Recomiendo leer ambos aspectos del tema del CLO2, lo bueno y lo malo, ver como llegaron esos investigadores a sus conclusiones, leer o ver los testimonios de las personas que ya lo estan usando y como obtuvieron esos resultados. Y asegurate de que lo que estan usando sea en verdad CLO2, ya que hay quienes dicen investigar o usar CLO2 pero no lo es, y por consiguiente, sus investigaciones y testimonios son erroneos.
Eso es lo que yo, como usuario del CDS, te recomendaria. Da temor salirse del paradigma establecido, pero una vez que te sales, encuentras un mundo de posibilidades que estaban ocultas, que te las habian ocultado. Algo que sin duda es absolutamente necesario es tratar de mantener en lo mayor posible, una vision de veracidad e imparcialidad, caiga quien caiga, sin descartar todas y cada una de las posibilidades, por muy improbables o imposibles, o descabelladas que estas parezcan.
Ve mas alla de lo que a simple vista se puede ver.
Te deseo mucha suerte y bendiciones.
El inicio del parrafo no debe aparecer el nombre del Andreas Kalcker... ese fue error de escritura. Lo siento.
Soy bioquímico y puedo afirmar que el dióxido de cloro es un producto muy efectivo para desinfectar. Por supuesto que siempre se lo debe adquirir en proveedores serios como dioxnatur. Es un producto que sirve mucho tener en el hogar.
Publicar un comentario