Pilot Study of Application of Combined Transbronchial and Intravenous Ultraviolet C (UVC) and Laser Beam Application for the Treatment of Critical COVID-19 Infection

Objective and background: Light-based antimicrobials, mainly ultraviolet C (UVC) and laser light irradiation, have a potential to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The aim of our study was to evaluate the effect of transbronchial and intravenous application of UVC and laser light irradiation on treatment of patients with severe COVID-19. Methods: The clinical outcome of six patients (age 42-69 years) with severe COVID-19 infection who were directly applied UVC (254 nm) transbronchially, and UVC plus green (630 nm) and red laser (535 nm) lights to the blood circulation in addition to standard pharmacotherapy (UVC group) were prospectively evaluated in comparison to six patients (age 50-69 years) treated only with pharmacotherapy (standard treatment group). Results: The patients in UVC group had shorter stay in intensive care unit (median length of stay 1 vs. 8.5 days;p=0.015), more negative PCR results after treatment (5/6 vs. 0/6 patients;p=0.003), higher discharge rate (5/6 vs. 3/6 patients), and lower mortality (1/6 vs. 3/6 patients), as compared to patients in standard treatment group. Serum D-dimer level, which reached up to 2500 ng/mL (six times of baseline value) seven days after treatment in standard treatment group, was much lower in UVC group (1000 ng/mL). Serum ferritin level was 1.5 to 1.9-fold higher and CRP level was up to 1.7-fold higher in standard treatment group during ten days after treatment as compared to UVC group. No adverse effects have been observed. Conclusions: Combined transbronchial and intravenous UVC and laser irradiation may improve outcome of severe COVID-19 cases. [ABSTRACT FROM AUTHOR] Copyright of Journal of Clinical & Experimental Investigations is the property of Modestum Publications and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)


INTRODUCTION
Coronavirus disease 2019 (COVID- 19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a respiratory disease with high transmission and mortality rate responsible for ongoing global pandemic. While lungs are the most commonly involved organs, extrapulmonary systems, including the cardiac, gastrointestinal, hepatic, renal etc., are also affected during COVID-19 disease [1]. Analysis of published databases reporting angiotensin-converting enzyme-2 (ACE2) expression indicated that, ACE2 expression is quite highly positive in various types of human tissues, mostly in respiratory tract, heart, kidney and gastrointestinal tract, making these tissues susceptible to COVID infection [2][3][4].
The cardiac manifestations accompanying COVID-19 include cardiac arrhythmias, myocarditis, pericarditis, acute coronary syndrome and heart failure [1]. In a meta-analysis of six studies involving more than 1,500 patients, prevalence of cardiovascular diseases in COVID-19 was reported as 16.4% [5]. In another metaanalysis based on 16 studies with more than In contrary to long wavelength UVB (290-340 nm) and UVA (340-400 nm) which may have damaging effects on healthy tissues on chronic exposure, UVC causes minimum DNA damage in mammalian cells at its effective wavelength range of 250-270 nm that can be quickly repaired by DNA repair enzymes [19,20]. Lachert even suggested that 254 nm wavelength irradiation is not absorbed by proteins, therefore conventional toxicity tests are not required [21].
Some authors have suggested that photobiomodulation or photodynamic therapies may be promising in the management of COVID-19 [27,28]. Fekrazad proposed that non-invasive or minimally invasive photobiomodulation or photodynamic therapy administered by intratracheal or intravenous routes may have a potential as an adjuvant to pharmacotherapy or even alternative therapy for COVID-19 [27]. Having minimum side effects and drug interactions, light-based therapies may be beneficial to patients with COVID-19 infection. Laser lights with different wavelengths may be effective against COVID-19 infection by increasing oxygenation of red cells and improving immune system [27]. Domínguez discussed that regular transdermal application of laser therapy 30 minutes per day for 3-5 days, can also control the cytokine storm in patients with COVID-19 [28]. Ferreira [29] and Camacho [30] emphasized that clinical studies are clearly needed to evaluate whether it is possible to use minimally invasive photobiomodulation therapy into the tissues to produce a systemic antimicrobial effect in the treatment of COVID-19. Because of the lack of effective and safe UVC-based technologies to deliver UVC into the tissues in vivo, these claims made in previous studies have not been tested in clinical studies yet. UVC has been used exclusively for superficial infections, transplant organs, or blood products [13][14][15].
In this study, we aimed to evaluate the effect of UVC and laser light, applied by an innovative UVC-generator device, directly into bronchial system and blood circulation.

Study Design and Patients
This study is a prospective, two-arm, open-label, randomized, controlled, multicenter trial conducted in Turkey. Study population included patients who have applied to the emergency service with symptoms of fever, weakness, cough, and shortness of breath; whose COVID-19 PCR test positive (Abbott Biorad CFX 96 RT-PCR) or those having findings of atypical pneumonia on computed tomography; and treated in the intensive care unit. Pregnant and lactating women and those with suspected pregnancy, and patients previously diagnosed with mental disorders were excluded from the study. Twelve patients complying with selection criteria among 16 subjects were randomly assigned into two groups (see Figure 1 for the study flow diagram). Patients in control group (standard treatment group) were administered standard treatment for COVID-19, which is a combination of antiviral, antimalarial and antibacterial drugs (i.e., favipiravir, hydroxychloroquine, azithromycin) according to the Guidelines of Turkish Ministry of Health [31]. Patients in the UVC group were applied transbronchial and intravenous UVC light and laser therapy with a newly developed UVC-generator device in addition to standard COVID-19 therapy based on the Guidelines of Turkish Ministry of Health.
For transbronchial UVC therapy, the fiberoptic catheter system was slowly pushed through the trachea down into the lower bronchia, and 20 mW/cm 2 power beam energy was applied for 30 sec every 5 cm segment (Figure 2). The procedure was performed on the closed-circuit laryngeal mask airway under general anesthesia.
For intravenous UVC and laser therapy, the fiberoptic catheter system was slowly pushed through the antecubital vein by using an intravenous catheter, and approximately 5 cm of the tip of the catheter was adjusted to remain in the intravascular area. UVC and laser therapy was applied to the blood for 30±5 minutes with a 200 mW/cm 2 power beam energy for virus inactivation, and the fiberoptic catheter was then slowly withdrawn.
The dose and the duration of exposure were kept as minimum as possible during the study. The patients had been monitored for possible lung toxicity very closely during and after the procedure for 48 hours, with complete equipment and experienced medical staff for noninvasive and invasive ventilation.
Considering the clinical condition of the patient, the maximum time for application of the beam was 30±5  minutes for intravenous route, 5±1 minutes for transbronchial route.

Study Outcome Endpoints
Primary endpoint to be analyzed was defined as conversion of positive PCR test to negative in bronchoalveolar lavage fluid (BAL) within 24 hours following the procedure.
Secondary end-points we intended to analyze were shortening of length of stay in intensive care unit, in medical ward, and total length of stay after treatment, lowering the elevated levels of serum D-dimer, ferritin and CRP values, not worsening of serum biochemistry values (blood urea nitrogen, creatinine, electrolytes, transaminases, bilirubin), hemostatic parameters (prothrombin time and activated partial thromboplastin time) and hematologic parameters (hemoglobin, hematocrit, red blood cell count, white blood cell count, neutrophil percentage, platelet count).
Patients' clinical status were defined using World Health Organization (WHO) R&D Blueprint Ordinal Clinical Scale scores at baseline and daily during the 10-days' follow-up [32]. Faster change in this clinical status was another secondary study endpoint.

Safety Issues
The patients had been monitored for possible lung toxicity (vital signs and clinical symptoms, physical examination, chest X-ray, chest computed tomography as needed, arterial oxygen, carbon dioxide and pH monitoring) very closely during and after the procedure for 48 hours, with complete equipment and experienced medical staff for noninvasive and invasive ventilation. Ophthalmologic examinations of the patients were performed daily for ten days following the procedure, in order to diagnose any eye toxicity finding.
Statistical Analysis D-dimer, ferritin, C-reactive protein (CRP), serum biochemistry tests, and hematologic tests were performed every 1-3 days, depending on the clinical status of the patients. Last-observation-carried-forward method was implemented in order to impute the missing data between two consequent measurements, i.e., in case of any missing data the valid measurement at the nearest previous time point was copied to the day with missing data.
The measurements during follow-up period were summarized with geometric mean and 95% confidence interval (CI) for D-dimer, ferritin and CRP levels, and median and 95%CI or minimum-maximum values for other tests. Increase in D-dimer, ferritin and CRP levels at followup days as the ratio over baseline values were also calculated (in order to avoid daily random fluctuations, moving threeday averages were used). These variables showed dispersion within very wide ranges. Therefore, geometric means were calculated, in order to avoid the disproportionate influence of extreme values on the average values.
Since the number of subjects was quite low, bootstrapping technique was applied to calculate CIs. Biascorrected accelerated 95%CIs were calculated.
Chi-square test or Fisher's exact test, when needed, was used to compare the proportions of categorical variables (conversion of positive PCR test to negative and death) between study groups. Nonparametric approach for the analysis of numerical variables, since the number of cases is low and/or the distribution of the values are non-normal. Mann-Whitney U test used to compare the geometric means or medians of numerical variables, and also median WHO R&D Blueprint Ordinal Clinical Scale scores between study groups. SPSS v22 was used to perform the statistical analysis.

Baseline Patient Characteristics
Individual patient data including demographic, hospital stay and clinical outcomes are presented in Table 2. Patients in the UVC group (median age: 50.5 years (42 to 69), . Patients in the UVC group and standard treatment group had been in hospital for 2 to 10 days when they were enrolled. Median length of stay before enrollment were 8 and 4.5 days, in the study groups, respectively (p=0.31; Mann-Whitney U test) ( Table 3).

Clinical Outcome and PCR Test Results
While no PCR test result for COVID-19 converted to negative in any patient in standard treatment group, five out of six patients in the UVC group had negative PCR test results after UVC treatment in all samples of BAL fluid, trachea and blood (p=0.003; chi-square test) ( Table 3).
Five of six patients in UVC group could be transferred to medical ward (two of them on the same day and other three one day after the UVC treatment). These patients stayed in medical ward for 3-6 more days and were discharged from the hospital with negative PCR test. The sixth patient in the UVC group died with positive PCR test result after staying eight days in the intensive care unit following the UVC treatment. Her peripheral oxygen saturation (SpO2) value increased from 56% to 90% just after the study procedure. Although PCR in BAL fluid did not convert to negative, the viral load decreased as evidenced with improvement in quantitative PCR from 16 to 30 cycle threshold.
On the other hand, all patients in the standard treatment group continued to stay in the intensive care unit for 4-22 days. None of them could reach to a clinical status good enough to be transferred to medical ward. PCR test of all patients in the standard treatment group continued to be positive-three were discharged and three were dead ( Table  3).
Median WHO R&D Blueprint Ordinal Clinical Scale scores at baseline were 4 in both study groups (3 to 5 in UVC group and 3 to 6 in standard treatment group; p=0.82, Mann-Whitney U test). The median score declined gradually during the ten days following the study procedure and happened to be 2 on Day-10, in UVC group. On the other hand, the median score in standard treatment group did not show any downward improvement, and on Day-10 median score was 4.5. The p values corresponding to differences of  Figure 3).

D-dimer, Ferritin and CRP Levels
The geometric mean of serum D-dimer level was initially almost similar in UVC and standard treatment groups on treatment day (Day-0), but the course was quite different in   the following ten days. Mean D-dimer level showed a remarkable increase from 414 ng/mL at baseline to higher than 2500 ng/mL during the first post-treatment week, in standard treatment group (Table 4, Figure 4). The ratio of D-dimer level at Day-7 over Day-0 was higher than six times. This course was then followed by a slow decline, but mean D-dimer level was still higher than 1000 ng/mL at Day-10 (almost three times higher than Day-0 value). On the other hand, this incline was quite suppressed in UVC group. Mean D-dimer level showed increase from 323 ng/mL at baseline to about 1100 ng/mL maximum during the first posttreatment week. This corresponded to almost three times increase as compared to Day-0. This course was then followed by a decline. At Day-10, mean D-dimer level was almost twice the Day-0 value. D-dimer level was 1.3 to 2.6- fold higher during to ten days after Day-0, in standard treatment group as compared to UVC group.
The course of serum ferritin level was quite different between UVC and standard treatment groups. In standard treatment group, mean serum ferritin level showed up to 33% increase in the first three days following Day-0, followed by a slow decline (Table 4, Figure 4). Then it stayed in between 900 and 1000 ug/L from Day-3 to Day-10. On the other hand, mean ferritin level did not show any increase after Day-0. It even declined slowly during ten days after Day-0. Mean ferritin level was 1.5 to 1.9-fold higher during ten days after Day-0, in standard treatment group as compared to UVC group.
Serum CRP level declined in both UVC and standard treatment groups (except the very first day in standard treatment group). In Day-7 and Day-10, mean serum CRP level was declined down to 32% and 21% of the CRP level at Day-0, respectively, in standard treatment group (Table 4, Figure 4). Corresponding figures were 19% and 16% in UVC group. CRP level was up to 1.7-fold higher during ten days after Day-0, in standard treatment group as compared to UVC group.

Safety
Patients in study groups were comparable with regards to baseline serum biochemistry values, hemostatic parameters and hematologic parameters (Supplementary Table). The courses of white blood cell count and platelet count were similar throughout the ten days following the study procedure. Neutrophil percentage, which was similar in study groups at baseline, show a continuous decline starting the next day after the procedure and lasting until Day-10. However, neutrophil percentage did not show any change during the ten days.
Patients did not experience any deterioration in their clinical symptoms, physical examinations, laboratory findings, or any ophthalmologic diagnosis, which could not be explained by the natural course of COVID-19. No other undefined adverse effects related with UVC application have been observed.

DISCUSSION
In this report, we presented for the first time transbronchial and intravenous UVC and laser light by an innovative UVC and laser generator device in six patients with severe COVID-19, and proposed that UVC and laser light therapy added to available pharmacotherapy has a potential to improve the clinical outcome of patients. UVC light has long been known to have antimicrobial properties, thus suggested to be used for treatment of superficial and catheter-related infections, and sterilization of surfaces, indoor environments, blood products and donor organs [13,[33][34][35][36][37]. In addition to its antibacterial and antiprotozoal effects, previous studies reported promising findings on the inhibitory effect of UVC light on viruses including human coronavirus. UVC light was shown to inactivate enveloped or nonenveloped, single-stranded RNA or DNA viruses in platelet concentrates [38], hepatitis C virus ex vivo in donor lungs during preservation [33][34][35][36][37], and aerosolized H1N1 influenza virus [39]. SARS-CoV-2 is also an enveloped, positive-sense single-stranded RNA coronavirus [40].
There are some studies showing evidence of viral inactivation induced by UVC on coronaviruses. Also there have been a couple of papers, in which scientists experienced on light therapy, have noted their opinions, comments and recommendations A summary of these publications are presented in Table 5.
In their model, Banerjee et al tested the effectiveness of UVC 254 nm at a dose of ≥1 J/cm 2 , on disinfecting respirators and personal protective equipment contaminated with SARS-CoV-2. They reported that 3-, 4-and 5-log reduction targets were reached in 19, 30 and 80 minutes, respectively. The average exposure of UV-C applied in 20 minutes was 3.5 kJ (11.5 J/cm 2 ), which corresponded to 3.1 log reduction of virus. Total exposure needed to reach 5-log reduction was 46.6 J/cm 2 in 80 minutes [23].
There are also several studies on the effects of UVC on coronaviruses other than SARS-CoV-2 published in recent years. As all human coronaviruses have similar genomic sizes, UVC would be expected to show similar inactivation efficiency against other human coronaviruses including SARS-CoV-2. It has been reported that UVC at half of the full UVC dose (0.1 J/cm 2 ) reduced the infectivity of SARS-CoV and virus reduction factor ≥3.4 for SARS-CoV was achieved with the UVC-based pathogen inactivation system applied on platelet concentrates [18]. Buonanno et al. showed that low doses of UVC (222 nm, 1.2 mJ/cm 2 ) inactivated aerosolized human betacoronavirus HCoV-OC43, which is a member of beta coronaviruses, as SARS-CoV-2 is, too [24]. Based on the results of UVC on betacoronavirus HCoV-OC43, which is in the same genus as SARS-CoV-2, it has been proposed that continuous far-UVC exposure at dose of 3 mJ/cm 2 /hour is expected to result in 90%, 99% and 99.9% viral inactivation in 8, 16 and 25 minutes. Thus, while staying within current regulatory dose limits, low-dose-rate far-UVC exposure can potentially safely provide a major reduction in the level of coronaviruses [24].
In a report released by Heimbuch and Harnish for Applied Research Associates in 2019, the efficiency of UV on MERS (EM/2012 strain) and SARS (200300592 strain) on personal protective equipment was reported. Log reduction rates reached by UV dose of 1 J/cm 2 , were ≥4.50 and ≥4.81 (equivalent to no detectable viable virus) for MERS and SARS, respectively. These values were still in the range between ≥3.87 and ≥4.28, even in the presence of artificial skin oil (sebum) and artificial saliva (mucin buffer), resembling real tissue conditions [22].
In their experiment, when Darnell et al. applied UVC to the SARS-CoV Urbani strain containing wells at a distance of 3 cm, partial inactivation started in 1 minute of UVC exposure with increasing efficiency up to 6 minutes and complete inactivation in 15 minutes [25].
UVC has also been suggested to be used for sterilization of N-95 masks commonly used for protection from COVID-19 and decontamination of respirators [41,42]. In their model, Sagripanti and Lytle claimed that coronaviruses are estimated to be least twice as sensitive to UVC (254 nm) as influenza viruses [43].
In a very recent review, Fernandes et al. reported that, other than UVC, photobiomodulation by application of laser light with various wavelength might be considered to be applied in COVID-19, expecting the effect on promoting immune system by increasing the production of adenosine triphosphate and oxygenation of red blood cells, and eventually increasing defense mechanisms of body against intense inflammatory processes induced by COVID-19 [44]. However, application of UVC irradiation and laser light in COVID-19 patients has not been reported yet.
UVC and laser light generator device used in the present study was first developed in 2017 for the treatment of infections within body with a semiflexible catheter and a custom-made light source. Upon rapid spread of COVID-19 cases, the device was adapted to be applied by transbronchial and intravenous route for severe COVID-19 cases. The germicidal spectrum range of UVC is 200-280 nm [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38]. Therefore, UVC-generator that we used in our study generates UVC at a wavelength of 200-280 nm. An effective dose of 2-3.7 mJ/cm2 UVC is required to destroy the SARS-CoV virus population in infected blood products [18,45]. We determined the duration and dose of UVC irradiation for the lungs and blood, based on this dose. The time it takes to sterilize the blood from the virus depends on the duration of circulation of the whole blood. Therefore, duration of the circulation of blood in the body as well as the volume of blood in the body are the main factors in determining the energy level of UVC and laser therapy. In intravenous application, the dose was calculated by converting the blood volume to the surface area. For the transbronchial route, the dose was calculated by the surface area of respiratory tract and alveoli.
Positive PCR tests converted to negative just after the procedure in five patients who were applied UVC. We think that the single patient whose PCR in BAL fluid did not convert to negative, might benefit from repeated applications of UVC irradiation, if we had done, considering the decrease in viral load in this patient. UVC patients also had shorter stay in intensive care unit, higher discharge rate and lower mortality compared to six patients who received only pharmacotherapy. On the basis of these critical findings, we suggest that UVC therapy may improve the patients' outcome in severe cases of COVID-19 without causing any side effects.
It is also remarkable that serum D-dimer levels declined faster and stayed at lower level in UVC group as compared to standard treatment group. There was no increase in ferritin levels in UVC group in contrary to significant increase in standard treatment group. High levels of D-dimer and ferritin have been suggested to be predictors of severe clinical course and poor prognosis in patients with . On the other hand, CRP level showed a parallel decline in both UVC and standard treatment groups, suggesting that UVC and laser light applied directly into the circulation had no obvious additional anti-inflammatory effect, contrary to what previous authors proposed [44]. However, it should be noted that UVC did not cause any extra inflammation in patients with COVID-19. Therefore, the biochemical findings of our case series may support the effectiveness and safety of UVC in COVID-19.

CONCLUSIONS
In conclusion, transbronchial and intravenous application of UVC (254 nm) and laser light (green laser 630 nm; red laser 535 nm) may improve the outcome of severe COVID-19 cases. Further studies involving more patients are needed to confirm the promising effect of UVC and laser light and to consider the use of light-based therapies in combination with pharmacotherapy, particularly for critical cases of COVID-19.
We suggest that our treatment protocol might be implemented on other organs/tissues, including wound or nasal mucosa, skin ulcers, teeth, toenail, stomach, intestines, i.e. tissues where light can be delivered safely and effectively [13].

Limitation
The major limitation of our study is the low number of patients. Actually, we had the opportunity to increase the number of patients easily before the publication, but since we observed that the preliminary results of our study in the UVC group were significantly better than the control group, we have decided to share the interim results of the first cases as a rapid contribution to the literature, because we are going through an extraordinary pandemic course and dealing with severe patient groups who need urgent treatment options.

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Before moving to clinical studies with larger sample size, we wanted our findings to be discussed openly within the scientific literature frame.
Author contributions: All authors have sufficiently contributed to the study, and agreed with the results and conclusions.
Funding: This is an investigator-initiated study. UVC-generator device and catheters were funded by RD Global Inc. (Florida, USA), which had no influence on the study design, findings, data interpretation, and writing of this manuscript.

Summary
Subchronic Systemic Toxicity Test was performed according to "ISO 10993-11 Biological evaluation of medical device: Systemic toxicology", "ISO 10983-2 Biological evaluation of medical devices: Animal welfare Requirements" and "ISO 10993-12 Biological evaluation of medical devices Sample Preparation" protocols.

Methods
The test was carried out using 5 females and 5 male mice / BAB-C of 8-12 weeks old sample. As recommended in the ISO 10993-11 test protocol, 5 female and 5 male mice / BAB-C were used for the control group for negative control.
The method of preparing the extract was chosen because the product cannot be applied directly. Incubation was performed at 37°C for 72 hours, and the extraction preparation rate was accepted as 3 cm 2 /mL.
The systemic effects of the product were followed during the 90-day test according to the clinical observation criteria stated in in ISO10993-11 and ISO10993-12, and no clinical findings were found. Food and water consumption of all groups is normal. No mice weight change was recorded in any mouse within the start and end time of the experiment. The liver weights of the experimental animals were found within normal limits (4-6%).
The product was applied for 90 days according to the ISO 10993-11 test protocol. In the gross pathology examination, no anomaly was detected.

Results
Repeated dose subchronic systemic toxicity test was performed with the extract obtained from the test material, and the test was terminated after 90 days of observation. It has been observed that as a result of clinical observation and gross pathology examinations according to ISO 10993-11, the product does not have a subchronic systemic toxic effect.
The L929 healthy mouse fibroblast cells (ATCC, USA) were cultured in DMEM (Sgima ® D6429, Germany), supplemented with 10% FBS (Sigma® f7524, Germany) and 1% penicillin/streptomycin (GibcoTM, Germany) and at 37°C in 5% CO2. L929 cells at 2x104 cell/well were plated in to 96 well black plate (Costar™, NY, USA) and incubated for 24 hours. After incubation, cells were exposed to UVC/laser application for 1, 3 and 5 minutes. After exposure, cell medium was discarded. MTT solution (0.5 mg/mL) was added to wells and cells were incubated for an additional 2 h at 37°C. After incubation, cell culture medium was discarded and 100 μL of isopropanol was added to wells to dissolve formazan. The absorbance was measured at 570 nm wavelength by ELISA microplate reader (Thermo Multiskan Spectrum, Finland). The percentage of cell viability compared to the negative control was calculated by using the following equation: Viability% = (Absorbance treatment group) / (Absorbance control) × 100%.

Results
The proportion of viable cells did not change significantly with increasing exposure time. This figure was 95.4%±4.7% after first minute, as compared to 100%±5.2%. Corresponding figures were 93.2%±7.3% and 86.7%±6.7%, at third and fifth minutes, respectively.
There was no significant decrease in cell viability within the exposure duration. The Ames test is based on bringing the mutated cell into another state by applying another mutation or reverse mutation. Salmonella typhimurium is used as microorganism in the test.

Methods
The cultures of S. typhimurium used in the test were prepared as follows: As the metabolic activation system in the test, NADPH Regensys™ A (contains 0.1 M phosphate buffer, glucose-6phosphate, MgCl2'KCI in pH 7.4) and Aroclor 1254-induced male Sprague Dawley supported by NADPH Regensys TM B (NADPH) co-factors. The postmitochondrial S9 fraction prepared from the liver of the rat was used. The analysis was carried out using a %10 (v/v) S9 mixture, and 500 µL of S9 mixture was used per plate.
Sample extraction was performed by incubation at 37°C for 72 hours by applying the v/v volume ratio PBS was used for extraction The sample extract was tested without waiting and 100 µL extract was used per plate.
The analysis was carried out in duplicate for all samples. Revertant (reverse mutant) colonies formed in each plate after incubation were counted visually. The average number of colonies for each duplicate study was determined. For each strain, the frequency of spontaneous reverse mutant (spontaneous reverse mutation frequency) was determined.
The AMES test of the sample was evaluated as negative (-) since the number of His reverse mutant colonies of the sample did not increase twice or above compared to spontaneous and negative control.

Results
The product does not cause mutation (not mutagenic) under the tested test conditions and used bacterial strains.  Blood taken from the ear veins of 3 rabbits was collected in a heparinized tube, approximately 5 mL from each rabbit.  Plasma free hemoglobin and total blood hemoglobin concentrations were calculated according to ASTM 756-13 Standard Practice for Assessment of Hemolytic Properties of Materials protocol.
 The blood that was found appropriate for the standard was diluted with PBS to be approximately 10 mg/mL hemoglobin.
 Test sample, positive control, negative control extract; The blind solution was prepared so that three tubes from each were 7 mL in each tube. One ml of blood diluted with PBS was added to the prepared tubes and incubated for 3 hours at 37°C.  After incubation, the tubes were centrifuged at 700-800 g for 15 minutes.  One mL of the supernatants was treated with 1 mL of cyanomethemoglobin for 4-5 minutes, and their absorbance was measured with a spectrophotometer at 540 nm.

Results
When the results are evaluated according to TS EN ISO 10993-4: 2010 Selection of Blood Interaction Experiments" and "ASTM F756-13: 2013 Standard Practice For Assessment of Hemolytic Properties of Materials" standards, Hemolytic Degree of the product sample was determined to be "not hemolytic".

Summary
Skin sensitization test was performed according to "ISO 10993-10: 2010 Biological evaluation of medical device: Tests for irritation and delayed-type hypersensitivity", "ISO 10993-2: 2006 Biological evaluation of medical devices: Animal welfare requirements" and "ISO 10993-12".

Methods
The purpose of this test is to evaluate whether the sample tested in the animal model causes skin hypersensitivity.
Guinea pig was used for the experiments as recommended in the standard protocol.
Each animal was injected intradermally 0.1 ml of each of the following into the injection areas (A, B and C), as shown in Figure A1: Region A: Freud's full adjuvant with physiological saline is 50:50 vol.
Region B: Test sample (non-propelled extract); solvent alone is injected into control animals.
Region C: Test sample at the concentration used in the region, Freund's full adjuvant and physiological saline (50%) Figure A1. Application points. (1) Test animal head tip, (2) test region (0.1 mL intradermal injection site), (3) area between two clipped scapula, (4) caudal tip Seven days after the completion of the intradermal induction phase, superficial application was performed to each animal with test specimens impregnated with gaseous cloth of approximately 8 cm 2 . The skin was pretreated 48 hours prior to local application to avoid irritation with 10% sodium dodecyl sulfate. Local application was terminated after 48 hours. All competing test and test sample control animals were applied locally to control and test samples directly to the areas that could not be treated at the induction stage, using appropriate patches dipped in the test sample at the concentration in the C region 14 days after completion of the local induction phase. 24 hours later, dressings and patches were removed. Following removal of dressings in the application areas, the appearance of the control animals and competing skin areas of the test was observed between 24 and 48 hours. Skin reactions were evaluated under good lighting.

Results
Skin sensitization test was performed with the extraction solution obtained from the tested sample product, and the test was terminated after 27 days of observation. The test result of the product was evaluated as "0-No visible change" according to the grade scale given in Magnusson and Kligman Scale.