Rapid Clothing Disinfectant and Mildew Remover Bactericidal Effect and Performance Testing

Sun Shanjia   Quan Limin

       
       The rapid clothing disinfectant and mildew remover is a compound disinfectant of biguanides and ethanol, a colorless transparent liquid with no special odor. The average content of polyhexamethylene biguanide is 1317mg/L, ethanol content is 40.47% by volume, and pH is 6.15. To study the disinfection-related properties of this rapid clothing disinfectant and mildew remover, we conducted laboratory physicochemical property tests, bactericidal effect, toxicity, and on-site disinfection effect observations. The results are introduced as follows.
       
       1  Materials and Methods
       1.1 Physicochemical Property Determination
       1.1.1 Content Determination   The ethanol content in the rapid clothing disinfectant and mildew remover was measured by gas chromatography according to the 2002 edition of the "Disinfection Technical Specifications." The polyhexamethylene biguanide content was detected by spectrophotometry. The specific measurement steps are: (1) Preparation of standard curve: standard solutions of polyhexamethylene biguanide at concentrations of 0.8, 1.6, 3.2, 4.8, 6.4 mg/L were prepared. The spectrophotometer wavelength was set to 545nm to measure the absorbance of these standard solutions and plot the standard curve. (2) Sample measurement: 10ml of unknown concentration polyhexamethylene biguanide solution was pipetted into a 25ml volumetric flask, added with 1ml acetic acid solution and 2.5ml indicator solution, diluted to 25ml with distilled water, shaken well, and absorbance measured by spectrophotometer. (3) Calculation: The polyhexamethylene biguanide content of the unknown solution was calculated based on the standard curve.
       1.1.2 Stability Test   Three sealed packages of the rapid clothing disinfectant and mildew remover were placed in a 37°C incubator for 90 days. Samples before and after storage were taken to detect ethanol content and polyhexamethylene biguanide content by the above methods, and the degradation rate was calculated.
       1.2  Preparation of Bacterial Suspension
       Test bacteria included Staphylococcus aureus (ATCC 6538), Escherichia coli (8099), Candida albicans (ATCC 10231), and Pseudomonas aeruginosa (ATCC 15442), all provided by the Disinfection Testing Center of the Military Medical Academy. Typical colonies of each bacterium were inoculated on nutrient agar slants (Sabouraud agar for Candida albicans) and cultured for 24h to obtain fresh slant cultures. The bacterial film was washed off with 1000mg/L tryptone physiological saline (TPS) diluent and diluted to prepare bacterial suspensions. During testing, the bacterial suspension was diluted with 30g/L bovine serum albumin solution to achieve a recovery bacterial count of 1×10^7 to 5×10^7 cfu/ml.
       1.3  Neutralizer Identification Test 
       Test bacteria were Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. Six parallel groups were set up according to the quantitative suspension bactericidal test procedure for neutralizer identification. Results showed: Group 1 had no bacterial growth or few bacteria; Group 2 had bacterial counts exceeding 100 cfu/ml; Groups 3, 4, and 5 had intergroup error rates not exceeding 15%; Group 6 had no bacterial growth. Three consecutive repeated tests were consistent, indicating the selected neutralizer and its concentration were appropriate.
       1.4  Quantitative Suspension Bactericidal Test
       4.0ml of the test concentration of the rapid clothing disinfectant and mildew remover was dispensed into sterile test tubes (TPS as positive control), placed in a 20°C water bath, and 1.0ml of bacterial suspension was added and reacted for the specified time. Then 0.5ml of the sample was added to 4.5ml of neutralizer tube and mixed. After 10 minutes of neutralization, viable bacteria were counted to calculate the log reduction. The test was repeated at least three times.
       1.5  Potency Test
       The rapid clothing disinfectant and mildew remover was diluted with hard water to prepare three concentrations: A (1.5x), B (x), and C (0.5x) for testing. The test method and result evaluation were conducted according to the 2002 edition of the "Disinfection Technical Specifications" potency test regulations.
       1.6  On-site Disinfection Test
       Thirty staff members were randomly selected as volunteers. Before disinfection, sterile cotton swabs moistened with sampling solution were used to swab the fingers and forearm skin areas according to specified areas. The swabs were placed into sampling tubes as positive controls. Then the rapid clothing disinfectant and mildew remover stock solution was applied with cotton swabs to the other side for wiping disinfection and acted for 1.0 min (3.0 min for forearm skin). Sterile cotton swabs moistened with sampling solution containing neutralizer were used for swabbing as above, and the swab tips were cut into sampling tubes containing neutralizer as the post-disinfection test group. Sampling tubes were shaken thoroughly to elute bacteria, and the eluate was used for viable bacteria counting to calculate the natural bacterial reduction log value.
       1.7  Toxicity Test
       1.7.1  Acute Oral Toxicity Test  A single maximum dose test was used. Twenty SD rats weighing 150g each, half male and half female, were selected. Before dosing, rats were fasted overnight with water allowed. The dose was 5000mg/kg (body weight), administered once by gavage at 2.0ml/100g (body weight). Animals were observed daily for toxic symptoms and mortality for 14 consecutive days, and the LD50 was calculated.
1.7.2 Mouse Bone Marrow Polychromatic Erythrocyte Micronucleus Test Select 50 ICR mice, divided into 5 groups, 10 mice per group, half male and half female. The test substance is the original disinfectant solution, with three dose groups designed at 5000, 2000, and 500 mg/kg (body weight). The positive control group uses 40 mg/kg (body weight) cyclophosphamide, and the negative control is distilled water. The animals are dosed by oral gavage for 30 hours, with two gavage administrations at 24-hour intervals, each dose volume 0.2 ml/10 g (body weight). Six hours after the last dose, animals are euthanized by cervical dislocation, and bone marrow from the sternum is collected, diluted with calf serum, smeared, fixed with methanol, and stained with Giemsa. Under an optical microscope, 1000 polychromatic erythrocytes are counted per animal to observe the number of polychromatic erythrocytes containing micronuclei, calculating the micronucleus rate (per thousand), statistically processed by Poisson distribution; 200 polychromatic erythrocytes are counted to calculate the ratio of polychromatic erythrocytes to mature erythrocytes (PCE/NCE).
1.7.3 Repeated Complete Skin Irritation Test Select 3 Japanese big-eared white rabbits. 24 hours before the test, shave the hair on both sides of the spine on the rabbit's back, with a hair removal area of about 3cm×3cm on each side. The next day, apply 0.5 ml of the original disinfectant solution directly to a 2.5cm×2.5cm area on the left side of the rabbit's skin, and apply physiological saline on the other side as a control. Four hours after application, wash off residual test substance with water. Apply once daily for 14 consecutive days. Observe results 24 hours after each application, scoring irritation response and evaluating irritation intensity.
1.7.4 Subacute Toxicity Test Select 40 SD rats weighing 150–180 g, randomly divided into 4 groups, 10 rats per group, half male and half female. Based on the oral toxicity LD50 value in mice, design three dose groups at 40, 200, and 1000 mg/kg (body weight), with distilled water as the negative control. Administer by gavage at 1.0 ml/100 g (body weight) once daily for 30 consecutive days. During the test, observe animals daily for toxic symptoms, clinical signs, and activity; weigh weekly. Twenty-four hours after the last dose, euthanize by cervical dislocation, collect eyeball blood for routine blood and biochemical tests. Routine blood tests include hemoglobin (Hb), hematocrit (HCT), red blood cell (RBC), white blood cell (WBC), platelet (PLT) counts, and WBC classification. Biochemical indicators measured by OLYMPUS AU400 automatic biochemical analyzer include total protein (TP), albumin (ALB), creatinine (Cr), blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol (CHOL), triglycerides (TG), and glucose (GLU). Perform gross necropsy, weigh liver, kidney, spleen, heart, lung, testis, and ovary, calculate organ-to-body weight ratios, and prepare pathological sections of liver, kidney, spleen, stomach, duodenum, testis, ovary, and brain for examination.

2 Results
2.1 Physicochemical Property Test Results
Test results show that the ethanol content of the original Rapid Clothing Disinfectant and Mildew Remover in three batches averaged 40.8%, 39.9%, and 40.7% by volume, with an overall average of 40.47% by volume. The polyhexamethylene biguanide content averaged 1320, 1331, and 1300 mg/L, with an overall average of 1317 mg/L. Stability test results indicate that when the Rapid Clothing Disinfectant and Mildew Remover is sealed and stored in a 37°C incubator for 90 days, the average ethanol decrease rate is 0.23%; the average polyhexamethylene biguanide decrease rate is 0.67%.
2.2 Neutralizer Identification Test
Test results demonstrate that a diluent containing 6 g/L sodium thiosulfate, 2.5 g/L sodium sulfite, 50 g/L Tween 80, and 7 g/L lecithin as a neutralizer can effectively neutralize the residual effects of the Rapid Clothing Disinfectant and Mildew Remover original solution on Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans, and that the neutralizer and its products have no effect on the culture medium or test bacteria (Table 1).

Table 1 Rapid Clothing Disinfectant and Mildew Remover Neutralizer Identification Test Results

Group Average Recovered Bacteria Count (cfu/ml)
Staphylococcus aureus Pseudomonas aeruginosa Candida albicans

1                        0                             0                            0
2                        81                           79                          63
3                        33 600 000             16 800 000            3 840 000
4                        35 700 000             23 900 000            3 420 000
5                        34 500 000             22 400 000            2 830 000
6                        0                             0                            0

2.3 Suspension Quantitative Bactericidal Test
Results show that the Rapid Clothing Disinfectant and Mildew Remover original solution acts on Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans in suspension for 0.5 minutes, with the average log kill value in each test greater than 5.00 (Table 2).
2.4 Efficacy Test Results
Three repeated tests demonstrate that the minimum effective concentration of this Rapid Clothing Disinfectant and Mildew Remover is 20% by volume, equivalent to 343 mg/L polyhexamethylene biguanide and 11% ethanol.
2.5 On-site Disinfection Test Results
Hand and skin tests with 30 subjects each proved that wiping hands with the original solution of this rapid clothing disinfectant and mildew remover for 1.0 min and wiping the forearm skin for 3.0 min can eliminate natural bacteria on the skin by more than 1 logarithmic value.
2.6 Toxicity Test Results
2.6.1 Acute Oral Toxicity Test Results After gavage administration of the original solution of this rapid clothing disinfectant and mildew remover, no obvious toxic symptoms were observed in the test animals during the observation period, and no animals died. All animals were euthanized at the end of the observation period for gross anatomical examination, and no visible pathological changes were found in the liver, kidney, spleen, stomach, intestines, heart, lungs, or other organs. The LD50 values for both male and female animals were >5000 mg/kg (body weight), classifying it as a non-toxic substance.

Table 2 Quantitative Bactericidal Test Results of Rapid Clothing Disinfectant and Mildew Remover Suspension

Test Bacteria Control Group Recovered Bacteria Log Value Log Reduction Values at Different Exposure Times (min)

                                        0.5             1.0             1.5

Staphylococcus aureus 6.83 5.62 6.82 6.83
                                     6.82                             5.64            6.82            6.82
                                     6.87                             5.26            6.87            6.87
Pseudomonas aeruginosa 6.62 4.92 6.62 6.62
                                     6.66                             4.88            6.66            6.66
                                     6.69                             4.88            6.69            6.69
Escherichia coli 7.34 5.28 7.34 7.34
                                     7.25                             5.32            7.25            7.25
                                     7.21                             5.36            7.21            7.21
Candida albicans 7.08 7.08 7.08 7.08
                                     7.22                             7.22            7.22            7.22
                                     7.04                             7.04            7.04            7.04
 

2.6.2 Mouse Bone Marrow Polychromatic Erythrocyte Micronucleus Test Results The micronucleus rates of all dose groups showed no significant difference compared with the negative control group, while the cyclophosphamide group showed a highly significant difference compared with the negative control group (P<0.01). The PCE/NCE ratios of all groups were within the normal range. The rapid clothing disinfectant and mildew remover showed negative results in the mouse bone marrow polychromatic erythrocyte micronucleus test for both male and female mice (Table 3).

Table 3 Mouse Bone Marrow Polychromatic Erythrocyte Micronucleus Test Results of Rapid Clothing Disinfectant and Mildew Remover

Dose Group Micronucleus Rate PCE/NCE

(mg/kg•bw) Tested Containing Micronuclei Micronucleus Rate (‰) Tested NCE PCE/NCE
PCE Count PCE Count (x±s) PCE Count (x±s)

5000 5000 7 1.3±0.5 1000 899 1.118±0.032
2000 5000 8 1.5±0.5 1000 895 1.113±0.032
500 5000 7 1.4±0.5 1000 897 1.116±0.020
0                5000       7       1.3±0.5        1000   892       1.112±0.021
40(cp)             5000       128       24.6±5.0        1000   1087       0.913±0.021

Note: "cp" inside the table represents the positive control group cyclophosphamide; each test group included 5 male and 5 female animals.

      
2.6.3 Subacute Toxicity Test Results During the test period, there were no significant differences in body weight and weight gain between the test animals in each dose group and the control group (P>0.05). No obvious changes were observed in the blood routine and biochemical indicators of rats. No obvious lesions were found during gross anatomical examination of animals in each dose group; pathological section examination showed no pathological changes related to the sample.
2.6.4 Single Complete Skin Irritation Test Results The results showed that wiping the skin with the original solution of this rapid clothing disinfectant and mildew remover caused no irritation in multiple skin irritation tests on white rabbits, with a score of 0, judged as non-irritating.

3 Discussion
In recent years, the disinfection and mildew removal of clothes and fabrics have attracted attention from disinfection experts and the public. This study's rapid clothing disinfectant and mildew remover contains 1300 mg/L polyhexamethylene guanidine and 40% ethanol by volume. Its usage concentration is lower than generally reported in the literature, but tests have proven it has good killing effects on bacterial propagules and Candida albicans [1-3]. There have been no comprehensive reports on the efficacy, stability, and toxicity tests of the rapid clothing disinfectant and mildew remover made from a compound of polyhexamethylene biguanide and ethanol. Test results show it is non-toxic, non-irritating to skin, negative in micronucleus tests, and subacute toxicity tests meet standard requirements. This rapid clothing disinfectant and mildew remover has good bactericidal effects, stable performance, and can be widely used for disinfection of clothes and fabrics.

(Sun Shanjia is Chairman of Qingdao Jieshen Investment Group Co., Ltd.      Quan Limin is an expert at Qingdao Center for Disease Control and Prevention, Shandong Province)