Disinfection Standards for Hygienic Wipes

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In the hygiene standards for disposable sanitary products C3.1, the neutralizing agent identification test is required; to conduct tests on bactericidal performance, it is necessary to pass this neutralizing agent identification test. C3.1.1 Test groups 1) Infected sample + 5 mL PBSGB 15979‑2002 Hygienic standard for disposable sanitary products, replacing GB 15979-1995. Preface: This standard is mandatory in its entirety. Since its issuance in 1996, GB15979－1995 \"Hygienic Standards for Disposable Hygiene Products\" has helped manufacturing enterprises clarify their hygienic requirements and goals, while also providing regulatory authorities with a basis for supervision and monitoring. It has played a positive role in promoting the healthy development of this industry and improving its hygienic standards. At the same time, with the development of product types and materials, there are some aspects of this standard that need to be improved. Therefore, a revised version of this standard is proposed. This standard replaces GB15979－1995 as of the date of its implementation. Appendices A to G of this standard are the appendices to the standard. This standard was proposed by the Ministry of Health of the People’s Republic of China. The entity responsible for drafting this standard: Shanghai Center for Disease Control and Prevention ； Participating drafting units: Procter & Gamble (China) Co., Ltd., Johnson & Johnson (China) Co., Ltd. The main drafters of this standard are Shen Wei, Lu Min, Yang Hongping, Zhou Mi, Pan Xihé, and Liu Yujing. 1 Scope This standard specifies the environmental hygiene standards for products and production processes related to disposable sanitary products, the biological monitoring criteria for evaluating disinfection effectiveness and the corresponding testing methods. It also outlines the hygiene requirements for raw materials as well as for the production, disinfection, storage, and transportation of products, along with the requirements regarding product labeling. In this standard, disposable sanitary products refer to: This standard applies to departments, organizations, or individuals in China that are engaged in the production and sales of disposable sanitary products, as well as to those who distribute imported disposable sanitary products. 2 Referenced Standards The provisions contained in the following standards become part of this standard by being referenced herein. At the time of publication of this standard, all versions listed are valid. All standards will be revised, and those using this standard should consider the possibility of using the latest versions of the following standards. GB 15981-1995 Methods and Standards for Evaluating the Efficacy of Disinfection and Sterilization 3 Definitions This standard adopts the following definitions: Single-use sanitary products – Various daily household items that come into direct or indirect contact with the human body and are discarded after being used once; they are used to achieve physiological hygiene or health care purposes (antibacterial or bacteriostatic effects), and can be in solid or liquid form. For example, disposable gloves or finger cots (excluding medical gloves or finger cots), tissues, wet wipes, sanitary wipes, phone covers, hats, masks, **, menstrual hygiene products for women (including sanitary pads), and waste management products such as diapers (excluding toilet paper such as crumpled paper), ** are collectively referred to as “hygiene products” in this standard. 4 Product hygiene indicators 4.1 The appearance must be neat and in line with the inherent characteristics of such sanitary products; there should be no abnormal odors or foreign substances. 4.2 It shall not cause adverse irritation, allergic reactions, or other harmful effects on the skin and mucous membranes. 4.3 The product must meet the microbiological criteria in Table 1. Table 1 Product Types, Microbial Indicators, Initial Contamination Bacteria1) cfu/g, Bacteria, Total Colony Count cfu/g or cfu/mL, E. coli Group, Pathogenic Pyogenic Bacteria2) Fungi, Total Colony Count cfu/g or cfu/mL. Gloves or finger cots, Tissues, Wet wipes, Hats**, Phone covers: ≤200, Not detectable, Not detectable; ≤100 for Antibacterial (or Antifungal) Liquid Products; ≤200, Not detectable, Not detectable; ≤100 for Hygienic Wet Wipes. ≤20, Not detectable, Not detectable, Not detectable. Masks:. General grade: ≤200, Not detectable, Not detectable; ≤100 for Disinfection-grade products; ≤10,000, ≤20, Not detectable, Not detectable, Not detectable. Women’s menstrual hygiene products:. General grade: ≤200, Not detectable, Not detectable; ≤100 for Disinfection-grade products; ≤10,000, ≤20, Not detectable, Not detectable, Not detectable. Diaper and other excrement-related hygiene products:. General grade: ≤200, Not detectable, Not detectable; ≤100 for Disinfection-grade products; ≤10,000, ≤20, Not detectable, Not detectable, Not detectable. **:. ≤20, Not detectable, Not detectable, Not detectable. 1) If the initial contamination level exceeds the values listed in the table, the sterilization index should be increased accordingly to ensure that the limits for bacteria and fungi specified in these standards are met. 2) Pathogenic pyogenic bacteria refer to Pseudomonas aeruginosa, Staphylococcus aureus, and hemolytic streptococci. 4.4 In addition to meeting the microbiological standards specified in Table 1, hygiene wipes must have a kill rate of ≥90% against Escherichia coli and Staphylococcus aureus. If the effect on fungi is to be indicated, a kill rate of ≥90% against Candida albicans is also required; their bactericidal effect must remain effective at room temperature for at least 1 year. 4.5 In addition to meeting the microbiological standards for products of the same type and grade as listed in Table 1, antibacterial (or bacteriostatic) products must have a bacteriostatic rate of ≥50% (for soluble products) or >26% (for non-soluble products) against Escherichia coli and Staphylococcus aureus. If the effect on fungi is to be indicated, the bacteriostatic rate against Candida albicans must also be ≥50% (for soluble products) or >26% (for non-soluble products), and this bacteriostatic effect must remain effective for at least 1 year at room temperature. 4.5 For any sanitary products that have been disinfected with ethylene oxide, the residual amount of ethylene oxide at the time of shipment must be ≤250μg/g. 5 Environmental Health Indicators in Production 5.1 The total bacterial colony count in the air of assembly and packaging workshops should be ≤2,500 cfu/m3. 5.2 The total bacterial colony count on the surface of the workbench should be ≤ 20 cfu/cm2. 5.3 The total bacterial colony count on the workers’ hands should be ≤ 300 cfu per hand, and no pathogenic bacteria should be detected. 6 Biological monitoring and evaluation of disinfection efficacy 6.1 Ethylene oxide disinfection: The kill index against Bacillus subtilis var. niger spores (ATCC 9372) should be ≥103. 6.2 Disinfection by ionizing radiation: The kill index for Bacillus brevis spores E6d (ATCC 27142) should be ≥103. 6.3 Pressure steam sterilization: The kill index for Bacillus stearothermophilus spores (ATCC 7953) should be ≥103. 7 Testing Methods 7.1 Product Testing Methods 7.1.1 Product Appearance: Visual inspection; it shall meet the requirements specified in 3.1 of this standard. 7.1.2 Product toxicology testing methods: See Appendix A. 7.1.3 Product microbial testing methods: See Appendix B. 7.1.4 Test methods for the sterilization performance, antibacterial performance, and stability of products: See Appendix C. 7.1.5 Test method for ethylene oxide residue in products: See Appendix D. 7.2 Sampling and testing methods for the production environment: See Appendix E. 7.3 Biological monitoring evaluation methods for disinfection efficacy: See Appendix F. 8 Hygiene requirements for raw materials 8.1 Raw materials should be non-toxic, harmless, and free from pollution ； The packaging of raw materials should be clean, with the name of the contents, the manufacturer, the production date, or the batch number clearly indicated ； Raw materials that affect hygiene quality should not be exposed ； Raw materials with special requirements should indicate their storage conditions and shelf life. 8.2 For raw materials that affect the hygiene quality of the product, corresponding inspection reports or certification documents shall be available; where necessary, microbial monitoring shall be carried out and appropriate measures taken. 8.3 The use of discarded sanitary products as raw materials or semi-finished products is prohibited. 9 Hygiene requirements for the production environment and processes 9.1 The area surrounding the production site should be clean, free of trash, and free from breeding grounds for pests such as mosquitoes and flies. 9.2 The production area should have sufficient space to meet production needs; its layout must comply with the requirements of the production process, with proper separation between people and materials, and no reverse or cross-flow in the product flow. There should be pollution prevention measures and strict operating procedures for the intake of raw materials and the output of finished products, in order to reduce microbial contamination in the production environment. 9.3 The production area shall be equipped with effective dust, insect, and rodent control measures. The floors, walls, and work surfaces shall be flat, smooth, free of dust, and easy to clean and disinfect. There shall be sufficient lighting as well as air disinfection or purification measures to ensure that the production environment meets the requirements specified in Chapter 5 of these standards. 9.4 Equip necessary production and quality inspection facilities, maintain complete records of production and quality checks, to ensure the hygiene and quality of products. 9.5 Where flammable, explosive materials are used or harmful substances are generated during the production process, appropriate safety protection measures must be in place to comply with **relevant standards or regulations. 9.6 Raw materials and finished products should be stored separately; raw materials and finished products that are pending inspection, qualified, or unqualified must be stored apart with clear markings. The warehouse should be dry, clean, and well-ventilated, equipped with pest and rodent control measures as well as pallets, to meet the requirements for product storage. 9.7 To enter the production area, one must change into work clothes and work shoes, as well as wear a work hat. Those who come into direct contact with bare products need to wear masks, and they should wash and disinfect their hands or wear gloves ； In front of the production area, there should be **rooms, washbasins, disinfection tanks, and buffer zones. 9.8 Personnel involved in the production of sanitary products must maintain personal hygiene; they should not have long nails, must not wear jewelry while working, and their long hair should be tucked inside work hats. Patients or carriers of dysentery, typhoid fever, viral hepatitis, active pulmonary tuberculosis, condyloma acuminata, gonorrhea, and suppurative or exudative skin diseases shall not participate in production activities that involve direct contact with the product. 9.9 Personnel engaged in the production of sanitary products must undergo health checks as well as training on hygiene knowledge (including production hygiene, personal hygiene, and relevant standards and regulations) before starting work and on a regular basis (once a year); only those who pass these assessments may take up their positions. 10 Requirements for the disinfection process 10.1 For products intended for disinfection purposes, effective disinfection methods such as ethylene oxide, ionizing radiation, or pressure steam must be used for the final disinfection step; the disinfection equipment employed must comply with relevant health standards. 10.2 Disinfection procedures, technical parameters, and operating protocols shall be established in accordance with product hygiene standards, criteria for evaluating initial contamination levels, and biological monitoring of disinfection effectiveness; once verified, operations must be carried out strictly in accordance with the established disinfection processes. When there are changes in the disinfection procedure, technical parameters, or the raw materials or production processes that affect the disinfection efficiency, the disinfection process must be re-verified. 10.3 During each disinfection process, appropriate process (physical) and chemical indicators must be monitored, and corresponding biological indicators must be used for monitoring on a monthly basis. Only when the process monitoring, chemical monitoring, and biological monitoring meet the specified requirements can the disinfected items be released for distribution. 10.4 After disinfection, the appearance and performance of the product should show no visible differences compared to those before disinfection. 11 Packaging, Transportation, and Storage Requirements 11.1 The entities or individuals responsible for transporting or storing sanitary products must do so in strict accordance with the transportation and storage requirements provided by the manufacturer. 11.2 Packaging materials that come into direct contact with the product must be non-toxic, harmless, and clean. All packaging materials for the product must have sufficient sealing properties and strength to ensure that the product remains free from contamination under normal conditions of transportation and storage. 12 Product labeling requirements 12.1 Product labeling shall comply with the provisions of the Product Quality Law of the People’s Republic of China, and the applicable health standard numbers, as well as the production date and expiration date (validity period) or production batch number and limited use date, shall be indicated on the product packaging. 12.2 Disinfection-grade products shall also bear the inscription “Disinfection Grade” on their sales packaging, along with the date of disinfection and expiration date, or the disinfection batch number and limited use date. The transportation packaging shall display the inscription “Disinfection Grade” as well as the name and address of the disinfection facility, the disinfection method, the date of disinfection and expiration date, or the disinfection batch number and limited use date. Appendix A (Standard Appendix) Product Toxicology Testing Methods A1 Toxicology testing indicators for various products: When changes in raw materials, production processes, etc. may affect the toxicity of the products, valid toxicology test reports for the finished products (prepared by **accredited third parties**) should be provided according to Table A1, depending on the type of product. Table A1 Product Types: Skin Irritation Test **Mucosal Irritation Test. Skin** Reaction Test: Gloves or finger covers, ** √ 　 √ Antibacterial (or bacteriostatic) liquid products √ Choose according to use 1) √ Wet wipes, sanitary wet wipes √ Choose according to use 1) Choose according to material. Masks √ 　 Women’s menstrual hygiene products √ √ Diaper-like waste management products √ 　 √ ** √ √ 1) Products intended for** mucous membranes must undergo a **mucosal irritation test**, but not a skin irritation test. A2 Test methods: The skin irritation test, **mucosal irritation test, and skin** reaction test methods are carried out in accordance with the corresponding test procedures outlined in the section on \"Toxicological testing of disinfectants\" in Volume 1 of the \"Technical Specifications for Disinfection\" (3rd edition), namely \"Experimental Technical Specifications\" (1999), issued by the Ministry of Health. The sample preparation method for solid products follows A3. Note 1: The control groups used in skin irritation tests should be normal saline and patch stickers. 2 In skin **reactions, the doses used for sensitization and challenge treatments remain the same. A3 Sample Preparation A3.1 Skin irritation test and skin** reaction test: Cut a piece of the product the size of a patch in a transverse manner. For dry products such as diapers and menstrual hygiene products, moisten them with saline solution, apply them to the skin, and then cover them with adhesive patches. Wet products, such as wet wipes, can be cut to the appropriate size as needed, applied directly to the skin, and then covered with a patch. A3.2 **Mucosal irritation test A3.2.1 For dry products (such as menstrual hygiene products), an adequate amount of the product is cut transversely, added to sterilized saline at a ratio of 1g/10mL, sealed in an extraction container, stirred, and then placed at 37°C ± 1°C for 24 hours. Cool to room temperature, stir, and then take a sample of the solution for testing. A3.2.2 For wet products (such as wet wipes), on the day of the **mucosal irritation test, the additive solution contained in the wipes is extracted as the sample. A4 Judgment Criteria: The relevant sections on \"Final judgment of toxicological test results\" in Volume 1 of the Ministry of Health’s \"Disinfection Technical Specifications\" (3rd edition), namely \"Experimental Technical Specifications\" (1999), are used as the principles for judging test results. 　 Appendix B (Standard Appendix) Methods for Microbial Testing of Products B1 Product Collection and Sample Processing At least 12 minimum selling unit samples shall be taken from three transport packages within the same batch; 1/4 of these samples are used for testing, 1/4 are kept as reference samples, and the remaining 1/2 can be sealed on site for use in retesting if necessary. The minimum sales packaging for sampling shall be intact and must not be opened prior to inspection. Under 100-grade purification conditions, at least 3 packages intended for testing were opened using aseptic methods. Samples were taken from each package, 10 g ± 1 g of the sample was accurately weighed, chopped up, and added to 200 mL of sterilized saline, after which it was thoroughly mixed to obtain a saline sample solution. For liquid products, the original solution is used directly as the sample solution. If the sample under inspection contains a large amount of water-absorbing resin material, preventing sufficient sample solution from being drawn out, the amount of diluent can be increased by 50 mL each time until enough sample solution for testing can be obtained. The dilution factor should be adjusted when calculating the total bacterial colony count and the total fungal colony count. B2 Method for detecting the total bacterial colony count and initial contaminating bacteria. This method is applicable to the detection of initial contaminating bacteria in products as well as the total bacterial colony count (hereinafter collectively referred to as bacterial colony count). B2.1 Procedure: After the aforementioned saline sample has settled naturally, the supernatant is collected for colony counting. A total of 5 petri dishes were used for inoculation; 1 mL of the sample solution was added to each dish, after which 15–20 mL of melted nutrient agar medium cooled to around 45°C was poured into each dish and mixed thoroughly. After the agar has solidified, the petri dish is turned over and incubated at 35°C ± 2°C for 48 hours, after which the number of colonies on the plate is counted. B2.2 Result reporting: Plates with colony growth in patches should not be used ； Count the colonies on the plates that meet the requirements, and calculate the result using equation (B1): X1 = A × K -------------------------------------------------------------------------------- . . . (B1) Where: X1 – total number of bacterial colonies, in cfu/g or cfu/mL ； A – Total number of bacterial colonies on 5 plates of nutrient agar medium ； K――Dilution factor. When the colony count is 100 or less, it is reported as the actual value; when it exceeds 100, two significant figures are used. If the total colony count of the sample exceeds the requirements of this standard, retesting and result reporting shall be carried out in accordance with B2.3. B2.3 Reinspection Method: The retained reinspection samples are tested again twice using the previous method; if the average of the results from these two tests meets the requirements specified in this standard, then the sample under inspection is considered qualified ； If the average value for any one of these tests exceeds the value specified in this standard, the sample under test is considered unqualified. B3 Method for testing Escherichia coli colonies B3.1 Procedure: Take 5 mL of the sample and inoculate it into a 50 mL lactose-cholesterol fermentation tube. Incubate at 35°C ± 2°C for 24 hours; if no acid or gas is produced, the result is reported as negative for Escherichia coli colonies. 　　If acid and gas are produced, make a streak inoculation on Eosin Methylene Blue agar plates, incubate at 35°C ± 2°C for 18–24 hours, and observe the colony morphology on the plates. Typical E. coli colonies are black-purple or red-purple in color, round in shape with smooth, even edges; their surface is moist and often has a metallic luster. Some colonies are purple-black, without or with only a slight metallic luster, or pink in color, with a darker center. 　　Take 1–2 suspected E. coli colonies for Gram staining and microscopic examination; simultaneously inoculate them into lactose fermentation tubes and incubate at 35°C ± 2°C for 24 hours to observe gas production. B3.2 Result Report: If the lactose-bile salts fermentation tube shows acid production and gas production, the lactose fermentation tube also shows acid production and gas production, typical E. coli colonies are present on the Eosin Methylene Blue plate, and it is a Gram-negative non-spore-forming bacterium, then it can be reported that E. coli was detected in the sample under examination. B4 Detection method for Pseudomonas aeruginosa B4.1 Procedure: Take 5 mL of the sample solution and add it to 50 mL of SCDLP culture medium, mix thoroughly, then incubate at 35°C ± 2°C for 18–24 hours. If Pseudomonas aeruginosa grows, a thin film of bacteria forms on the surface of the culture medium, which often appears yellow-green or blue-green. Cultures were picked from the thin bacterial film on the culture medium and streaked onto hexadecyltrimethylammonium bromide agar plates. These plates were incubated at 35°C ± 2°C for 18–24 hours to observe the characteristics of the colonies. Pseudomonas aeruginosa grows well on this medium; its colonies are flat with irregular edges, and the medium surrounding the colonies appears slightly pink. No other bacteria grow on it. 　　A smear of the suspicious colony is prepared for Gram staining; if Gram-negative bacteria are observed under the microscope, the following test should be conducted: Oxidase test: A small piece of clean white filter paper is placed in a sterilized petri dish, and the suspicious colony is transferred onto the filter paper using a sterile glass rod. Then, a drop of freshly prepared 1% dimethyl p-phenylenediamine solution is added to it. If a pink or purplish-red color appears within 30 seconds, the oxidase test is positive; no color change indicates a negative result. 　　Pseudomycin test: Take 2–3 suspect colonies and inoculate them separately onto slants containing medium for pseudomycin determination. Incubate at 35°C ± 2°C for 24 hours, then add 3–5 mL of chloroform. Shake thoroughly to dissolve any pseudomycin present in the culture. Once the chloroform turns blue, transfer it using a pipette to another test tube and add 1 mL of 1 mol/L hydrochloric acid. Shake and let it stand for a while. A positive result is indicated by a pink or purplish-red color in the upper layer, indicating the presence of pyocyanin. 　　Nitrate reduction gas production test: A pure culture of the microorganism under examination is inoculated into nitrate peptone broth, and incubated at 35°C ± 2°C for 24 hours; a positive result is indicated by the presence of gas in the small tube attached to the medium. 　　Gelatin liquefaction test: A pure culture of the suspected colony is inoculated by puncture into gelatin medium, and then incubated at 35°C ± 2°C for 24 hours. After that, it is placed at 4–10°C; a positive result is indicated if the medium remains liquid, while a negative result is obtained if it solidifies. 　　42°C growth test: A suspect culture is picked and inoculated on ordinary agar slant medium, then incubated at 42°C for 24–48 hours; the presence of Pseudomonas aeruginosa growth indicates a positive result. B4.2 Result Report: After enrichment, isolation, and culture of the sample under examination, if it is confirmed to be a Gram-negative bacillus and both the oxidase test and the Pseudomonas test yield positive results, then it can be reported that Pseudomonas has been detected in the sample. If the pyocyanin test is negative, but the tests for gelatin liquefaction, nitrate reduction with gas production, and growth at 42°C are all positive, it is still possible to report the detection of Pseudomonas aeruginosa in the sample under examination. B5 Detection method for Staphylococcus aureus B5.1 Procedure: Take 5 mL of the sample solution and add it to 50 mL of SCDLP culture medium, mix thoroughly, then incubate at 35°C ± 2°C for 24 hours. 　　1–2 inoculation loops are taken from the aforementioned enrichment broth and streaked onto blood agar medium, which is then incubated at 35°C ± 2°C for 24–48 hours. On blood agar plates, the colonies of this bacterium are golden yellow, large and raised, round, opaque, with a smooth surface, and surrounded by a hemolytic zone. 　　Typical colonies were selected, spread on a slide for Gram staining and microscopic examination. Staphylococcus aureus is a Gram-positive coccus that forms grape-like clusters, and it lacks spores and a capsule. If microscopic examination confirms the above findings, the following tests should be performed: Mannitol fermentation test: Inoculate the aforementioned colonies into a mannitol culture medium and incubate at 35°C ± 2°C for 24 hours; a positive result is indicated by acid production as a result of mannitol fermentation. 　　Plasma coagulase test: Slide method: Take a clean and dry slide, drop a drop of normal saline at one end and a drop of rabbit plasma at the other end. Pick up colonies and mix them with the normal saline and plasma respectively. If clumps or granular coagules appear in the plasma after 5 minutes, while the saline drop remains uniformly turbid without coagulation, the result is positive; if no coagulation occurs in either mixture, the result is negative. Coagulation occurs in both saline drops and plasma drops; then a tube coagulase test is performed ； Test tube method: 0.5 mL of 1∶4 fresh plasma is drawn and placed in a sterilized small test tube, to which 0.5 mL of an equal volume of the 24-hour broth culture of the microorganism to be tested is added. Mix well, place it in an incubator or water bath at 35°C ± 2°C, and observe every half hour; a clot forming within 24 hours indicates a positive result. Meanwhile, 0.5 mL each of the broth cultures from known plasma coagulase-positive and negative strains were used as positive and negative controls. B5.2 Result Report: If suspicious colonies grow on the agar plate, they are identified as Gram-positive staphylococci under microscopic examination, they can ferment mannitol to produce acid, and the plasma coagulase test is positive, then it can be reported that Staphylococcus aureus was detected in the sample being tested. B6 Test method for hemolytic streptococci B6.1 Procedure: Add 5 mL of the sample solution to 50 mL of glucose broth, and incubate at 35°C ± 2°C for 24 hours. 　　The culture was streaked onto blood agar plates and incubated at 35°C ± 2°C for 24 hours to observe the colony characteristics. Haemolytic streptococci appear grayish-white on blood agar, are semi-transparent or opaque, have needle-like protrusions, a smooth surface, neat edges, and a colorless, transparent hemolytic zone around them. 　　Typical colonies are selected for smear Gram staining and microscopic examination; they should be Gram-positive cocci arranged in chains. If microscopic examination shows the above findings, the following tests should be performed: Lactate kinase test: 0.2 mL of potassium oxalate plasma is taken (by mixing 0.01 g of potassium oxalate with 5 mL of rabbit plasma, followed by centrifugation to isolate the supernatant); 0.8 mL of sterile saline is added to this mixture, after which 0.5 mL of the 24-hour broth culture of the bacterium under investigation and 0.25 mL of 0.25% calcium chloride are added. The mixture is then stirred and placed in a water bath at 35°C ± 2°C. Observations are made every 2 minutes (coagulation usually occurs within 10 minutes); once the plasma has coagulated, further observations are conducted to record the time it takes for it to dissolve. If it does not dissolve within 2 hours, leave it for another 24 hours to observe; if the clot dissolves completely, the result is positive, while if it still does not dissolve after 24 hours, the result is negative. 　　Bacitracin sensitivity test: The test bacterial suspension is applied to blood agar plates, and sterile forceps are used to place paper discs containing 0.04 units of bacitracin on the surface of the plates. A known positive strain is used as a control. The plates are incubated at 35°C ± 2°C for 18–24 hours; a zone of inhibition indicates a positive result. B6.2 Result report: Microscopic examination reveals Gram-positive cocci arranged in chains; hemolytic zones are observed on blood agar plates, and the streptokinase and bacitracin tests are positive. It can be reported that hemolytic streptococci were detected in the sample under examination. B7 Method for determining the total number of fungal colonies B7.1 Procedure After the saline sample has settled naturally, the supernatant is taken for fungal counting. A total of 5 petri dishes are used, with 1 mL of the sample added to each dish. Then, 15–25 mL of melted Schaeffer’s agar medium cooled to around 45°C is added to each dish and mixed thoroughly. Once the agar has solidified, the dishes are turned over and incubated at 25°C ± 2°C for 7 days. Observations are made on days 3, 5, and 7, and the number of colonies on each plate is calculated. If colony spread is observed, the count from the previous measurement is used. B7.2 Result Report: Plates on which colonies are produced in patches should not be used ； Count the colonies on the plates that meet the requirements, and calculate the result using equation (B2):
K
X2 = B × --------------------------------------------------------------------------------
……(B2)
5
Where: X2 – total number of fungal colonies, in cfu/g or cfu/mL ； B – Total number of fungal colonies on 5 plates of Schaeffer’s agar medium ； K――Dilution factor. When the colony count is 100 or less, it is reported as the actual value; when it exceeds 100, two significant figures are used. If the total colony count of the sample exceeds the requirements of this standard, retesting and result reporting shall be carried out in accordance with B7.3. B7.3 Reinspection Method: The retained reinspection samples are tested again twice using the previous method. If the results of both tests meet the requirements of this standard, the sample in question is considered qualified; if either result exceeds the standards specified, the sample is deemed unqualified. B8 Qualitative detection method for fungi B8.1 Procedure: Add 5 mL of the sample solution to 50 mL of Sabouraud medium, and incubate at 25°C ± 2°C for 7 days, observing daily for any fungal growth. B8.2 Result Report: If the culture tube becomes turbid, it should be transferred to Sabouraud agar medium; if fungal growth is confirmed, it can be reported that fungi were detected in the sample under examination. 　 Appendix C (Standard Appendix) Test Methods for Product Sterilization Performance, Bacteriostatic Performance, and Stability C1 Sample Collection To ensure that the samples are representative, at least 20 samples from the smallest sales packaging units should be randomly selected from three shipping packages of the same batch; 5 of these samples should be retained, 5 used for testing bacteriostatic or sterilization performance, and 10 used for stability testing. C2 Test Organisms and Preparation of Bacterial Solutions C2.1 Test Organisms C2.1.1 Bacteria: Staphylococcus aureus (ATCC 6538), Escherichia coli (8099 or ATCC 25922). C2.1.2 Yeast: Candida albicans (ATCC 10231). Bacterial solution preparation: Take fresh cultures from the 3rd to 14th generation of the strain on nutrient agar slants (18–24 hours old), wash the mycelium with 5 mL of 0.03 mol/L phosphate buffer (hereafter PBS) to suspend the bacteria evenly, and then dilute them to the desired concentration using the aforementioned PBS. C3 Bactericidal Performance Test Method: The sampling location for this test is determined in accordance with the instructions provided by the manufacturer of the product under test. 2) Contaminated sample slide + 5 mL neutralizer 3) Contaminated control slide + 5 mL neutralizer 4) Sample slide + 5 mL neutralizer + contaminated control slide 5) Contaminated control slide + 5 mL PBS 6) PBS from the same batch 7) Neutralizer from the same batch 8) Medium from the same batch. C3.1.2 Evaluation criteria: 1) No test bacteria are present in Group 1, or only very few colonies of test bacteria grow. 2) Group 2 had more test bacteria growing than Group 1, but fewer than Groups 3, 4, and 5, and met the requirements. 3) Groups 3, 4, and 5 showed similar amounts of bacterial growth, ranging from 1×104 to 9×104 cfu per slice, with an error rate in the number of colonies between groups not exceeding 15%. 4) Groups 6–8 showed sterile growth. 5) Pass the evaluation in 3 consecutive tests. C3.2 Bactericidal Test C3.2.1 Procedure: Wash the 24-hour slant culture of the test bacteria with PBS to prepare a bacterial suspension (the required concentration is such that 100 μL of this suspension applied to the control sample yields 1×104–9×104 cfu per sample). Four test sample pieces (2.0 cm×3.0 cm) and four control sample pieces (made of the same material as the test samples, of the same size, but without antibacterial materials and sterilized) were taken and divided into 4 groups, which were placed in 4 sterilized petri dishes. Take the aforementioned bacterial suspension and apply 100 μL of it onto each of the test samples and control samples, spreading it evenly. Start timing; after 2, 5, 10, and 20 minutes, use sterile forceps to place the samples into test tubes containing 5 mL of the corresponding neutralizing agent, mix thoroughly, and dilute appropriately. Then, select 2–3 dilution levels, take 0.5 mL from each level, and place it in two petri dishes. Pour in 15 mL of nutrient agar medium (for bacteria) or Sabouraud agar medium (for yeasts) that has been cooled to 40–45°C. Rotate the petri dishes to ensure even distribution of the contents. Once the agar has solidified, turn the plates over and incubate them at 35°C ± 2°C for 48 hours (for bacteria) or 72 hours (for yeasts), followed by counting the viable bacterial colonies. The experiment was repeated 3 times, and the sterilization rate was calculated using equation (C1): X3 = (A – B)/A × 100%……(C1) Where: X3 – Sterilization rate, % ； A – Average colony count of the control sample ； B – Average colony count of the test sample. C3.2.2 Evaluation criteria: The bactericidal rate must be ≥90%, indicating that the product has a bactericidal effect. Test method for antibacterial activity of C4 soluble antibacterial (bacteriostatic) products C4.1 Procedure: Wash the 24-hour slant culture of the test bacteria with PBS to prepare a bacterial suspension (the required concentration is such that 100 μL of this suspension is applied to the control sample or added to 5 mL of the sample, resulting in a bacterial count of 1×104–9×104 cfu per sample piece or per mL). Take 4 test sample pieces (2.0 cm×3.0 cm) or sample solutions (5 mL), as well as 4 control sample pieces or sample solutions (made of the same material as the test samples, of the same size, but without antibacterial materials and sterilized) (placed in sterilized petri dishes) or 4 tubes each. Take the aforementioned bacterial suspension and add 100 μL of it to each test sample or solution, as well as to the control samples or solutions, either on their surface or inside them; spread it evenly/mix it thoroughly. Start timing, and after 2, 5, 10, and 20 minutes, use sterile forceps to place 0.5 mL of each sample or solution into a test tube containing 5 mL of PBS. Mix well to achieve appropriate dilution. Then, take 2–3 different dilutions, draw 0.5 mL from each, and place them in two petri dishes. Pour 15 mL of nutrient agar medium (for bacteria) or Sabouraud agar medium (for yeasts) that has been cooled to 40–45°C over these samples. Rotate the petri dishes to ensure even distribution of the contents. Once the agar has solidified, turn the plates over and incubate them at 35°C ± 2°C for 48 hours (for bacteria) or 72 hours (for yeasts), in order to count the viable bacterial colonies. The experiment was repeated 3 times, and the bacteriostatic rate was calculated using equation (C2): X4 = (A – B)/A × 100%……(C1) Where: X4 – Bacteriostatic rate, % ； A – Average colony count of the control sample ； B – Average colony count of the test sample. C4.2 Evaluation criteria: The antibacterial rate is ≥50%–90%, indicating that the product has an antibacterial effect; when the antibacterial rate is ≥90%, the product has a strong antibacterial effect. C5 Test Method for Antimicrobial Performance of Non-leachable Antimicrobial (Bacteriostatic) Products C5.1 Procedure Weigh 0.75 g of the test sample pieces (cut to 1.0 cm × 1.0 cm size) and package them separately. A 0.75 g sample was placed in a 250 mL Erlenmeyer flask, to which 70 mL of PBS and 5 mL of the bacterial suspension were added, resulting in a concentration of the bacterial suspension in PBS ranging from 1×104 to 9×104 cfu/mL. The Erlenmeyer flask was fixed on an oscillating shaker and shaken at 300 r/min for 1 hour. Take 0.5 mL of the shaken sample solution, or a properly diluted sample solution using PBS, and inoculate it onto petri dishes using the agar pouring method for colony counting. At the same time, a control sample group and a no-sample group were established. The control samples in the control sample group were of the same size as the test samples but contained no antibacterial components; all other procedures were identical to those used for the test sample group. For the no-sample group, 5 mL of the bacterial suspension and 70 mL of PBS were added to a 250 mL Erlenmeyer flask and mixed together. At time 0 and after shaking for 1 hour, 0.5 mL each of the mixture of bacterial suspension and PBS was taken, appropriately diluted, and then used for colony counting. The experiment was repeated 3 times, and the bacteriostatic rate was calculated using equation (C3): X5 = (A – B) / A × 100%……(C1) Where: X5 – Bacteriostatic rate, % ； A――Average colony count of the test sample before shaking ； B – Average colony count after shaking the test sample. C5.2 Evaluation criteria: The colony count in the no-sample group should be between 1×104 and 9×104 cfu/mL, and the difference in the average colony counts before and after shaking the sample should be within 10%; in this case, the test is valid ； The difference between the antibacterial rate of the test sample group and that of the control sample group is >26%, indicating that the product has antibacterial properties. C6 Stability testing method C6.1 Testing conditions C6.1.1 Natural storage: Place the sample in its original packaging at room temperature for at least 1 year, with antibacterial or bactericidal performance tests conducted every six months. C6.1.2 Accelerated test: Place the samples in their original packaging in an incubator at a temperature of 54–57°C for 14 days, or in an incubator at 37–40°C for 3 months, with a relative humidity of >75%, in order to test their antibacterial or bactericidal properties. C6.2 Evaluation Criteria: When the product is subjected to natural aging, and its bactericidal or antibacterial rate meets the standard values specified in Appendix C3, Appendix C4, or Appendix C5, the duration for which the product’s bactericidal or antibacterial effect persists at room temperature is considered the natural aging time. After undergoing accelerated testing at 54°C, the product achieves a sterilization or bacteriostatic rate that meets the standard values specified in Appendix C3, Appendix C4, or Appendix C5; its sterilizing or bacteriostatic effect persists at room temperature for at least one year. After undergoing accelerated testing at 37°C, the product achieves a sterilization or bacteriostatic rate that meets the standard values specified in Appendices C3, C4, and C5; its sterilizing or bacteriostatic effect persists at room temperature for at least two years.