ISO 21702:2019 download free.Measurement of antiviral activity on plastics and other non-porous surfaces.
Antibacterial-treated porous and non-porous products have been widely accepted and used among general consumers as their new choices to purchase for the additional function, which are different from what traditional materials had in terms of material protection.
Recently, antiviral-treated porous and non-porous products have been also in the market.
The measuring test method of antibacterial activity on non-porous products Is described in Iso 22196.
The measuring test method of antibacterial activity on porous products (textiles) is described in
ISO 20743.
The measuring test method of antiviral activity on porous products (textiles) is described in ISO 18184.
ISO 21702 is the test method of antiviral activity on non-porous products. It is written based on ISO 22196 and ISO 18184.
In ISO 22196, the scope has been expanded to include surfaces made of plastics and other non-porous materials, thus ISO 21702 is intended to be applicable to products such as plastics, coating materials, ceramics, natural and artificial leathers, stainless, rubbers, etc.
4.3.4 7,5 % sodium bicarbonate solution
Select and prepare the solution using one of the two following options:
— Option 1: Prepare a 7.5 % sodium bicarbonate solution by dissolving 75 g of sodium bicarbonate in 1 000 ml of water. Sterilize the solution by using a 0.22 lIm membrane filter.
— Option 2: Prepare a 7,5 % sodium bicarbonate solution by sterilizing 75 g of sodium bicarbonate in a culture container with a cap closed tightly in an autoclave. Sterilize 1 000 ml of water in the autoclave. Dissolve the sterilized sodium bicarbonate in the sterilized water well.
If the solution is not intended to be used immediately after preparation, then preserve it at 5 °C to 10 °C. Never use 7,5 % sodium bicarbonate solution that has been kept for longer than one month after preparation.
4.3.5 Formaldehyde solution
Prepare the formaldehyde solution by adding 100 ml of 37 % formaldehyde solution to 900 ml atwater. If it is not intended to be used immediately after preparation, preserve it at 20°C to 25 DC. Never use the formalin solution that has been kept for longer than one month after preparation.
NOTE The other solution for cell lixation can be used after appropriate validation for cell fixation.
4.3.6 Methylene blue solution
Prepare the methylene blue solution by dissolving 0,375 g of the methylene blue and 62.5 itl of 1 N sodium hydroxide solutions in 1 000 ml of water. If it is not intended to be used immediately after preparation, then preserve it at 20 °C to 25 °C. Never use the methylene blue solution that has been kept for longer than one month after preparation.
4.3.7 Inactivated fetal bovine serum (FBS)
Put a freezed cryopreserved fetal bovine serum in a package into a water bath at 37 °C and keep it until it defrosts. Then, raise the temperature of the water bath to 56 °C and keep it for 30 mm to inactivate. Divide it into several tubes. Put them in a freezer at a temperature less than —20 °C. Just before using, put it in the water bath at 37 °C and keep it until it defrosts.
Add 15 ml of 7,5 % sodium bicarbonate solution in the solution and mix well, if it is not intended to be used Immediately after preparation, then preserve it at 5 °C to 10 °C. Never use a maintenance medium that has been kept for longer than one month after preparation.
4.3.10 Double concentration of the maintenance medium
Dissolve 19,06 g of the Eagle’s minimum essential medium and 120 mg of kanamycin sulfate in 800 ml of water. Add water till there are 1 000 ml of solution in total. Sterilize the solution by using a 0,22 pm membrane filter. ii jt is not intended to be used immediately after preparation, then preserve it at 5 °C to 10 °C. Never use a growth medium that has been kept for longer than one month after preparation. When L-glutamine is not included in the EMEM purchased on market, sterilizing by autoclaving could be applied. Then, before use, add L-glutamine as listed in the example of composition in Annex A.
4.3.11 Phosphate buffered saline (PBS (-)J
Prepare PBS (-) by dissolving 8,0 g of sodium chloride, 0,2 g of potassium chloride, 2,9 g of phosphoric acid hydrogen 2 sodium 12 hydrate and 0,2 g of phosphoric acid 2 hydrogen potassium in 1 000 ml of water. Sterilize by autoclaving (see fz.2). If it Is not intended to be used immediately after preparation, preserve it at 5 °C to 10 °C. Never use a PBS (..) that has been kept br longer than one month after preparation.
4.3.12 Trypsln derived from beef pancreas and PBS () solution
4.3.12.1 Dissolve 1,0 g of trypsin derived from pancreas in 100 ml of PBS (-) and mix well for 2 h by using a mixer. Sterilize the solution by using 0,22 im membrane filter. if it is not intended to be used immediately after preparation, divide the solution in test tubes and preserve them in the freezer at a temperature less than —80 °C. Just before using, put it in the water bath at 37 °C and keep it until it defrosts.
4.3.12.2 Add 1,0 ml of the solution of 4.3.12.1 to 9,0 ml of PBS (-) and mix well. Divide the solution in test tubes and preserve them in the freezer at a temperature less than —20 O Just before using, put it in the water bath at 37 DC and keep it until it defrosts.
6.2 Subculture of host cell
Remove the growth medium from the flask and wash the cell monolayer with 5 ml of PBS (-) two times. After removing an extra PBS (-), add 0.5 ml of trypsin EDTA solution to the flask and spread the solution over the whole surface. Incubate the flask at 37 °C in the CO2 incubator for 10 mm to 20 tnin. Then. observe the flask if the cells are starting to come off, tap the side of the flask and disperse the cells. Add S ml of the growth medium to the flask and pipette the cell suspension gently to avoid the damage to the cells. Transfer I ml of the cell suspension into a new flask for cell culture use with 20 ml of the growth medium. The ratio of the suspension and the growth medium may be changed as needed. Incubate the flask at 37 °C in the CO2 Incubator for 3 days to 5 days until confluent cell monolayer is confirmed. The culture period may be changed as needed.
In case of continuously subculturing the cell, repeat the same procedure from the beginning of 62.
6.3 Cell culture for measuring virus infectivity titer
The cell culture in a 6 wells plate is required for measuring the infectivity titer of virus by the plaque assay. Transfer I ml of the subcultured cell suspension prepared in 62 into a sterile media bottle with 20 ml of the growth medium.
The ration of the suspension and the growth medium could be changed as needed.
Transfer 3 ml of the cell suspension into each hole of the 6 wells plastic plate for the plaque assay. Incubate the plate at 37°C In the CO2 incubator for 3 days to 5 days. The culture period could be changed as needed. Observe the condition of the cells under the inverted microscope and confirm if the cells are cultured as a confluent cell monolayer on the bottom of each hole.
6.4 Preparation of test inoculums
6.4.1 Influenza virus
Put the cryopreserved stock virus suspension in the water bath at 37 °C and defrost rapidly. Dilute the defrosted stock influenza virus suspension with the maintenance medium to obtain a virus suspension of IO PFtJ/ml to 104 PFU/ml.
7.3 Contact of the inoculated test specimens
Unless otherwise specified, keep each of the Petri dish with the inoculated test specimens (including the untreated test specimens) at (25 ± 1) °C and a relative humidity of not less than 90% for 24 h.
Other contact time up to 24 h may be applied if agreed upon by the relevant parties. In this case, the modified contact time shall be included in the test report.
7,4 Recovery of virus from test specimens
7.4.1 Test specimens immediately after inoculation
Immediately after inoculation, process the three untreated test specimens by adding 10 ml of either the SCDLP broth (4.3.16) or a suitable, validated neutralizer to the Petri dish. The number of the infectivity titer of virus recovered from the untreated test specimens will be used to determine the recovery rate of the virus. It is Important to ensure that the neutralizer completely washes the specimens with pipetting the SCDLP broth at least four times.
If there are any changes on volume or component to be applied to the neutralizer, it shall be included in the test report. The volume change shall be taken account of the calculation described In &1.
Special considerations may be required to achieve a sufficient recovery, in case of option 2 in 72 and the viscosity of the inoculum is increased. In this case, mechanical agitations may be required, such as stomaching, vortexing or sonicating. If one of these shows a recovery rate equivalent to or superior to that of obtained when tested without adding viscosity, such methods may be used. If one of the alternative recovery methods is used, it shall be included in the test report. Use of alternative recovery methods may affect the antiviral activity and shall therefore be fully validated.
7.4.2 Test specimens after contact
After the contact as described in LI. process three untreated test specimens and three treated test specimens with the same procedure described in Z4J. Proceed immediately to measure the infectivity titer of virus recovered from the test specimen (see Z).