The following alcohol-based hand rubs were tested: Preparation A, based on isopropanol (45%, w/w), n-propanol (30%, w/w) and mecetronium etilsulfate (0.2%, w/w); preparation B, based on ethanol (80%, w/w); and preparation C, based on ethanol (95%, w/w). All hand rubs were manufactured by Bode Chemie GmbH & Co. KG, Hamburg, Germany.
Virus suspensions that allowed a reduction of at least 4 log10-steps to be measured were used in all experiments. Infectivity assays were performed between 2003 and 2006 according to the test method of the Bundesgesundheitsamt (BGA, Federal Office of Health, now Robert Koch-Institute) and the German Association for the Control of Virus Disease (DVV) , which uses the same test principle as EN 14476, with the following test viruses:
• Vaccinia virus strain Elstree, passaged and cultured in buffalo green monkey cells (BGM cells) (range of virus titres: 7.6–9.9 log10TCID50/ml)
• Bovine viral diarrhea virus (BVDV) strain NADL, ATCC VR-534, passaged and cultured in KOP-R cells (range of virus titres: 5.5–6.4 log10TCID50/ml)
• Herpes simplex virus (HSV) type 1 MacIntyre, ATCC VR-539, passaged and cultured in Vero cells (African green monkey kidney, ATCC CCL-81) (range of virus titres: 7.5–8.1 log10TCID50/ml)
• Herpes simplex virus type 2, ATCC VR-540, passaged and cultured in Vero cells (range of virus titres: 7.6–8.5 log10TCID50/ml)
• Human influenza A virus, Panama strain 2007/99 (H3N2), passaged and cultured in Madin-Darby canine kidney epithelial cells (range of virus titres: 5.5–5.9 log10TCID50/ml)
• Avian influenza A virus/duck/Ukraine/1/63 (H3N8), passaged and cultured in Madin-Darby canine kidney epithelial cells (range of virus titres: 6.5–6.9 log10TCID50/ml). This virus was used as surrogate for H5N1 owing to biosafety considerations.
Tests were conducted in accordance with BGA/RKI and DVV guidelines in a waterbath at 20°C . Eight parts by volume of the disinfectant were mixed with one part by volume of the virus suspension and one part by volume of aqua bidest. This test mixture was investigated at exposure times of 15, 30 and 60 seconds. In tests with different organic loads, one part by volume of the interfering substance was added instead of aqua bidest. The disinfectant was inactivated immediately at the end of the chosen exposure time by serial dilution with ice-cold cell culture medium or by gel filtration with MicroSpin™ S-400 HR columns (Amersham Biosciences Europe GmbH, 79021 Freiburg, Germany), which were used according to the manufacturer's instructions, to reduce the cytotoxicity of the product when BVDV or either of the influenza viruses was tested. Virus controls without columns were run in parallel.
Owing to the addition of virus suspension and organic load, disinfectants could only be evaluated as 80% solutions.
Virus controls were incorporated after the longest exposure time (60 s). One part by volume of virus suspension was mixed with nine parts by volume of aqua bidest. or with one part by volume of organic load and eight parts by volume of aqua bidest.
One experiment was performed for each hand rub, each exposure time, each virus and each type of organic load.
Types of organic load
Different types of organic load were used (final concentrations):
▪ No organic load (aqua bidest.)
▪10% fetal calf serum
▪ 0.2% bovine serum albumin
▪ 0.03% bovine serum albumin ("clean conditions" according to EN 14476:2005)
▪ 0.3% bovine serum albumin with 0.3% washed sheep erythrocytes ("dirty conditions" according to EN 14476:2005).
Determination of cytotoxicity
To determine the cytotoxicity of the disinfectants, two parts by volume of aqua bidest. were mixed with eight parts by volume of the disinfectant, diluted with ice-cold cell culture medium and inoculated into permissive cells. Controls for the different organic loads consisted of one part by volume of aqua bidest, one part by volume of organic load and eight parts by volume of the disinfectant. Any microscopic changes in the cells were recorded when the tests were read for cytopathic effects (CPE). This control allowed cytotoxicity and viral CPE to be clearly differentiated.
Determination of infectivity
Infectivity was determined in a micro-procedure by end-point dilution titration. At the end of each exposure time, the test mixture was immediately diluted with ice-cold cell culture medium, and 100 μl of each dilution were placed in 8 wells of a sterile polystyrene flat-bottomed 96-well microtitre plate (Nunc A/S, 4000 Roskilde, Denmark) with a preformed cell culture monolayer. The microtitre plates were incubated at 37°C with 5% CO2 for the appropriate incubation time (3–10 days). Cultures were observed for the presence or absence of CPE. The infective dose (TCID50) was calculated according to the method of Spearman  and Kärber . Titre reduction was calculated as the difference between the virus titres of the water control (60 s) and the products after the contact times, and is presented as the reduction factor (RF). The following formula was used:
RF = a - b
a = log10 TCID50/ml of the control titration
b = log10 TCID50/ml of the test virus titration (after hand rub exposure)
A reduction of infectivity of ≥ 4 log10-steps (inactivation ≥ 99.99%) was regarded as evidence of sufficient virucidal activity against the tested virus according German and European guidelines .