Study N 286V AGRI GERM /14
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1 Study N 286V /14 TEST REPORT VIRUCIDAL ACTIVITY OF THE PRODUCT AGAINST THE ENTERIC CYTOPATHOGENIC BOVINE ORPHAN VIRUS (ECBO) Delivered to: For : Mme FELBACQ-SERRANO Laboratoires CEETAL 1, rue des Touristes CS SAINT-ETIENNE Cedex 1 Study number: n 286V VIRUCIDAL TESTS: According to the NF EN standard (May 2015) chemical antiseptics and disinfectants virucidal quantitative suspension tests for chemical disinfectants and antiseptics used in veterinary area. Tests using the product batch n against the enteric cytopathogenic bovine orphan virus (ECBO). This test report included 14 pages. Study completion date: 12/15/2015 Stephanie MOROT-BIZOT PhD in microbiology Study director Professeur Georges HERBEIN Professor of Medicine MD-PhD in virology Scientific consultant
2 Study N 286V /14 SUMMARY 1 PERFORMING LABORATORY 2 PRODUCT IDENTITY 3 EXPERIMENTAL CONDITIONS 4 VALIDATIONS 5 VIRUCIDAL ASSAYS 6 VALIDATION OF THE METHODOLOGY 7 CONCLUSION 8 TECHNICAL APPENDIX
3 Study N 286V /14 1 PERFORMING LABORATORY APEX BIOSOLUTIONS 18, rue Alain SAVARY BESANÇON FRANCE 2 PRODUCT IDENTITY Reference N lot Expiration date: 11/01/2017 Manufacturer: CEETAL Manufacturing date: non communicated Storage conditions: room temperature Active compounds: glutaral, alkydimethyl benzyl ammonium chloride, didecyl dimethyl ammonium chloride, nitrilotrimethylenetris phosphonic acid Appearance of the product: clear Product diluent recommended by the manufacturer for use: tap water Date of delivery of the product: 11/09/2015 Date of tests: from 11/02/2015 to 12/07/ EXPERIMENTAL CONDITIONS Temperature used during the assays: 10 C ± 1 C Titration unit: log TCID 50 Exposure Time: utes Tested concentration:, 0.8% and 1.0% Diluent used for the product: carbonated water Viral strain: Enteric cytopathogenic bovin orphan ATCC VR-248 (ECBO), grown on VERO cells, at 37 C, under 5% CO2 atmosphere Organic soil load: BSA at 3g/L (low soiling level) Product stability: stable Stop solution: cold shock Viral titer: Viral titers are expressed in TCID 50, according to the Spearman-Kärber method = 6,25 log TCID 50.
4 Study N 286V /14 4 VALIDATIONS a) Cytotoxicity The product has been tested on the VERO cells and no toxicity was observed (after microspin filtration for the 1.0% concentration). b) Cells sensitivity to the virus For each viral suspension, comparative titers of the virus were performed on cells treated or untreated with the product. Product dilution Viral suspension on untreated cells Viral titer (log TCID 50 ) Viral suspension on treated cells Viral titer difference (log TCID 50 ) ,50 6,125 0,375 The product do not affect the infectious capacity of the virus: the differences in viral titers between the virus inoculated on VERO cells and the virus inoculated on the VERO cells treated with the product was 1,0 log (0,375 log). c) Inactivation of the virus Viral titer (log TCID 50 ) Viral suspension 6,250 Formaldehyde 0,7% Inactivation after 5 min 6,150 Inactivation after 15 min 5,500 Viral titer reduction (log TCID 50 ) Inactivation after 4,500 1,750 The virus is inactivated with the control solution of 0,7 % formaldehyde after of exposure if the reduction is comprised between -0,5 and -2,5 log. The reduction observed was of 1,750 log for the ECBO virus.
5 Study N 286V /14 5 VIRUCIDAL ASSAYS TRIAL 1 The viral suspension control was titered at 6,250 log TCID 50. PRODUCT Final concentration (v/v) Exposure Temperature Titer after trial (log TCID 50 ) Viral titer reduction 5,500 0, % 10 C 2,125 4, % 1,750 4,500 TRIAL 2 The viral suspension control was titered at 6,375 log TCID 50. PRODUCT Final concentration (v/v) Exposure Temperature Titer after trial (log TCID 50 ) Viral titer reduction 5,250 1, % 10 C 2,125 4, % 2,000 4,375 The product has a virucidal effect if the viral titer reduction is 4,0 log. 6 VALIDATION OF THE METHODOLOGY The assays were validated as required by the European standard EN 14675:2015 : The viral titers of the suspension tests were sufficient in order to observe a reduction of 4 log after exposure with the product. The viral titer of the ECBO virus was 6,25 log TCID 50. The virus was inactivated with the control solution of 0,7 % formaldehyde after of exposure : the reduction observed was of 1,750 log for the ECBO virus. The product has a weak cytotoxic effect on the VERO cells, after microspin column treatment. The product do not affect the infectious capacity of the virus: the differences in viral titers between the virus inoculated on VERO cells and the virus inoculated on the VERO cells treated with the product was 1,0 log (0,375 log).
6 Study N 286V /14 7 CONCLUSION The assays performed with the batch n product demonstrated that: The product demonstrated a virucidal activity against the ECBO virus from the concentration 0.8%, as required by the European standard EN :2015, following a utes exposure period, at 10 C, in low soiling level.
7 Study N 286V /14 TECHNICAL APPENDIX 1 Cell line for ECBO virus: VERO cells (R&D Biotech, ref , batch n V) Viral strain: Enteric cytopathogenic bovin orphan ATCC VR-248 (ECBO) (batch n ) Buffer and reagents: Buffer PBS: sodium chloride, Dominique DUTSCHER, ref ; sodium phosphate dibasic, Sigma Aldrich, ref. S5136, batch n BCBC7067V; sodium phosphate monobasic, Sigma Aldrich, ref. S5011, batch n 1019K01021V MEM media, PAN Biotech, ref. P , batch n horse serum, Sigma Aldrich, ref. H1270, batch n 12J395 Organic soil load: Bovine Sera Albumin, Sigma Aldrich, ref. A5479, batch n STBB7838V Inactivation solution: Formaldehyde, Sigma Aldrich, ref. F-1635, batch n BCBB3510
8 Study N 286V /14 TECHNICAL APPENDIX 2 Table A1 Viral titer of ECBO virus calculated with the Spaerman-Kärber (cytopathic effect method): log TCID 50 = 6,25 Dilution (- log) Result % positive results Sum of the % of the positive wells 375
9 Study N 286V /14 Chart 1 graphical chart of the results : Trial 1 Trial 2
10 Study N 286V /14 Table A2 results with the product against the ECBO virus in low soiling level TRIAL 1 TRIAL 2 Trial 1 Product Concentration Organic soil load cytotoxicity level Lg TCID ,5 6,250 5, % 3 g/l BSA 0,5 6,250 2, % 0,5 6,250 1,750 0,625 6,375 5, % 3 g/l BSA 0,625 6,375 2, % 0,625 6,375 2,000 Formaldehyde 0,70% MEM+2% HS 2,5 6,375 4,500 Trial 2 2,5 6,125 4,500 Reduction R = 0,750 R = 4,125 R = 4,500 R = 1,125 R = 4,250 R = 4,375 R = 1,750 R = 1,875 Trial 1 N.A 6,375 6,500 Infectivity N.A. MEM+2% HS control Trial 2 N.A 6,250 6,375 Trial 1 N.A 6,500 6,500 Infectivity N.A. 3 g/l BSA control Trial 2 N.A 6,375 6,375 HS : Horse Serum
11 Study N 286V /14 Table A3 Raw data for the product against the ECBO virus, in low soiling level (cytopathic effect; 8 wells) Trial 1 AGRI'GERM trial 1 Concentration organic soil load 0.8% 3 g/l BSA exposure Dilutions viral AGRI'GERM trial 1 1.0% 3 g/l BSA N.A. control N.T. cytotoxicity N.T. viral control of infectivity N.A. N.A. MEM+2% HS 3 g/l BSA Formaldehyde 0,70% MEM+2% HS Formaldehyde (cytotoxicity) 0,70% MEM+2% HS N.A N.T. N.T. N.T. N.T. N.T. N.T
12 Study N 286V /14 Trial 2 AGRI'GERM trial 2 Concentration organic soil load 0.8% 3 g/l BSA exposure Dilutions viral AGRI'GERM trial 2 1.0% 3 g/l BSA N.A. control N.T. cytotoxicity N.T. viral control of infectivity N.A. N.A. MEM+2% HS 3 g/l BSA Formaldehyde 0,70% MEM+2% HS Formaldehyde (cytotoxicity) 0,70% MEM+2% HS N.A N.T. N.T. N.T. N.T. N.T. N.T
13 Study N 286V /14 VALIDATIONS Raw data Other concentrations tested Trial 1 Concentration 1.0% organic soil load 3 g/l BSA exposure dilutions Concentration organic soil load exposure Log DICT min Reduction 6,25 5,5 0,75 inactive 3 g/l BSA 1.0% 6,25 1,75 4,5 active Trial 2 Concentration organic soil load exposure dilutions % 3 g/l BSA Concentration organic soil load exposure Log DICT 50 0 Reduction 6,375 5,25 1,125 inactive 3 g/l BSA 1.0% 6, ,375 active
14 Study N 286V /14 Sensitivity of the cells to the virus Trial 1 Product dilution organic soil load Dilutions g/l BSA Untreated cells Treated cells Trial 2 Product dilution organic soil load g/l BSA Untreated cells Treated cells Dilutions Validation of the stop method (microspin filtration and cold shock) Product dilution organic soil load Dilutions MEM+2% HS Cold dilution Viral control Cold dilution : viral titer = 6,5 R = 0 Viral control : viral titer = 6,5 The stop method is validated (R 0,5 log)
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