Test Report: BS EN 14476:2013 + A2:2019 Chemical disinfectants and

antiseptics – Quantitative suspension test for the evaluation of virucidal activity in the
medical area‐ Test method and requirements (Phase 2/Step 1)

Test Laboratory BluTest Laboratories Ltd

5 Robroyston Oval, Nova Business Park, Glasgow, G33 1AP
Identification of sample
Name of the product Alcohol Anti‐Viral Hand Sanitiser
Batch number Not supplied
Client Assured products Limited
Client Address Units 16-17 Hawkley Brook Trading Estate, Worthington Way
Wigan, WN3 6XE
Project Code
Date of Delivery BT‐COV‐07FT(2)
Storage conditions 12 March 2020
Active substances Ambient
Appearance Ethanol
Condition upon receipt Liquid
Undamaged
Test Method and its validation
Method
1 part interfering substance + 1 part virus suspension + 8 parts
biocide were mixed and incubated at the indicated
contact temperature for the indicated contact times.
Assays were validated by a cytotoxicity control,
interference control, neutralisation control and a formaldehyde
Neutralisation internal standard.

Dilution‐neutralisation/gel filtration
Eagles Minimum Essential Medium + 5.0% v/v foetal bovine serum
Experimental Conditions at 4°C

Period of analysis 14 March 2020 to 01 April 2020

Product diluents used Sterile distilled water
Product test concentrations 10% v/v; 50.0% v/v; 80.0% v/v
Appearance product dilutions Solution became more viscous at 50.0% v/v and Viscous gel
becomes more fluid at 10.0% v/v
Appearance in test mixture Turbidity, sedimentation and viscous gel became more fluid at
80.0% v/v
Contact times (minutes) 2 ± 10s
Test temperature 20°C + 1°C
Interfering substances 0.3g/l bovine albumin
Temperature of incubation 37°C + 1°C + 5% CO2
Identification and passage (P) of virus Vaccinia virus VR‐1549 Elstree strain (P6)
Identification and passage (P) of cells Vero Cells (P 48) (Vaccinia Virus)

Page 1 of 6
SOP 11000
SOP 8003 EN14476 Vaccinia REPORT TEMPLATE V01
Effective Date: 23 March 2020 BT-COV-07FT(2)

BLUTEST LABORATORIES LIMITED, 5 Robroyston Oval, Nova Business Park, Glasgow, UK, G33 1AP
Telephone: +44 (0)141 558 2782. Email: [email protected]. Web site: www.blutest.com.
Company Registration Number: SC364409 VAT Registration Number: GB 979 1131 96 UKAS Number: 4597
 PROTOCOL SUMMARY

The basic virucidal efficacy test is set up with three concentrations of test product solution and a 2‐minute contact
time. Virus is exposed to disinfectant in 24‐well plates, then neutralised, serially diluted and virus titred in 96‐well
tissue culture plates to determine the tissue culture infectious dose 50 (TCID 50) of surviving virus. Vaccinia virus VR‐
1549 Elstree strain / Vero cells are assayed in parallel in each test. TCID50 is determined by the method of Karber1.

Cytotoxicity control
The test product solution is measured for its effects on the host cells used to propagate the virus, to determine the
sensitivity of the assay.

Interference control
The effect of the cells after treatment of the test product solution are verified to ensure the cells can show
susceptibility for virus infection. This is compared against cells that have not been treated with test product.

Disinfectant suppression control VS1

Virus is added to the highest concentration of test product solution and then the mixture immediately removed
and neutralised. The neutralised virus titre is then determined to assess the efficiency of the neutralisation
procedure.

Disinfectant suppression control VS2

Internal control which adds virus to neutralised test product solution to assess the efficiency of the neutralisation
procedure.

No column Control
Internal control on the highest contact time to assess any impact of the Microspin™ S 400 HR columns.

Virus recovery control

Virus titre is determined for virus in contact with sterile distilled water at t=0, t = 2 and at t =15. The virus titre after
2 minutes is then compared to the recovery of disinfectant‐treated virus to measure the log reduction in virus titre.
The virus titre at 15 minutes is compared to the reference virus inactivation control.

Reference virus inactivation control

Virus is exposed to 0.7% W/V formaldehyde and the recovery of virus determined by TCID50 after 5 and 15 minutes,
in order to assess that the test virus has retained reproducible biocide resistance. In addition, the formaldehyde
cytotoxicity of neutralised formaldehyde is determined, to measure assay sensitivity.

:1Kärber, G. Beitrag zur Kollektiven Behandlung Pharmakologischer Reihenversuche. Arch. Exp. Path.
Pharmak. 162 (1931): 480‐487.

Page 2 of 6
SOP 11000
SOP 8003 EN14476 Vaccinia REPORT TEMPLATE V01
Effective Date: 23 March 2020 BT-COV-07FT(2)

BLUTEST LABORATORIES LIMITED, 5 Robroyston Oval, Nova Business Park, Glasgow, UK, G33 1AP
Telephone: +44 (0)141 558 2782. Email: [email protected]. Web site: www.blutest.com.
Company Registration Number: SC364409 VAT Registration Number: GB 979 1131 96 UKAS Number: 4597
Vaccinia virus (VR‐1549) Elstree strain Test Results
EN14476:2013 + A2:2019 Suspension test for the efficacy of Anti‐Viral Hand Sanitiser,
BT‐COV‐07FT(2) from Assured Products Ltd against Vaccinia virus VR‐1549 under
CLEAN conditions
Test Results
Concentration 10.0% (v/v) 50.0% (v/v) 80.0% (v/v)

Exposure Time data TCID50/ml data TCID50/ml data TCID50/ml

t = 2 minutes 4.83 2.15E+06 3.83 2.15E+05 0.00 3.16E+01
Raw Data 666641 2.15E+06 666500 2.15E+05 000000 3.16E+01
log 6.33 5.33 1.50
log difference ‐0.17 0.83 4.67

EN14476:2013 + A2:2019 Suspension test for the efficacy of Anti‐Viral Hand Sanitiser, BT‐COV‐07FT(2) from Assured Products
Ltd against Vaccinia virus VR‐1549 under CLEAN conditions
Summary Table
Product: Interfe ri ng Concentration Level of lg TCID50 >4 lg reducti on
substance cytotoxi ci ty after 'X' Mi n

0 min 2 min 15 min 30 min 60 min

3.0g/l BSA + 80.0% (v/v) 1.50 6.17 1.50 n.a . n.a . n.a . <2 mins
Anti ‐ Viral
3.0ml /l
Hand 50.0% (v/v) 1.50 n.a. 5.33 n.a. n.a. n.a. >2 mins
Sanitiser erythrocyte s
10.0% (v/v) 1.50 n.a. 6.33 n.a. n.a. n.a. >2 mins
Virus Control CLEAN 6.17 6.17 6.17 n.a. n.a. n.a.
30 min 60 min
Formaldehyde PBS 0.7% (w/v) 3.50 4.50 3.50 >60 mins

Page 3 of 6
SOP 11000
SOP 8003 EN14476 Vaccinia REPORT TEMPLATE V01
Effective Date: 23 March 2020 BT-COV-07FT(2)

BLUTEST LABORATORIES LIMITED, 5 Robroyston Oval, Nova Business Park, Glasgow, UK, G33 1AP
Tel: +44 (0)141 558 2782. Email: [email protected]. Web site: www.blutest.com. Company No.: SC364409. VAT No.: GB 979 1131 96. UKAS No.: 4597
Vaccinia virus (VR‐1549) Elstree strain Control Data
EN14476:2013 + A2:2019 Suspension test for the efficacy of Anti‐Viral Hand Sanitiser, BT‐COV‐07FT(2) from Assured Products Ltd against Vaccinia
virus VR‐1549 under CLEAN conditions
Controls
Virus Recovery Virus Recovery Virus Recovery Cytotoxicity Disinfectant Disinfectant
0 min 2 min 15 min Suppression VS Suppression VS2
ra w data TCID50/ml raw data TCID50/ml raw data TCID50 /ml raw data TCID50/ml raw data TCID50 /ml raw data TCID50/ml
4.67 1.47E+06 4.67 1.47E+06 4.83 2.15E+06 0.00 3.16E+01 4.67 1.47E+06 5.17 4.64E+06
666640 1.47E+06 666640 1.47E+06 666650 2.15E+06 000000 3.16E+01 666640 1.47E+06 666661 4.64E+06
6.17 6.17 6.33 1.50 6.17 6.67
0.00 ‐0.50

Formaldehyde reference inactivation controls No column Control

Cytotoxicity Exposure time 0.7% Formaldehyde 2 mins
5 mins 15 mins ra w data TCID50/ml
ra w data TCID50/ml raw data TCID50/ml raw data TCID50/ml 5.00 3.16E+06
2.00 3.16E+03 3.00 3.16E+04 2.00 3.16E+03 666651 3.16E+06
660000 3.16E+03 666000 3.16E+04 660000 3.16E+03 6.50
3.50 log 4.50 3.50
log difference 1.83 2.83

Virus dilution Stock Virus (TCID50)

Interference control
‐3 ‐4 ‐5 ‐6 ‐7 ‐8 6.00
1 1 1 0.17 0 0 3.16E+07
PBS Control 3.16E+02 3.16E+02 3.16E+02 4.68E+01 3.16E+01 3.16E+01 6666660000
2.50 2.50 2.50 1.67 1.50 1.50
Raw Data 6 6 6 1 0 0
1 1 1 0.33 0.17 0
Product 3.16E+02 3.16E+02 3.16E+02 6.76E+01 4.68E+01 3.16E+01
2.50 2.50 2.50 1.83 1.67 1.50
Raw Data 6 6 6 2 1 0
Log Difference 0.00 0.00 0.00 ‐0.16 ‐0.17 0.00
Product Cyt Dilution ‐1 ‐1 ‐1 ‐1 ‐1 ‐1
PBS Dilution Neat Neat Neat Neat Neat Neat

Page 4 of 6
SOP 11000
SOP 8003 EN14476 Vaccinia REPORT TEMPLATE V01
Effective Date: 23 March 2020 BT-COV-07FT(2)

BLUTEST LABORATORIES LIMITED, 5 Robroyston Oval, Nova Business Park, Glasgow, UK, G33 1AP
Tel: +44 (0)141 558 2782. Email: [email protected]. Web site: www.blutest.com. Company No.: SC364409. VAT No.: GB 979 1131 96. UKAS No.: 4597
 CONCLUSION
Verification of the methodology
A test is only valid if the following criteria are fulfilled:
a) The titre of the test suspension of at least 108 TCID50 /ml is sufficiently high to at least enable a titre
reduction of 4 Ig to verify the method.
b) Detectable titre reduction is at least 4 log10.
c) Difference of the logarithmic titre of the virus control minus the logarithmic titre of the test virus in the
reference inactivation test is between:
 Between 0.75 and 3.5 after 5 min and between 2.0 and 4.0 after 15 min for Vaccinia virus
d) Cytotoxicity of the product solution does not affect cell morphology and growth or susceptibility for the test
virus in the dilutions of the test mixtures which are necessary to demonstrate a 4 log10 reduction of the virus.
e) The interference control result does not show a difference of < 1.0 log10 of virus titre for test product treated
cells in comparison to the non‐treated cells.
e) Neutralisation validation. This is called the disinfectant suppression test in this protocol. The disinfectant was
neutralised by column chromatography through an Illustra Microspin S‐400 HR column to achieve the best
possible neutralisation available for this test. The difference for virus is not greater than 0.5 log10 indicating
effective neutralisation of the virucidal activity of the disinfectant by dilution at a concentration of 80.0% v/v.

According to EN 14476:2013 + A2:2019, Anti‐Viral Hand Sanitiser POSSESSES VIRUCIDAL activity at a

concentration of 80.0% v/v of the working concentration as tested after 2 MINUTES at 20°C
under CLEAN conditions.
(0.3 g/l bovine albumin) against Vaccinia virus VR‐1549 Elstree strain / Vero cells.

This product therefore is effective against all enveloped viruses as defined in EN 14476:2013 + A2:2019 Annex
A*. This therefore includes all coronaviruses and SARS‐CoV‐2.

Authorised signatory

Dr Chris Woodall, Director

BluTest Laboratories Ltd
Glasgow, UK.
Date:

DISCLAIMER
The results in this test report only pertain to the sample supplied. BluTest (BT) has performed the testing detailed in this report using reasonable skill and care and
has used reasonable endeavours to carry out the testing in accordance with an EN 14476 protocol. All forecasts, recommendations and results contained in this repo
rt are submitted in good faith. However, other than as expressly set out in this report, no warranty is given (i) in relation to the testing or the use(s) to which any
results or deliverables produced in the course of the testing are or may be put by the Client or their fitness or suitability for any particular purpose or under any spe
cial conditions notwithstanding that any such purpose or conditions may have been made known to BT or (ii) that the intended results or deliverables from the testin
g can be achieved or (iii) that the Client can freely make use of the results or the deliverables without infringing any third party intellectual property rights and the
Client will be deemed to have satisfied itself in this regard. BT shall have no liability (which is hereby excluded to the fullest extent permissible by law) in respect
of any loss, liability or damage, including without limitation any indirect and/or consequential loss such as loss of profit or loss of business, market or goodwill, that
the Client may suffer directly or indirectly as a result of or in connection with: (i) the performance of the testing; (ii) the use of any materials, samples or other inf
ormation provided by the Client for use in the testing; and (iii) the Client’s reliance upon or use of any results or deliverables provided as part of the testing

Page 5 of 6
SOP 11000
SOP 8003 EN14476 Vaccinia REPORT TEMPLATE V01
Effective Date: 23 March 2020 BT-COV-07FT(2)

BLUTEST LABORATORIES LIMITED, 5 Robroyston Oval, Nova Business Park, Glasgow, UK, G33 1AP
Telephone: +44 (0)141 558 2782. Email: [email protected]. Web site: www.blutest.com.
Company Registration Number: SC364409 VAT Registration Number: GB 979 1131 96 UKAS Number: 4597
 *EN 14476 2013 + A2 2019 Annex A (informative – Enveloped viruses)

Poxviridae
Herpesviridae
Filoviridae (e.g. Ebola, Marburg)
Flavivirus
Hepatitis C Virus (HCV)
Hepatitis Delta Virus (HDV)
Influenza Virus
Paramyxoviridae
Rubella Virus
Measles Virus
Rabies Virus
Coronavirus (e.g. SARS, MERS)
Human Immunodeficiency Virus (HIV)
Human T Cell Leukemia Virus (HTLV)
Hepatitis B virus (HBV)

Reference: Van Regenmortel MHV et al.,Eds.: Virus Taxonomy, Classification and Nomenclature of Viruses, seventh report of the
international committee on taxonomy of viruses. Academic Press, San Diego, 2000

Page 6 of 6
SOP 11000
SOP 8003 EN14476 Vaccinia REPORT TEMPLATE V01
Effective Date: 23 March 2020 BT-COV-07FT(2)

BLUTEST LABORATORIES LIMITED, 5 Robroyston Oval, Nova Business Park, Glasgow, UK, G33 1AP
Telephone: +44 (0)141 558 2782. Email: [email protected]. Web site: www.blutest.com.
Company Registration Number: SC364409 VAT Registration Number: GB 979 1131 96 UKAS Number: 4597