R2012FSFOR001 Page 1/ 12

R2012FSFOR001
Antiviral activity of MARMOLEUM TOPSHIELD 2 surface against humancoronavirus HCoV-229E for contact times of 2 and 5 hours
according to ISO 21702 (2019) standard

CLIENTTEST LABORATORYTECHNICALCONTRIBUTIONFORBO FLOORING B.V.
Monsieur Leon van der Riet
Industrieweg 12
NL-1566 JP Assendelft
The Netherlands
S.A.S VIRHEALTH
Site Laennec-La Buire, 2éme étage, Bat B
7-11 rue Guillaume Paradin,
69372 Lyon Cedex 08FranceLéa Szpiro, technical manager
Loranne Durimel, laboratory technician

Quality validation

Name: Dr Vincent MOULES, CEO
Date: Lyon, 07/12/2020
Signature :Report includes 12 pages

REPRODUCTION AND DIFFUSION ARE FORBIDDEN WITHOUT PRIOR AUTHORISATION

R2012FSFOR001 Page 2/ 12SUMMARYI.

CONCLUSION ...................................................................................................................... 3

II.

CONTRACTUAL DOCUMENTS............................................................................................... 4

III. TEST CONDITIONS AND SAMPLES DATA ............................................................................... 4III.1 SAMPLES IDENTIFICATION ............................................................................................................... 4
III.2

EXPERIMENTAL CONDITIONS ............................................................................................................ 5IV. RESULTS ............................................................................................................................. 6
IV.1 ANTIVIRAL ACTIVITY OF THE MARMOLEUM TOPSHIELD 2 SURFACE AGAINST HUMAN CORONAVIRUS HCOV229E FOR A CONTACT TIME OF 2 HOURS ...................................................................................................... 6a.
b.
c.
d.

Cell susceptibility ............................................................................................................................. 6Cytotoxicity ...................................................................................................................................... 6
Inactivation of antiviral activity ....................................................................................................... 7
Test .................................................................................................................................................. 8V.

CONCLUSION ...................................................................................................................... 9

VI. ANNEXES .......................................................................................................................... 10
VI.1 MATERIALS AND REAGENTS ........................................................................................................... 10
VI.2

RAW DATA: TCID50 QUANTIFICATION AGAINST HUMAN CORONAVIRUS HCOV-229E AFTER 2 AND 5HOURS, VISUAL READING OF CYTOPATHIC EFFECTS (8 WELLS PER DILUTIONS) .................................................... 11

REPRODUCTION AND DIFFUSION ARE FORBIDDEN WITHOUT PRIOR AUTHORISATIONR2012FSFOR001 Page 3/ 12

I. CONCLUSION
Antiviral activities of the MARMOLEUM TOPSHIELD 2 surface and non-active surface have beentested under conditions defined by the ISO 21702 (2019) protocol for contact times of 2 and 5 hours
against human coronavirus HCoV-229E.
The stainless-steel surface is the control for this test.

MARMOLEUM TOPSHIELD 2 surface, 2 hours

Under experimental conditions (25°C, 2 hours, 90% RH), the MARMOLEUM TOPSHIELD 2 surface
shows an antiviral activity per cm2 associated with a logarithmic reduction of 0.8log10 (84.15%)
efficiency under the ISO 21702 protocol.

PRODUCT

Contact time

Antiviral activity R (log10/cm2)

Antiviral activity (%)

MARMOLEUMTOPSHIELD 22 hours

R=0.884.15

MARMOLEUM TOPSHIELD 2 surface, 5 hours

Under experimental conditions (25°C, 5 hours, 90% RH), the MARMOLEUM TOPSHIELD 2 surface
shows an antiviral activity per cm2 associated with a logarithmic reduction of 2.0 log10 (99.00%)
efficiency under the ISO 21702 protocol.

PRODUCT

MARMOLEUMTOPSHIELD 2Contact time

Antiviral activity R (log10/cm2)

Antiviral activity (%)

5 hours

R = 1.998.74

REPRODUCTION AND DIFFUSION ARE FORBIDDEN WITHOUT PRIOR AUTHORISATION

R2012FSFOR001 Page 4/ 12

II. CONTRACTUAL DOCUMENTS
The present service is defined by the following contractual documents:

DEV0236. Quotation. Order

Order 4501136357 from 09/11/2020

III. TEST CONDITIONS AND SAMPLES DATA
III.1 Samples identification
Surface

MARMOLEUM TOPSHIELD 2

Stainless steel

Appearance

GreygreySize (cm)

5 x 5 cm

5 cm x 5 cm

Thickness (mm)2.51Porous / non porous

Non porous

Non porous

Manufacturer: FORBO FLOORING SYSTEMS
Supplier: FORBO FLOORING SYSTEMSStorage conditions: room temperature
Evaluation period: 11/2020

REPRODUCTION AND DIFFUSION ARE FORBIDDEN WITHOUT PRIOR AUTHORISATION

R2012FSFOR001 Page 5/ 12

III.2 Experimental conditions
Test surface: MARMOLEUM TOPSHIELD 2Experimental Conditions

Date-25/11/2020

Viral strain

Human coronavirus HCoV-229E

Inoculum volume400 µL

Cover film

4 cm x 4 cm = 16 cm²Temperature
Humidity HR (%)

25°C ± 0.1
90% ± 5
2 hours
5 hoursn.a.
Submerging in 10mL of SCDLP medium
endpoint titration on permissive cells
834 ± 1 °C

Contact time
Interfering substance
Neutralisation
Quantification
Number of wells per dilution
Incubation temperature

REPRODUCTION AND DIFFUSION ARE FORBIDDEN WITHOUT PRIOR AUTHORISATION

R2012FSFOR001 Page 6/ 12

IV. RESULTS
IV.1 Antiviral activity of the MARMOLEUM TOPSHIELD 2 surface against
human coronavirus HCoV-229E for a contact time of 2 and 5 hours
a. Cell susceptibility
Surface

Log10 TCID50/mL

SCDLP medium

6.1

MARMOLEUM TOPSHIELD 2

6.1

Non active surface

6.3

Active Surface: Difference < 0.5 log10 ☒ yes ☐ noInactive Surface: Difference < 0.5 log10 ☒ yes ☐ nob. Cytotoxicity
The test surface cytotoxicity is determined by reading of cytopathic effect (CPE) on MRC5 permissivecells and quantified by TCID50 technique.
For viral recuperation on surface, the surfaces are submerging in in 10mL of SCDLP medium(recuperation buffer). The recuperation buffer cytotoxicity is determined by reading of cytopathiceffect (CPE).
Under test conditions, the recuperations buffers from MARMOLEUM TOPSHIELD 2 and reference
surfaces did not show cytopathic effects on MRC5 cells for a contact time of 2 and 5 hours, 90% RH.
The test results are dependent on and take into account the cytotoxicity results.

REPRODUCTION AND DIFFUSION ARE FORBIDDEN WITHOUT PRIOR AUTHORISATION

R2012FSFOR001 Page 7/ 12c.

Inactivation of antiviral activity
ProductLog10
TCID50/mL

Sn = SCDLP medium

5.1

St = active surface

4.9

Su = non Active surface (reference)

5.0

Sn - Su ≤ 0,5 ☒ yes ☐ no
Sn - St ≤ 0,5☒ yes ☐ no

Explanations:
Sn: the average of the common logarithm of the infectivity titer of virus from three specimens of theSCDLP broth for negative control.
Su: the average of the common logarithm of the infectivity titer of virus recovered from three untreatedtest specimens;
St: the average of the common logarithm of the infectivity titer of virus recovered from three test
specimens.

REPRODUCTION AND DIFFUSION ARE FORBIDDEN WITHOUT PRIOR AUTHORISATION

R2012FSFOR001 Page 8/ 12d. Test
Raw data for antiviral activity of MARMOLEUM TOPSHIELD 2 and reference surfaces against
human coronavirus HCoV-229E under test conditions (25°C, 2 hours, 90% RH) are presented inappendices.
Results have been determined by visual reading of cytopathic effects (CPE) and quantified by TCID50
technique on MRC5 cells.
Cytotoxicity(log10TCID50)

SpecimenUo (log10TCID50/cm2)

Ut2h (log10TCID50/cm2)

Stainless steel

0.5

L1L2
L3
Average

4,94,75,14,94,64,34,74,54,74,64,34,5

Surface

Cytotoxicity(log10TCID50)

Specimen

A0 (log10TCID50/cm2)

At2h (log10TCID50/cm2)

At5h (log10TCID50/cm2)

0.5L1L2
L3
AverageR (log10TCID50/cm2)

5,34,84,85,03,63,83,73,7/

0.82,62,72,42,61.9

Surface

MARMOLEUMTOPSHIELD 2

Ut5h (log10TCID50/cm2)

R is the antiviral activity
U0 is the average of the common logarithm of the number of TCID50 recovered from threeuntreated test specimens immediately after inoculation
Ut is the average of the common logarithm of the number of TCID50 recovered from threeuntreated test specimens
A0 is the average of the common logarithm of the number of TCID50 recovered from threetreated test specimens immediately after inoculationAt is the average of the common logarithm of the number of TCID50 recovered from threetreated test specimens.
The logarithmic value of the number of TCID50 recovered immediately after inoculation from
untreated test specimen (U0) satisfies the requirement below: (Lmax – Lmin) / (Lmean) ≤ 0.2.

REPRODUCTION AND DIFFUSION ARE FORBIDDEN WITHOUT PRIOR AUTHORISATION

R2012FSFOR001 Page 9/ 12

V. CONCLUSIONMARMOLEUM TOPSHIELD 2 surface shows antiviral activity of 0.8log10 TCID50/cm²(84.15%) against human coronavirus HCoV-229E after a contact time of 2 hours at 25°C,
90% RH.
MARMOLEUM TOPSHIELD 2 surface shows antiviral activity of 1.9log10 TCID50/cm²(98.74%) against human coronavirus HCoV-229E after a contact time of 5 hours at 25°C,
90% RH.

REPRODUCTION AND DIFFUSION ARE FORBIDDEN WITHOUT PRIOR AUTHORISATION

R2012FSFOR001 Page 10/ 12

VI. ANNEXES
VI.1

Materials and reagents
Cell line

Name : MRC5 ATCC® CCL-171™
Number of passages: 22
Culture medium: EMEM (Lonza) with 10% of FCS (Dutscher), 1% of antibiotics (Gibco) et 1% of Lglutamine (Gibco)

Viral strain

Name: human coronavirus 229E ATCC® VR-740™
Viral test suspension: 4.22x10^7 (batch number: 102020229-9)
Quantification technique :- Successive tenfold dilution in infection medium: EMEM (Lonza) with 2% of FCS (Dutscher), 1% ofantibiotics (Gibco) et 1% of L-glutamine (Gibco)- Add 100μL of every dilution on 8 wells on a 96 plate.
- Incubate 7 days at 34°C, 5% C02

REPRODUCTION AND DIFFUSION ARE FORBIDDEN WITHOUT PRIOR AUTHORISATION

R2012FSFOR001 Page 11/ 12

VI.2 RAW DATA: TCID50 quantification against human coronavirus HCoV229E after 2 and 5 hours, visual reading of cytopathic effects (8 wells perdilutions)

Table 1: Inactivation of antiviral activity
ProductSCDLP

supression of
prioduct's activity

Stai nl es s s teel

MARMOLEUM TOPSHIELD
2

contact time(h)/////////

dilutions (-log)
p

1

2

3

4

5

6

744444444

44444444

44444444

44444444

11020333

00044444444

44444444

44444444

44444444

11424444

00044444444

44444444

44444444

44444444

11202222

00044444444

44444444

44444444

44444444

00020222

00044444444

44444444

44444444

44444444

11423220

00044444444

44444444

44444444

44444444

00212111

00044444444

44444444

44444444

44444444

10102200

00044444444

44444444

44444444

44444444

21212000

00044444444

44444444

44444444

44444444

11423200000

Explanations:
1-4: degrees of CPE in 8 cell culture unit (microtiter plate)
0: no virus present
n.a: not applicable
n.d: not done

Table 2 : A0/U0ProductSta i nl es s s teel

A0/U0
MARMOLEUM TOPSHIELD
2contact time(h)dilutions (-log)
p1

2

3456

7

000044444444

44444444

44444444

44444444

11002301

00044444444

44444444

44444444

44444444

12100000

00044444444

44444444

44444444

44444444

14230021

00044444444

44444444

44444444

44444444

000004444444444444444

44444444

44444444

11203000

000044444444

44444444

44444444

44444444

11020300000Explanations:
1-4: degrees of CPE in 8 cell culture unit (microtiter plate)
0: no virus present
n.a: not applicable
n.d: not done

●Table 3 : cytotoxicity
Product

contact time(h)dilutions (-log)p

Cytotoxicity

Cytotoxicity

Sta i nl es s s teel
MARMOLEUM TOPSHIELD
2
Sta i nl es s s teel
MARMOLEUM TOPSHIELD
22030405070106200200000000500000000500000000Explanations:
1-4: degrees of CPE in 8 cell culture unit (microtiter plate)
0: no virus present
n.a: not applicable
n.d: not done

REPRODUCTION AND DIFFUSION ARE FORBIDDEN WITHOUT PRIOR AUTHORISATION

R2012FSFOR001 Page 12/ 12

Table 4 : cell susceptibility
ProductSCDLP

cell susceptibility

Stai nl es s s teel
MARMOLEUM TOPSHIELD
2

contact time(h)dilutions (-log)
p1234567//44444444

44444444

44444444

44444444

00111102

00044444444

44444444

44444444

44444444

00333212

000/44444444

44444444

44444444

44444444

01002222000p123456744444444

44444444

44444444

44444444

10000020

00044444444

44444444

44444444

44444444

000044444444

44444444

44444444

44444444

03302000

00044444444

44444444

44444444

100000020

000044444444

44444444

44444444

11020000

02000000

00044444444

44444444

44444444

20300001

000044444444

44444444

44444444

21111111

02011000

00044444444

44444444

44444444

44444444

03300000

00044444444

44444444

44444444

44444444

000044444444

44444444

10010000

0000044444444

44444444

11030000

0000044444444

44444444

02000000

00000

Explanations:
1-4: degrees of CPE in 8 cell culture unit (microtiter plate)
0: no virus present
n.a: not applicablen.d: not done

Table 5 : test
Product

Stai nl es s s teel

test
MARMOLEUM TOPSHIELD
2

Stai nl es s s teel

test
MARMOLEUM TOPSHIELD
2contact time(h)222222555555dilutions (-log)

Explanations:
1-4: degrees of CPE in 8 cell culture unit (microtiter plate)
0: no virus present
n.a: not applicable
n.d: not done

REPRODUCTION AND DIFFUSION ARE FORBIDDEN WITHOUT PRIOR AUTHORISATION