date of issue: September 8, 2020

Test report
Verification results of inactivating activity of ALTANT
against the COVID-19(SARS-CoV-2)

Experiment period: July 1, 2020-September 8, 2020
Obihiro University of Agriculture and Veterinary Medicine[E-Tech Co., Ltd. translated this report from "Japanese version" to "English version"]1

[Material]
・Virus solution
The JPN / TY / WK-521 strain provided by the National Institute of Infectious Diseases wasused. For the experiment,a viral growth medium (VGMCoV-2 ; see below for composition, but not amphotericin B)
containing SARS- CoV-2 was used as the virus solution (virus titer: approx. 6.75 log10 TCID50/ ml).
・Cells used, medium
VeroE6 / TEMPRSS2 cells donated by the National Institute of Infectious Diseases were used.
As the cell growth medium, modified Dalveco supplemented with 10% fetal bovine serum, 2mM L-glutamine, 100μg / ml canamycin, 2μg / ml amhotericin B, and 500μg / ml G418.
Eagle's medium (DMEM) was used. As VGM, DMEM supplemented with 1% fetal bovineserum, 2 mM L-glutamine, 100μg / ml kanamycin, and 2μg / ml amphotericin B was used.
On the day of the test, VereE6 / TEMPRSS2 cells were inoculated into a 96-well plate inadvance so that the cells became 90-100% confluent, washed once with serum-free MEM
immediately before the experiment, and then 180μl / well was added to VGM.

[Test solution]
・ALTANT2

[Evaluation method]
ALTANT and virus solution (SARS-CoV-2 / VGM) were mixed in a screw cap tube so thatthe liquid volume ratio was 1: 1 or 0.5: 1, and pipetting was performed 10 times or more. At
that time, a group in which distilled water (DDW) and virus solution were mixed was also
placed as a reference group. The test was conducted with 3 tubes in 1 group (n=3). Afterreacting the mixture at room temperature for 20 seconds, 20μl of the mixture was added toa 96 well plate pre-inoculated with VeroE6 / TMPRSS2 cells (from each tube to 2 wells).
After that, 10-fold step dilution was performed on a 96-well plate. After culturing in a CO2
incubator at 37 ° C for 3 days, the cytopathic effect caused by virus infection / proliferation
was observed under a microscope, and based on this, the virus titer (TCID50 / ml) wasobserved using the Behrenskelber method. Was calculated. The virus titers were compared
between the DDW group and the ALTANT group, and the virus inactivating activity of
ALTANT was evaluated.

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[Result]
Reaction time 20 seconds, test solution: virus solution = 1: 1, 0.5: 1(≤2.25: below the detection limit)
Mixing ratio 1: 1

Mixing ratio 0.5: 1

DDW

ALTANT

DDW

ALTANT

Virus titer

Tube 16.75

≦2.25

6.25

≦2.25

(log10TCID50/ml)

Tube 26.25

≦2.25

6.75

≦2.25Tube 36.75

≦2.25

6.25

≦2.25

Mean

6.58

≦2.25

6.42

≦2.25

±standard deviation

±0.29

±0.00

±0.29

±0.00

Difference between-≧4.33-≧4.17-≧99.995-≧99.993

the mean values ofthe DDW group andthe ALTANT group
Virus inactivation rate in the ALTANT
group compared to the DDW group (%)

Average value graph

When the mixture ratio of the test solution and the virus solution was 1: 1 or 0.5: 1, the virus
titer in the ALTANT group was below the detection limit in the reaction time of 20 seconds.
Over 99.99% of viruses were inactivated compared to the DDW group.

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