Test Report

Inactivation test of feline calicivirus by "ALTANT"

2017.9.22
Kitasato Research Center for Environmental Science1. Test purpose
The inactivation effect of Feline calicivirus (substitution for norovirus) by "Altant" provided
by your company.
2. Requester
name: E · Tech Co., Ltd.
location: Hyogo Prefecture Kobe City Chuo-ku Minatojima 9-1 Kobe Incubation Office 316
3. Laboratory
name: Kitasato Research Center for Environmental Sciencelocation: 252-0329Kanagawa prefecture Sagamihara-shi Minami-ku Kitasato 1-15-1
Responsible: Virus Department Virus Division
4. Test period
2017.9.5～2017.9.8
5. Grained goods
1) "ALTANT"
2) IPA (control)
Each stock solution was used
6. Test Condition
working temperature: 25±２℃
working time: control: 0(initial), 15 seconds、1minute、 5minutes
ALTANT、IPA; 15seconds、1 minute、5minutes
7. Test virus
Feline calcivirvs, F-9,ATCC VR-782
8. Cells for infectivity measurement
CRFK: Crandell- Rees feline kidney
9. Test method
1) How to prepare virus solution
The virvs was infected with CRFF,and when about 90% or more of the cell culture area
showed cytopathic effect, it was frozen in a freezer at -30° C.
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Thereafter, freeze-thaw operation was performed, and the supernatant was centrifugedat 2,380×g for 10minutes. The supernamant was collected,and the virus solution
concentrated with an ultrafiltration membrane was stored at -80% C as a storage virus.
For the test,virus solution diluted 10-fold with PBS(phosphate buffered saline) was usedas test virus solution.
2) Virus Inactivation test
The virus Inactivation effect test was conducted according to the following procedure.
0.1ml of the test virus solution was added to 0.9ml of the test product in a test tube,mixedgently by a test tube,mixed gently by a test tube mixer,and allowed to act at room
temperature for a predetermined time.
The stopping of the test on the virus was diluted 100-fold with 0.1% sodium thiosurfateadded SCDLP broth medium to prepare a sample for measuring the virus infectivity value.
In addition,PBS was used in place of the specimen for working time 0(initial) and"control". The effectiveness of suspension was confirmed by a separate test. The
procedure and results are shown on page 12.
3) Measure virus infect ivity value
CRFK cells were seeded in a 12-well plate and cultured for 4days in a CO2incubator.
Prior to ingesting the virus, the culture supernamant was removed and replaced withfresh medium.
Next,the stock solution of the virus infectivity value measurement sample was seriallydiluted 10times with PBS.
Wells from which the culture solution was removed wer inoculated with 0.1ml of a 10fold serial dilution of stock solution for infectivity titration sample or PBS, and cells wereinfected with virus at 37° C for 1hour.
After virus inoculation, the plate were shaken every 15minutes to prevent drying andabsorb the virvs evenly across the cells.
After 1 hour,a plaque forming meditm was added to each well and allowed tosolidify,followed by culturing in a CO2 incubator.
After cultivation,1ml of PBS (4% formalin／PBS ) with 4% formalin was added to eachwell of a 12-well plate and allowed to stand at room temperature for 30minutes.
Thereafter,4% formalin／PBS and agarose were removed and 5% crystal violet 1ml ofmethanol added was added to each well to stain the cells.
The wells were washed with tap water,air-dried, aod the number of plaques formed by
the growth of the virus was counted to obtain the virus infectivity value(PFU／ml) per
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1ml of the sample for measuring the virus infectivity value. In addition, when the solution
after stopping the operation of the test product shows toxicity to cells for infectivity
measurement, it becomes difficult to measure the infectivity value and the detection limit
value also changes, so a separate confirmatary test was conducted. Procedures and results
are shown in section 12.
4) Calculation of log infectivity value log reduction value.
Based on the initial infectivity value of the control and the infectivity value after theaction of the test product, the infectivity value log reduction value(LRV was calculatedusing the following calculated values) In addition, LRV is represented by one digit after
the decimal point (rounded down to the second place)

A formula
LRV = log10(Initial infectivity value of control÷ Infectivity value after test article action )10. Test results
The test results are shown in Table 1 and Table 2. Also, as reference data, pictures ofplaques are shown in Photo-1.
The test result of the virus inactivation test is the virus infectivity value per 1ml of thesample for measuring the virus infectivity value log reduction value (LRV : log reductionvalue).
Early viral infectivity was 7.2×10⁵PFU／ml.
No significant fluctuation in virus infectivity was observed ,even when "control(PBS )" wasallowed to act for a maximum of 5minutes.
On the otherhand, when the virus was allowed to act on "ALTANT" for15 seconds, 1minuteand 5minutes, it become less than the detection limit value(10 PFU／ml) in 15 seconds.
The LRV at each action time was > 4.8 in 15seconds.
Also, when IPA was given a virus for 15seconds, 1minute, and 5minutes, the LRV of eachaction time was 0.5, 0.9 and 1.2, respetively.

11. Comment
In this study, we evoluated the inactivation effect of feline calcivirvs( substitution fornorovirus) by the company supplied by your company.
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As a criterion for disinfecting effect according to EN14476:2005 ( Chemical disinfectants
and antiseptics. Virucidal quantitative suspension test for chemical disinfectants andantiseptics used in human medicine.
In the present study," ALTANT" had LRV greater than 4.0 with action for 15seconds, anda virus inactivating effect was observed.
In general, it is reported that the disinfecting effect is affected by the virus inactivating
effect by organic matter in the environment.
Depending on the use environment of the test, disinfection effect may be affected byorganic substances, and it is important to consider appropriate usage.
From now on, consider the effect of virus inactivation under conditions loaded with organicmatter.

Table-1
Grained goods

working time0(initial)

15sec

1min

5min

7.2×10⁵

7.8×10⁵

8.0×10⁵

7.9×10⁵

Altant＜10＜10＜10IPA

2.2×10⁵

8.5×10⁴

4.3×10⁴

control(PBS)

Infectious value unit : PFU/ml
Test virus infectivity : 4.7 × 10⁸ PFU/ml
Detection limit value : 10 PFU/ml

Table-2
The virus infectivity value log reduction value(LRV : log reduction value)

Grained goodsa)

15sec

1min

5min

control(PBS)

0.0

0.0

0.0

Altant

≻4.8

≻4.8

≻4.8

IPA

0.50.91.2a) Log10 (Early viral infectivity / Infectivity value after action for a predetermined time)

5 / 812. Efficacy confirmation test of ation stop solution1) The purpose
The effectiveness of the stopping solution used for stopping the in activating effect of thetest virus by the test product was confirmed.
2) Method
As a stop solution for the test product,a method of diluting 100times with a 0.1% sodium
thiosurfate- added SCDLP broth medium was adopted.
0.1ml of PBS was added to 0.9ml of the test product in stead of the virus solution, andthe solution diluted with the action stopping solution was designated as "test sample".
1ml of test sample was in oculated with 0.1m of virus solution and allowed to act at room
temperature for 5minutes.
This solution was used as a stock solution and virus infectivity titer was measured.
The effectiveness of the infectivity value did not decrease by more than 0.5log10 ascompared with the control(PBS ).
3) Result
The results are shown in Table3.
The infectivity value when the virus was applied to the "Efficacy test sample" was within
the criteria compared to "control(PBS )" . Based on the above result,it was judged thatthe stop solution was effective for the test specimen.
T able-3

Confirming the validity of the stop solution

Confirmation of Efficacystop effectivenessa)

Virus infectivity

Infection valuevaluelog reductionvalue

Effectiveness ofoutagec)b)

ALTANT

3.3 × 10⁵

0.0

Effectiveness

IPA

5.0 × 10⁵

0.0

Effectiveness

control(PBS )3.3 × 10⁵

Infectious value unit : PFU/mla) Samples to be tested were diluted 100-fold with 0.1% sodium thiosurfate added SCDLP broth mediumb) log10(Infectivity value of control/ infectivity value of test samplec) A case where it did not decrease by 0.5log10 or more relative to the control was judged as effective.

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13. Cytotoxicity confirmation test
1) the purpose
When the tested product shows cytotoxicity to the cells to be cultured of the virus to betested, it is difficult to measure the virus infectivity value. Therefore, the toxicity to CRFK
cells was confirmed using the test product after the inactivation, Detection limit value
was examined.
2) Method
0.1 ml of PBS was added to 0.9 ml of the test product instead of the virus solution, andthe solution diluted with the action stop solution was designated as "sample forcytotoxicity confirmation". This solution was inoculated into a CRFK cell previously
cultured on a 96-well plate with a sample stock solution for measurement or diluted virus
solution in an amount of 25 μL per well, and allowed to stand in a CO 2 incubator at
37 ° C. for 1 hour, then inoculated The solution was removed, 100 μL / well of DMEM
supplemented with 1% FBS was added and cultured for 1 day in a CO 2 incubator.
After culturing, the cells were stained with crystal violet and cytotoxicity was confirmedby the degree of staining of each well. Cytotoxicity is determined by determining theviable cell ratio of cells cultured by adding a sample for confirming cytotoxicity with theviable cell ratio set to 100% when cultured with addition of PBS, and when it is less than50%, there is cytotoxicity .
3) Results
The test results are shown in Table 4. In this test, cytotoxicity was not observed even in
the stock solution of the sample for cytotoxicity confirmation. Therefore, the detection
limit value in this test was 10 PFU / mL.
TABLE-4 Toxicity of test article to CRFK cells
Sample for confirming
cytotoxicity a)Viable cell ratio b) (mean ± standard deviation)

Determination ofStock solution

10 times diluted solutioncytotoxicity c)

ALTANT95 ± 0118 ± 2

No toxicity

IPA

111 ± 3116 ± 3

No toxicitya) Samples to be tested were diluted 100-fold with 0.1% sodium thiosulfate added SCDLP broth mediumb) The average value and standard deviation of 4 wells are shown.
c) When the viable cell ratio was less than 50%, it was judged that there was cytotoxicity .

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