Test report

Bactericidal efficacy test against spores by "ALTANT"

2017.9.14
Kitasato Research Center for Environmental Science1. The Purpose
The bactericidal effect on spores by the "Altant" provided by your company was investigated.
In this test, as a test system to investigate the basic bactericidal effect of the specimen, amethod of inoculating the specimen directly with the fungus solution was used.
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: Microbial Biotechnology Division
4. Test period
2017.9.4〜2017.9.6
5. Test item (provided by your company)
1) 「ALTANT」
2) IPA ( Control article)
Each stock sample was subjected to the test.

6. Test condition
1) Working time: 0( Initial, control only), 15seconds,1minute, 5minutes2) Working temperature : 25±2℃
7. Used medium and reagent
1) Culture medium① Tryptic Soy Agar( Difco, Hereinafter referred to as TSA)
② SCDLP broth medium( EIKEN CHEMICAL, Hereinafter referred to as SCDLP)
2) reagent① Sodium chloride( Wako Pure Chemical Industries、 Hereinafter, the 0.85% solution
is described as physiological saline)② sodium thiosulfate (Wako Pure Chemical Industries、First grade)

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8. Preparation of test bacteria and test bacteria
1) Test fungus
Bacillus subtilis ATCC6633( Spore fluid manufactured by CROSSTEX、 Lot.SB009,, 1.4×10⁹CFU/mL)
2) Preparation of test bacteria solution
Spore solution was diluted with sterilized ion-exchanged water to be about 10⁸ CFU /mL, and this was used as a test bacterial solution.
9. Test method1) Bactericidal efficacy test
10 ml of the test sample was collected in a centrifuge tube having a volume of 50 ml.
0.1 ml of the test bacterial solution was added thereto, mixed with a test tube mixer, andallowed to act at 0 (initial), 15 seconds, 1 minute, 5 minutes at 25 ± 2 ℃.
After a predetermined period of action, 9 mL of the test article was added to theinactivating agent * 1 to stop the bactericidal action on the test bacteria, which was usedas a sample solution for measuring the number of bacteria.
For working time 0 (initial) and control, sterile physiological saline was used instead ofthe specimen.
* 1: Sodium thiosulfate added SCDLP (hereinafter referred to as SCDLP with reagent
added) confirmed its effectiveness was used.
Confirmation of the effectiveness of the tested product as an inactivating agent The test
procedure and the test are shown on the last page.
2) Measurement of bacterial countA 10-fold serial dilution series was prepared with a physiological saline solution as a stocksolution, and 1 ml each of the sample stock solution and dilution solution was transferred
to a petri dish, mixed with about 20 ml TSA, and solidified to obtain 36 ± 2 ℃ for 42hours. The number of test bacteria per 1 ml of the test sample was counted by counting
the growth colonies after cultivation (lower limit of determination: 10 CFU).
3) Calculation of bacterial count log reduction value
Based on the initial number of control bacteria and the number of test bacteria in the test
product, the bacterial number log reduction value (= LRV: log reduction value) wascalculated using the following formula. In addition, LRV is represented by one digit after
the decimal point (the second digit after the decimal point is rounded down).
LRV (bacterial count log reduction value): log ₁₀ (initial number of control ÷ numberof bacteria after test article action)

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10. Test results
The test results are shown in Table 1.
The test results showed that "Altant" was less than the lower limit of quantification (<10CFU / ml, LRV> 5.2) at a time of action of 5 minutes, 6.8 × 10⁴ CFU / ml (LRV 1.3)
against Bacillus subtilis spores, The number of bacteria decreased.
On the other hand, the control product, IPA, did not change from the initial number ofbacteria even at the action time of 5 minutes.
As a reference, the culture medium after the culture is shown in Photo 1. In the photographs,
the petri dish after culturing the sample stock solution was photographed.
11. comment
In this test, the bactericidal efficacy against spores of your "Altant" was evaluated. In theevaluation test method of the disinfectant of the 17 th revised Japanese Pharmacopoeia, thebacterial count log reduction value before and after disinfectant action is defined as"efficacious" with the LRV of 2 or more in the spores.
Therefore, in this test, it was judged that there was a bactericidal effect when LRV was 2 ormore. As a result of the test, by acting "altant", the number of bacteria decreased dependingon the action time, LRV became> 5.2 when the action time was 5 minutes, and thebactericidal effect was Admitted.
This test is a test system for examining the basic bactericidal effect of the tested product,
which is a condition without load of organic matter. Ordinarily, microorganisms are present
together with organic substances (nutrient organic substances such as carbohydrates, fats
and oils, proteins, saliva, nasal discharge, feces, vomitus, etc.).
Therefore, as a future test, it is desirable to verify the effectiveness test based on EN 1276:
2009 loaded with organic matter and the effect on real life use model.
12. Reference Test · Reference
1) 17 th revised Japanese Pharmacopoeia, Reference information disinfection method anddecontamination method, 2414-2416.
2) EN 1276:20094 / 7

Table 1. Bactericidal effect of test article on Bacillus subtilis (spores)
Test condition

Working time

LRV *20(initial)

15sec

1min

5min

15sec

1min

5min

1.7×10⁶

1.6×10⁶

1.5×10⁶

1.4×10⁶---① Altant

4.8×10⁵

6.8×10⁴＜100.51.3≻5.2② IPA(control item)

1.5×10⁶

1.4×10⁶

1.5×10⁶---

Control(physiological saline)

Test bacteria: Bacillus subtilis ATCC 6633
(spore fluid, manufactured by CROSSTEX, Lot. SB009, 1.4 × 10⁹ CFU / ml)
Unit: CFU / ml
Lower limit of determination: 10 CFU / ml
* 2: LRV (bacterial log log reduction) = log 10 (initial number of control ÷ number of bacteria
after test article action) LRV truncates the second digit after the decimal point, and when
"LRV" is 0 or less, it is expressed as "-".

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6 / 70(initial)

13. Efficacy confirmation test of deactivator
1)the purpose
The effectiveness of the inactivating agent used for stopping the bactericidal action on
the test bacteria by the tested product was confirmed.
2) Method
1 ml of the test sample was added to 9 ml of deactivator (reagent added SCDLP) andmixed. To this was inoculated 0.1 ml of a bacterial solution of about 10³ to 10⁴ CFU /ml and allowed to act at room temperature for 20 minutes, then the number of bacteria
in this mixed solution was measured.
As a control, sterilized water was used instead of the specimen. The effectiveness of thedeactivating agent was judged according to the following judgment criteria in accordancewith 17 th revised Japanese Pharmacopoeia 4.05 - I - 3.5.
Judgment criteria: B (number of bacteria after deactivating agent treatment) / A (controlbacterial count × 100 = within 50 to 200%3) results
The test results are shown in Table 2. Since the ratio with the number of control bacteria
was 92 to 116% and it was within the judgment criteria shown in 13.2), the deactivator
was judged as effective for the test.
表-2. Effectiveness of inactivating agent on specimen
Inactivating

Number of bacteria

agent used

(CFU / ml)

examined(dilutiongoods

ratio of test

Controlsample)(A)① Altant
② IPA

SCDLP

(control(10 times)

Thedeactivator

A ratio *³

result of(%)

effectiveness*⁴(B)

3.5×10¹

Reagent
addedEvaluation

92

Effectiveness116

Effectiveness3.8×10¹
4.4×10¹item)* 3: B / A × 100* 4: Judged as valid when the ratio is within 50 to 200% (No. 17 Japanese Pharmacopoeia)
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