Test of bactericidal efficacy of Cleantop®WM-S under changing conditions

(4 Test Reports)

Tests of bactericidal activity by electrolyzed acid water under existence of protein

in vitro

Bactericidal activity by the electrolyzed acid water under existence of protein (blood serum) was
tested.

Summary of test:
After mixing the electrolyzed acid water produced by CLEANTOP WM-1 and the prepared
solution of bacteria solution + blood serum, it was added intermittently to the culture medium
and incubated at 35°C for 4 days. Then, the existence of bacteria growth was examined.

Test methods:
After mixing bacterial solutions adjusted to each viable cell number 108/ml with blood serum
(50,100, 200, 500 ml and 2 ml), each solution was mixed with the electrolyzed acid water (pH
2.7 or less, ORP 1000 mV or more, free residual chlorine concentration 3 ppm or more) to make
200 ml in total. Collected 1 ml each of these prepared solutions at 15, 30, 60, 120, 180 and 240
seconds after, and immediately after collection, the reaction was stopped by adding and mixing
of 9 ml of normal saline solution. 1 ml of each solution and its diluted (10 to 100 times) solution
were added to each culture medium to incubate at 35°C for 4 days. The existence of bacteria
growth was examined and bacteria numbers were counted.

(Examined bacteria) Pseudomonas aeruginosa

Staphylococcus aureus
Escherichia coli

(Examined blood serum) Human blood serum, Sigma, Inc, Lot.14H9305

(Medium for bacteria count) Soybean casein agar

Results:

As shown in the attached sheet.

In case of 50 ml to 2 ml of blood serum was added with electrolyzed acid water to make a total
quantity of 200 ml.

• pH changed little
• Oxydation-reduction potential (ORP) fell rapidly by adding blood serum, decreasing in
accordance with the increased quantity of added blood serum.
• Concentration of free residual chlorine showed the same tendency as ORP, decreasing
in accordance with the increased quantity of added blood serum.
• Viable cell count increased in accordance with decreased ORP and concentration of free
residual chlorine, showing the fall of bactericidal activity.

1
Discussion:

• In case of adding 50 ml of blood serum, all three types of bacteria were disinfected in 15
seconds. P. aeruginosa was disinfected up to 500 ml of serum added, and S. aureus up
to 200 ml. In case of E. coli, viable cells were detected by the addition of 100 ml blood
serum. It was be recognized that viable cell count increased and bactericidal effect
decreased according to the increase of added blood serum.
• In the reference ("Research Report of Electrolyzed Acid Water", Operative Medicine,
15:508-520, 1994), the electrolyzed acid water had the free residual chlorine
concentration of about 25 ppm, but this test used the electrolyzed acid water with the
initial free residual chlorine concentration of about 5 ppm. The fall of bactericidal effect by
the addition of blood serum showed the same tendency.

* This test was performed in order to examine how bactericidal performance was affected by the
degradation of electrolyzed acid water under existence of protein. The results showed the
performance of electrolyzed acid water decreased under existence of protein, failing to make a
sufficient bactericidal effect. However, CLEANTOP WM-1 maintained the proper physical
characteristics of electrolyzed acid water by constant supply of maintaining electricity.

2
Test results 1
1 Change of pH, ORP and free residual chlorine
2 Detected Bacteria
concentration

3
Test results 2

4
Performance of CLEANTOP WM-S

Test for determination of appropriate FRCC concentration (Primary

Report)

SRL Corporation
2-49-19, Akebonocho, Tachikawa, Tokyo

The above test was requested by Kaigen Co., Ltd. and was performed with witness
from them. This is the report of the tests.

Client Kaigen Co., Ltd.

Cleantop WM-S
Specimen Endoscopes XQ30 and XQ200 made by Olympus
Corporation
Address:153, Komiya-cho, Hachioji, Tokyo
Place of testing
Name: The Third Laboratory, Hachioji
Person tested and Tested by: Mr. Nobuya Sakagami, Bacteria Inspection
Responsible Section
person Responsible person: Mr. Kazunori Hochito
Date of testing March 29 and 30, 2001

Outline of testing
For disinfection of endoscopes with Cleantop WM-S, this test aimed to determine
most appropriate concentration of free residual chlorine and necessary time period
for disinfection. After adhering prepared bacteria solution on outside of endoscopes
and inside of hole for forceps, this endoscopes were disinfected and the presence or
absence of bacteria adhered to the endoscopes after disinfection were verified.

Testing method
Injecting the prepared bacteria solution into forceps hole by means of a sterilized
syringe as well as dipping the endoscopes into prepared bacteria solution with more
than 108 of number of live bacteria and adhered bacteria on the outside of the
endoscopes. After this processing, disinfection as described below will be performed
using Cleantop WM-S and with electrolytic acid water of less than pH 2.7 and with
more than 1000 mV ORP.

Free residual chlorine concentration in electrolytic acid water: 10, 15, 20, 30 and 40
ppm
Disinfection time: 1, 2, 3, 4, 5, 6 and 7 minutes

After disinfection, wiping off the outside of endoscopes with sterilized gauze and
injecting 40 ml of sterilized physiological salt solution into forceps hole, solution in the
hole and the gauze shall be incorporated in a sterilized beaker. The extracted
solution from the beaker will be tested.

5
Bacteria tested

Concentration of bacteria used

Tested on March 29 Tested on March 30

(No. of testing (No. of testing
bacteria:1 - 30) bacteria: 31 - 70)

Staphylococcus aureus ATCC 2.1 × 1010 (cfu/ml) 2.0 × 1011 (cfu/ml)

29213

Enterococcus faecalis ATCC 7.0 × 109 (cfu/ml) 1.4 × 1010 (cfu/ml)

29212

Escherichia coli ATCC 25922 1.4 × 1010 (cfu/ml) 2.5 × 1010 (cfu/ml)

Pseudomonas aeruginosa 1.5 × 1010 (cfu/ml) 1.5 × 1010 (cfu/ml)

ATCC 10145

Bacillus subtilis ATCC 19659 3.0 × 109 (cfu/ml) 2.0 × 109 (cfu/ml)

Culture medium for bacteria determination and culture conditions

Culture medium used:

Blood agar (Poa medium by Eiken)

Culture conditions:
The extracted solution as original solution shall be diluted into × 10 and × 102 with
sterilized physiological salt solution respectively. Dropping 100 ml of the original and
diluted solutions onto blood agar and expanded with sterilized Conrage bars. After
cultivating the agar at 35±2°C for 48 hours, the number of colonies have been
counted and determined the final number of bacteria for judgment.

Test results
As shown in the separate paper attached.

Conclusion
Except one result, no bacteria have been found under all conditions. The detected
bacteria of a test (Device No. 002, 30 ppm, 6 minutes for original solution) were 9×10
cfu/ml of Staphylococcus aureus and 3×10 cfu/ml of Pseudomonas aeruginosa and
those are the limit quantities for determination. At the double testing under the same
conditions no bacteria have been found. From these results, disinfection effect is
considered to be recognized for all tests.

6
Test result 1: CLEANTOP WM-S (Device 001)
Endoscopes used for disinfection: Olympus XQ200
No. of bacteria Properties of acid
No. of
Disinfection (Number of colonies) water
FRCC bacteria
time (min) x 10 x 100 FRCC ORP
used Original PH
dilution dilution (ppm) (mV)
10cfu/ml 10cfu/ml 10cfu/ml
1 61 10.8 2.63 1127
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
2 63 10.5 2.50 1139
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
3 65 11.0 2.54 1121
or less or less or less
10 10cfu/ml 10cfu/ml 10cfu/ml
4 67 10.5 2.52 1128
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
5 69 9.7 2.40 1140
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
6 70 9.7 2.34 1142
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
7 21 10.7 1.82 1142
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
1 58 14.7 2.14 1136
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
2 57 15.7 2.13 1136
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
3 55 15.8 2.14 1148
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
15 4 53 14.2 2.26 1157
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
5 51 15.9 2.17 1162
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
6 48 15.6 2.54 1152
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
7 30 16.6 2.09 1127
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
1 46 19.4 2.20 1140
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
2 45 20.6 2.22 1143
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
3 28 21.4 2.14 1133
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
20 4 20 20.0 1.87 1148
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
5 18 20.4 1.86 1164
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
6 10 21.6 1.90 1153
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
7 8 22.0 2.24 1161
or less or less or less

7
 10cfu/ml 10cfu/ml 10cfu/ml
1 44 29.8 2.13 1149
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
2 41 31.4 2.04 1144
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
3 39 31.0 1.89 1170
or less or less or less
30 10cfu/ml 10cfu/ml 10cfu/ml
4 26 32.2 2.22 1141
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
5 6 29.2 2.01 1168
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
6 4 29.2 1.82 1163
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
7 2 29.6 2.01 1152
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
1 36 42.0 2.10 1153
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
2 34 43.4 2.24 1165
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
3 32 42.2 2.29 1171
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
40 4 24 42.2 2.09 1152
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
5 16 29.4 1.83 1153
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
6 14 41.2 1.95 1153
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
7 12 41.4 2.36 1146
or less or less or less

8
Test result 2: CLEANTOP WM-S (Device 002)
Endoscopes used for disinfection: Olympus XQ30
No. of bacteria Properties of acid
No. of
Disinfection (Number of colonies) water
FRCC bacteria
time (min) x 10 x 100 FRCC ORP
used Original PH
dilution dilution (ppm) (mV)
10cfu/ml 10cfu/ml 10cfu/ml
1 59 9.8 2.61 1148
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
2 60 11.0 2.66 1127
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
3 62 11.0 2.59 1128
or less or less or less
10 10cfu/ml 10cfu/ml 10cfu/ml
4 64 9.5 2.55 1147
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
5 66 9.7 2.50 1136
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
6 68 9.9 2.46 1135
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
7 19 11.2 1.91 1156
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
1 56 16.3 2.26 1147
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
2 54 15.5 2.18 1153
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
3 52 16.0 2.26 1172
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
15 4 50 15.1 2.35 1148
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
5 49 14.6 2.39 1143
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
6 47 16.2 2.41 1155
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
7 29 15.6 2.07 1141
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
1 40 21.6 2.04 1165
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
2 37 20.8 2.02 1148
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
3 27 21.2 2.00 1141
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
20 4 25 19.2 2.00 1169
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
5 23 21.6 2.15 1157
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
6 17 18.4 2.00 1145
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
7 11 21.0 2.01 1152
or less or less or less

9
 10cfu/ml 10cfu/ml 10cfu/ml
1 38 30.0 1.90 1187
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
2 35 30.4 2.03 1140
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
3 13 30.8 2.11 1159
or less or less or less
30 10cfu/ml 10cfu/ml 10cfu/ml
4 9 29.4 1.96 1169
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
5 5 29.4 1.88 1155
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
6 3 32.6 1.89 1116
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
7 1 28.8 2.23 1148
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
1 43 39.6 2.18 1159
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
2 42 39.6 2.22 1151
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
3 33 40.0 2.14 1175
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
40 4 31 39.6 2.32 1170
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
5 22 40.6 2.26 1135
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
6 15 39.6 2.02 1155
or less or less or less
10cfu/ml 10cfu/ml 10cfu/ml
7 7 38.8 2.00 1174
or less or less or less

10
Test for determination of disinfection time
(Secondary Report)

SRL Corporation
2-49-19, Akebonocho, Tachikawa, Toky

The above test was requested by Kaigen Co., Ltd. and was performed with witness
from them. This is the report of the tests.

Client Kaigen Co., Ltd.

Cleantop WM-S
Specimen Endoscopes XQ30 and XQ200 made by Olympus
Corporation
Address:153, Komiya-cho, Hachioji, Tokyo
Place of testing
Name: The Third Laboratory, Hachioji
Person tested and Tested by: Mr. Nobuya Sakagami, Bacteria Inspection
Responsible Section
person Responsible person: Mr. Kazunori Hochito
Date of testing March 29 and 30, 2001

Outline of testing
For disinfection of endoscopes with Cleantop WM-S, this secondary test aimed to
determine most appropriate disinfection time periods under a fixed free residual
chlorine concentration of 10 ppm, as the disinfection effect under the conditions of
free residual chlorine concentrations of 10 - 40 ppm have been found to be effective
at the primary test. At this test, Bacillus subtilis was cultured at 37°C for 3 days and
bacteria with spore was used.

Testing method
Injecting 1 ml of the prepared bacteria solution with number of live bacteria of more
than 108 into the forceps hole by means of a sterilized syringe as well as dipping the
endoscopes into the prepared bacteria solution, the endoscopes into the prepared
bacteria solution, the endoscopes were disinfected with Cleantop® WM-S under the
condition of electrolyzed acid water of less than pH 2.7, with more than 1000 mV
ORP and 10 ±2 ppm free residual chlorine concentration. Also, considering the
possible deterioration in properties of electrolyzed acid water, conditions with and
without horse blood serum have been added.

Disinfection time: 1, 2, 3, 4, 5, 6 and 7 minutes

Horse blood serum: Horse blood serum of 5 % was added to bacteria solution to be
1 %.

After disinfection, wiping off the outside of endoscopes with sterilized gauze and
Injecting 40 ml of sterilized physiological salt solution into forceps hole, solution in the
hole and the gauze shall be incorporated in a sterilized beaker. The extracted
solution from the beaker will be tested.

11
Bacteria tested

Concentration of bacteria used

Bacillus subtilis ATCC 19659 (sporous) 1.0 x 105 (cfu/ml)

Culture medium for bacteria determination and culture conditions

Culture medium used: Blood agar (Poa medium by Eiken)

Culture conditions:
The extracted solution as original solution shall be diluted into × 10 and × 102 with
sterilized physiological salt solution respectively. Dropping 100ìml of the original and
diluted solutions onto blood agar and expanded with sterilized Conrage bars. After
cultivating the agar at 35±2°C for 48 hours, the number of colonies have been
counted and determined the final number of bacteria for judgment.

Test results
As shown in the separate paper attached.

Conclusion
Except two results, no bacteria have been found under all conditions. The two
detected cases (Device No. 002, withiout horse blood serum, a quarter original
solution and with horse blood serum, a half original solution) after disinfection were
10 cfu/ml limit quantity respectively. At the double testing under the same conditions,
no bacteria have been found. From these results, disinfection effect is considered to
be recognized for all tests.

12
Test result 1: Without horse blood serum
No. of measured Properties of
Disinfection bacteria No. of Endoscope acid water
FRCC
time (min) x 10 x 100 device type FRCC ORP
Original PH
dilution dilution (ppm) (mV)
10 10 10
Olympus
1 cfu/ml cfu/ml cfu/ml 001 9.8 2.53 1132
XQ30
or less or less or less
10 10 10
Olympus
2 cfu/ml cfu/ml cfu/ml 001 10.2 2.59 1130
XQ30
or less or less or less
10 10 10
Olympus
3 cfu/ml cfu/ml cfu/ml 001 10.6 2.50 1140
XQ30
or less or less or less
10 10 10
Olympus
10 4 cfu/ml cfu/ml cfu/ml 001 9.9 2.40 1132
XQ30
or less or less or less
10 10 10
Olympus
5 cfu/ml cfu/ml cfu/ml 001 9.1 2.46 1125
XQ30
or less or less or less
10 10 10
Olympus
6 cfu/ml cfu/ml cfu/ml 001 10.0 2.37 1134
XQ30
or less or less or less
10 10 10
Olympus
7 cfu/ml cfu/ml cfu/ml 001 9.1 2.29 1147
XQ30
or less or less or less
10 10 10
Olympus
1 cfu/ml cfu/ml cfu/ml 002 9.8 2.65 1125
XQ200
or less or less or less
10 10 10
Olympus
2 cfu/ml cfu/ml cfu/ml 002 9.8 2.50 1119
XQ200
or less or less or less
10 10 10
Olympus
3 cfu/ml cfu/ml cfu/ml 002 10.2 2.44 1126
XQ200
or less or less or less
10 10 10
Olympus
10 4 cfu/ml cfu/ml cfu/ml 002 9.8 2.49 1133
XQ200
or less or less or less
10 10 10
Olympus
5 cfu/ml cfu/ml cfu/ml 002 9.0 2.43 1111
XQ200
or less or less or less
10 10 10
Olympus
6 cfu/ml cfu/ml cfu/ml 002 9.4 2.30 1118
XQ200
or less or less or less
10 10 10
Olympus
7 cfu/ml cfu/ml cfu/ml 002 10.6 2.22 1146
XQ200
or less or less or less

13
Test result 2: With 1 % of 5 % horse blood serum against bacteria sol.
No. of measured Properties of
Disinfection bacteria No. of Endoscope acid water
FRCC
time (min) x 10 x 100 device type FRCC ORP
Original PH
dilution dilution (ppm) (mV)
10 10 10
Olympus
1 cfu/ml cfu/ml cfu/ml 001 10.9 2.47 1123
XQ30
or less or less or less
10 10 10
Olympus
2 cfu/ml cfu/ml cfu/ml 001 10.9 2.50 122
XQ30
or less or less or less
10 10 10
Olympus
3 cfu/ml cfu/ml cfu/ml 001 10.6 2.43 1108
XQ30
or less or less or less
10 10 10
Olympus
10 4 cfu/ml cfu/ml cfu/ml 001 9.6 2.34 1125
XQ30
or less or less or less
10 10 10
Olympus
5 cfu/ml cfu/ml cfu/ml 001 9.3 2.32 1134
XQ30
or less or less or less
10 10 10
Olympus
6 cfu/ml cfu/ml cfu/ml 001 9.2 2.22 1133
XQ30
or less or less or less
10 10 10
Olympus
7 cfu/ml cfu/ml cfu/ml 001 10.3 2.18 1145
XQ30
or less or less or less
10 10 10
Olympus
1 cfu/ml cfu/ml cfu/ml 002 9.5 2.58 1121
XQ200
or less or less or less
10 10 10
Olympus
2 cfu/ml cfu/ml cfu/ml 002 9.8 2.44 1128
XQ200
or less or less or less
10 10 10
Olympus
3 cfu/ml cfu/ml cfu/ml 002 10.3 2.37 1105
XQ200
or less or less or less
10 10 10
Olympus
10 4 cfu/ml cfu/ml cfu/ml 002 10.6 2.33 1136
XQ200
or less or less or less
10 10 10
Olympus
5 cfu/ml cfu/ml cfu/ml 002 9.4 2.38 1114
XQ200
or less or less or less
10 10 10
Olympus
6 cfu/ml cfu/ml cfu/ml 002 9.6 .230 1138
XQ200
or less or less or less
10 10 10
Olympus
7 cfu/ml cfu/ml cfu/ml 002 10.2 2.27 1138
XQ200
or less or less or less

14
Test for determination of possible number of continuous applications (3rd
Report)

SRL Corporation
2-49-19, Akebonocho, Tachikawa, Tokyo

The above test was requested by Kaigen Co., Ltd. and was performed with witness
from them. This is the report of the tests.

Client Kaigen Co., Ltd.

Cleantop WM-S
Specimen
Endoscopes Olympus XQ30 and Fujinon EG-400HR
Address:153, Komiya-cho, Hachioji, Tokyo
Place of testing
Name: The Third Laboratory, Hachioji
Tested by: Mr. Nobuya Sakagami, Bacteria Inspection
Person tested and
Section
Responsible person
Responsible person: Mr. Kazunori Hochito
Date of testing May 31, 2001

Outline of testing
The properties of electrolytic acid water will be gradually degraded due to the
contaminations of protein and others during the continuous applications. Cleantop
WM-S is designed to maintain the specified properties by flowing conservation
current for intervals of disinfections. The purpose of this test is to verify the
maintenance of the specified properties and to determine the number of possible
applications, when Cleantop WM-S is continuously applied for disinfections of
endoscopes. At the test, after adhering prepared bacteria solution on outside of
endoscopes, operational part and the inside of forceps channel, the endoscopes
were disinfected with Cleantop WM-S and presence or absence of the bacteria
adhered to the endoscopes was verified.

Testing method
After having soaked endoscopes into prepared bacteria solution with more than 108
of live bacteria and adhering the bacteria on outside and operational part of the
endoscopes as well as injected the prepared solution of 1 ml into the forceps channel
with a sterilized syringe, the endoscopes have been disinfected 23 times
continuously with Cleantop WM-S under the electrolytic acid water conditions of less
than pH 2.7, ORP more than 1000 mV ORP and free residual chlorine density of 10 ±
2 ppm.

After these disinfections, the outside of endoscopes were wiped off with sterilized
gauze and several places of the operational part were also wiped with sterilized
swabs. Those gauze and swabs were individually flushed with 40 ml of sterilized
physiological salt solution in separate sterilized beaker, and the extracted solution
from the beakers have been tested. For the forceps channel, after injecting 40 ml of
sterilized physiological salt solution into the forceps hole and extracted the wash from
the tip of the forceps channel, and the extracted wash was tested. In addition, to
verify that the electrolytic acid water in the electrolytic cell has not been contaminated
with bacteria, the electrolytic acid water in the cell had been extracted at 5th, 10th,
15th, 20th and 23rd disinfections for each test for the verification.

15
In those tests, 5 % horse serum was added to bacteria solution to be 1 %, assuming
that the properties of the electrolytic acid water could be degraded due to contacts
with protein.

Bacteria tested
Concentration of bacteria used
Staphylococcus aureus ATCC 29213 2.0×1010 (cfu/ml)
Enterococcus faecalis ATCC 29212 3.0×1010 (cfu/ml)
Esccherichia coli ATCC 25922 8.4×101 (cfu/ml)
Pseudomonas aeruginosa ATCC 10145 2.0×1010 (cfu/ml)
Bacillus subtilis ATCC 19659 (*sporous) 1.3×101 (cfu/ml)

* Inoculating bacillus subtillis ATCC 19659 in a test tube, this has been cultivated for
three days at 37 ± 2 °C and the culture in bouillon shall be taken out from the
incubator. The bacillus subtillis shall be placed in a triturator to crush the pellicle
lightly. Next, filtering this cultures with a sterilized funnel with wet cotton cloth, the
filtrate was collected in a sterilized test tube.

Culture medium for bacteria determination and culture conditions

Culture medium used: Blood agar (Boa medium by Eiken)

Culture conditions:
The extracted solution as original solution shall be diluted into × 10 and × 102 with
sterilized physiological salt solution respectively. Dropping 100 ml of the original and
diluted solutions onto blood agar and expanded with sterilized Conrage bars. After
cultivating the agar at
35±2 °C for 48 hours, the number of colonies have been counted and determined the
final number of bacteria for judgment.

Test results
As shown in the separate paper attached.

Conclusion
Except two results, no bacteria have been found under all conditions. The two
detected cases (Device No. 002, withiout horse blood serum, a quarter original
solution and with horse blood serum, a half original solution) after disinfection were
10 cfu/ml limit quantity respectively. At the double testing under the same conditions,
no bacteria have been found. From these results, disinfection effect is considered to
be recognized for all tests.

16
Test result 1: CLEANTOP WM-S (Device 001)
Endoscopes: Fujinon EG-400HR
Properties of acid Operational
Insertion part Inside channel In electrolytic cell
No. of water part
disinfections FRCC ORP x 10 x 100 x 10 x 100 x 10 x 100
PH Original Original Original Original
(ppm) (mV) dilution dilution dilution dilution dilution dilution
10 10 10 10 10 10
10 cfu/ml or
1 10.8 2.68 1120 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
2 10.4 2.65 1125 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
3 9.4 2.66 1125 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
4 9.2 2.65 1125 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10 10 10 10
10 cfu/ml or
5 8.2 2.63 1120 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
6 8.8 2.62 1120 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
7 8.7 2.64 1120 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
8 10.0 2.64 1135 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
9 9.4 2.58 1135 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10 10 10 10
10 cfu/ml or
10 9.7 2.54 1140 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less or less or less or less
10 10 10 10
10 cfu/ml or
11 8.3 2.50 1135 less
cfu/ml cfu/ml cfu/ml *1 *2 cfu/ml
or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
12 9.2 2.46 1135 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
13 9.3 2.40 1140 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
14 8.9 2.36 1140 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10 10 10 10
10 cfu/ml or
15 8.1 2.34 1140 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
16 9.5 2.30 1145 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
17 9.5 2.28 1145 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
18 8.4 2.26 1140 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
19 10.6 2.24 1140 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less

17
 Properties of acid Operational
Insertion part Inside channel In electrolytic cell
No. of water part
disinfections FRCC ORP x 10 x 100 x 10 x 100 x 10 x 100
PH Original Original Original Original
(ppm) (mV) dilution dilution dilution dilution dilution dilution
10 10 10 10 10 10 10 10 10
20 9.0 2.20 1140 *3 cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
21 8.2 2.20 1135 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
22 8.3 2.16 1140 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10 10 10 10
10 cfu/ml or
23 8.7 2.12 1140 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less or less or less or less

*1 Staphylococcus aureus 3.2 x 101 cfu/ml / Pseudomonas aeruginosa 1.8 x 101 cfu/ml
*2 Staphylococcus aureus 2 x 101 cfu/ml / Pseudomonas aeruginosa 2 x 101 cfu/ml
*3 Staphylococcus aureus 10 cfu/ml

18
Test result 2: CLEANTOP WM-S (Device 002)
Endoscopes: Olympus XQ30
Properties of acid Operational
Insertion part Inside channel In electrolytic cell
No. of water part
disinfections FRCC ORP x 10 x 100 x 10 x 100 x 10 x 100
PH Original Original Original Original
(ppm) (mV) dilution dilution dilution dilution dilution dilution
10 10 10 10 10 10
10 cfu/ml or
1 11.0 2.68 1125 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
2 9.2 2.66 1130 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
3 8.2 2.68 1135 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
4 8.3 2.64 1130 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10 10 10 10
10 cfu/ml or
5 8.8 2.60 1130 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
6 9.1 2.54 1130 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
7 9.3 2.48 1150 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
8 9.5 2.40 1150 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
9 8.2 2.40 1150 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10 10 10 10
10 cfu/ml or
10 8.3 2.34 1150 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
11 8.0 2.30 1150 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
12 8.1 2.24 1150 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
13 8.2 2.20 1150 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
14 8.1 2.18 1150 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10 10 10
10 cfu/ml or
15 8.2 2.14 1150 less
*4 cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
16 8.0 2.10 1150 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
17 8.3 2.06 1150 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
18 8.1 2.02 1150 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
19 8.2 1.98 1155 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10 10 10 10
10 cfu/ml or
20 8.6 1.94 1155 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
21 8.4 1.92 1150 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10
10 cfu/ml or
22 9.0 1.90 1150 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less
10 10 10 10 10 10 10 10 10
10 cfu/ml or
23 8.5 1.90 1150 less
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
or less or less or less or less or less or less or less or less or less

*4 Stphylococcus aureus 10 cfu/ml

19
Test for disinfection effect on endoscopes with city water
(4th Report)

SRL Corporation
2-49-19, Akebonocho, Tachikawa, Tokyo

The above test was requested by Kaigen Co., Ltd. and was performed with witness
from them. This is the report of the tests.

Client Kaigen Co., Ltd.

Cleantop WM-S
Specimen
Endoscopes Fujinon EG-400HR
Address:153, Komiya-cho, Hachioji, Tokyo
Place of testing
Name: The Third Laboratory, Hachioji
Tested by: Mr. Nobuya Sakagami, Bacteria Inspection
Person tested and
Section
Responsible person
Responsible person: Mr. Kazunori Hochito
Date of testing May 31, 2001

Outline of testing
Cleantop WM-S can be expected to have washing away effect of bacteria by running
water as well as disinfections effect. The purpose of this test were to verify the
disinfections effect in the case that endoscopes are disinfected only by city water with
Cleantop WM-S, and to compare the results with those of the tests with electrolytic
acid water in the series of primary to 3rd tests.
After having adhered bacteria on outside, operational part and forceps channel of
endoscopes, endoscopes were disinfected by circulating city water with Cleantop®
WM-S in electrolytic cell and it was confirmed whether bacteria were adhered on the
endoscopes or not.

Testing method
After having soaked endoscopes into prepared bacteria solution with more than 108
of live bacteria and adhering the bacteria on outside and operational part of the
endoscopes as well as injected the prepared solution of 1 ml into the forceps channel
with a sterilized syringe, the endoscopes have been disinfected for 3 minutes
circulating non-electrolytic city water with Cleantop WM-S in a electrolytic cell.

After these disinfections, the outside of endoscopes was wiped off with sterilized
gauze and several places of he operational part were also wiped with sterilized
swabs. Those gauze and swabs were individually flushed with 40 ml of sterilized
physiological salt solution in separate sterilized beaker, and the extracted solution
from the beakers have been tested. For the forceps channel, after injecting 40 ml of
sterilized physiological salt solution into the forceps hole and extracted the wash from
the tip of the forceps channel, and the extracted wash was tested.
20
Bacteria tested
Concentration of bacteria used
Staphylococcus aureus ATCC 29213 2.0×1010 (cfu/ml)
Enterococcus faecalis ATCC 29212 3.0×1010 (cfu/ml)
Esccherichia coli ATCC 25922 8.4×101 (cfu/ml)
Pseudomonas aeruginosa ATCC 10145 2.0×1010 (cfu/ml)
Bacillus subtilis ATCC 19659 (*sporous) 1.3×101 (cfu/ml)

* Inoculating bacillus subtillis ATCC 19659 in a test tube, this has been cultivated for
three days at 37 ± 2 °C and the culture in bouillon shall be taken out from the
incubator. The bacillus subtillis shall be placed in a triturator to crush the pellicle
lightly. Next, filtering this cultures with a sterilized funnel with wet cotton cloth, the
filtrate was collected in a sterilized test tube.

Culture medium for bacteria determination and culture conditions

Culture medium used: Blood agar (Boa medium by Eiken)

Culture conditions:
The extracted solution as original solution shall be diluted into × 10 and × 102 with
sterilized physiological salt solution respectively. Dropping 100 ml of the original and
diluted solutions onto blood agar and expanded with sterilized Conrage bars. After
cultivating the agar at 35±2 °C for 48 hours, the number of colonies have been
counted and determined the final number of bacteria for judgment.

Test results
As shown in the separate paper attached.

Conclusion
At all tests, bacteria were detected. From the above results, 3 minutes circulating
disinfections with only city water with Clean-Top WM-S could not expect disinfections
effects, and this method is considered clearly inferior to the 3 minutes circulating
disinfections with electrolytic acid water (10 ppm).

21
Test result 1: CLEANTOP WM-S (Device 001 by top water)
Endoscopes: Fujinon EG-400HR
Properties of
No. of Operational part Insertion part Inside channel
No. of acid water
bacteria
disinfections ORP x 10 x 100 x 10 x 100 x 10 x 100
used PH Original Original Original
(mV) dilution dilution dilution dilution dilution dilution
10 10
10 cfu/ml 10 cfu/ml
1 48 7.28 755 *1 cfu/ml
or less *6 *7 cfu/ml *11 *13 or less
or less or less
10 cfu/ml
2 49 7.26 705 *2 *4 or less a lot *8 *9 *12 *14 *16
10 cfu/ml
3 50 7.26 650 *3 *5 or less a lot a lot *10 a lot *15 *17

*1 Enterococcus faecalis 10cfu/ml, Pseudomonas aeruginosa 10cfu/m

*2 Enterococcus faecalis 10cfu/ml, Pseudomonas aeruginosa 1×101cfu/ml
*3 Staphylococcus aureus 6×10cfu/ml, Enterococcus faecalis 4×101cfu/ml,
Pseudomonas aeruginosa 8×101cfu/ml
*4 Pseudomonas aeruginosa 2×101cfu/ml
*5 Enterococcus faecalis 4×101cfu/ml, Pseudomonas aeruginosa 8×101cfu/ml
*6 Staphylococcus aureus 4×10cfu/ml, Escherichia coli 1.3×101cfu/ml,
Enterococcus faecalis 5×10cfu/ml, Pseudomonas aeruginosa a lot
*7 Enterococcus faecalis 1×101cfu/ml, Escherichia coli 2×101cfu/ml, Pseudomonas
aeruginosa 8×101cfu/ml
*8 Staphylococcus aureus 2×101cfu/ml, Enterococcus faecalis 2×101cfu/ml,
Pseudomonas aeruginosa 2.9×101cfu/ml
*9 Pseudomonas aeruginosa 4×101cfu/ml
*10 Staphylococcus aureus 1×101cfu/ml, Enterococcus faecalis 1×101cfu/ml,
Pseudomonas aeruginosa 5.6×101cfu/ml
*11 Staphylococcus aureus 1.4×101cfu/ml, Enterococcus faecalis 7×10cfu/ml,
Pseudomonas aeruginosa 2.9×101cfu/ml
*12 Staphylococcus aureus 6×10cfu/ml, Enterococcus faecalis 6×10cfu/ml,
Escherichia coli 9×10cfu/ml, Pseudomonas aeruginosa 2.8×101cfu/ml
*13 Staphylococcus aureus 2×101cfu/ml, Enterococcus faecalis ×101cfu/ml,
Pseudomonas aeruginosa 2×101cfu/ml
*14 Enterococcus faecalis 2×101cfu/ml, Pseudomonas aeruginosa 4×101cfu/ml
*15 Staphylococcus aureus 4×101cfu/ml, Others a lot
*16 Pseudomonas aeruginosa 1×101cfu/ml
*17 Enterococcus faecalis 2×101cfu/ml, Pseudomonas aeruginosa 1.9×101cfu/ml

22