Test Report No.

: VX-TR-20-0432
Copy No.: 1

DETERMINATION OF THE BACTERICIDAL ACTIVITY (EN 1500) OF A333B

Lab No.: VX-91-20-0001

Sample Name: A333B

Method: EN 1500:2013 (E)

Chemical disinfectants and antiseptics – Hygienic handrub – Test method and

requirements (phase 2, step 2)

Client: Afterworx - Hygiene Labs

3/85 Chipping Norton
Sydney, NSW 2170
Australia

Sample Receipt Date: 8 July 2020

Report Date: 21 August 2020

Page 1 of 17

Kuala Lumpur, 21 August 2020

Digitally signed by
PETER CHEONG
CHIEW HING
Date: 2020.08.28
17:03:12 +08'00'
Dr Peter Cheong
Head of Microbiological Testing
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 2 of 17
Materials and Method

Qualitative test method for the evaluation of bactericidal activity of hygienic handrub used in the medical
area according to EN 1500:2013 (E)

1. Testing laboratory identification Viroxy Sdn. Bhd.

6th Floor, Menara RKT
50300 Kuala Lumpur, Malaysia

2. Sample identification

2.1 Sample name: A333B

2.2 Batch no.: A001

2.3 Product appearance: Clear, colourless solution

2.4 Manufacturer: Afterworx - Hygiene Labs

3/85 Chipping Norton
Sydney, NSW 2170
Australia

2.5 Active substances per 100 g: <1.00 % Benzalkonium chloride (ADBAC)

<1.00 % Proprietary
0.50 % Dimethyloctadecyl [3-(trimethoxysilyl)propyl]ammonium chloride

2.6 Sample receipt date: 8 July 2020

2.7 Storage conditions: Room temperature

2.8 Product diluent: Distilled water

3. Experimental conditions

3.1 Testing period: 14 August 2020

3.2 Test organism(s): Escherichia coli K12 NCTC 10538

3.3 Reference product (RP): 60.00 % v/v Propan-2-ol

Apply 3.0 ml every 30 seconds for 1 minute

3.4 Test product (PP): 100.00 %

Apply 3.0 ml every 30 seconds for 1 minute

3.5 Test temperature: 20 °C ± 1 °C

3.6 Counting method: Pour plate

3.7 Incubation period: 24 hours, 36 °C ± 1 °C

3.8 Number of volunteers: 22

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 3 of 17
4. Test method and its validation

4.1 Testing method: Dilution-neutralization

4.2 Inactivation combination: 60.00 g/L Tween 80

60.00 g/L Saponin
6.00 g/L Lecithin
2.00 g/L L-Histidine
2.00 g/L L-Cysteine
in tryptone soya broth

The results of validation tests A, B, and C proved the viability of the method in all cases.

5. Test results

The results are stated in Tables A and B.

6. Conclusion

The mean reduction of the release of the test strain Escherichia coli K12 NCTC 10538 achieved by A333B in accordance with
EN 1500:2013 (E) at 100.00 % concentration following the application of 3ml every 30 seconds for 1 minute is not inferior to
that of the reference hygienic handrub (60.00 % v/v propan-2-ol).

Kuala Lumpur, 21 August 2020

Digitally signed by
PETER CHEONG
CHIEW HING
Date: 2020.08.28
17:03:34 +08'00'
Dr Peter Cheong
Head of Microbiological Testing

7. Note

Bactericidal activity – the capability of a product to produce a reduction in the number of viable bacteria belonging to reference
strains under defined conditions.

R = Prevalue / Postvalue = the reduction in viability, or lg R = lg Prevalue - lg Postvalue

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 4 of 17
Table A: Evaluation of the bactericidal activity of A333B on test strain according to EN
1500

Product: A333B
Test strain: Escherichia coli K12 NCTC 10538

N VC1 VC2 Test suspension, N

10-6 >330 >330

N: 4.30 x 108
lg N: 8.63
10-7 45 41

Volunteer Reference product (RP) Test product (PP)

lg lg lg lg lg lg
No Sequence
Prevalues Postvalues Reduction Prevalues Postvalues Reduction
1 RP -> PP 6.35 2.65 3.70 6.31 2.38 3.94
2 RP -> PP 6.13 2.26 3.87 6.34 3.33 3.01
3 RP -> PP 5.76 2.00 3.75 6.07 2.59 3.48
4 RP -> PP 6.39 2.24 4.16 6.85 2.77 4.08
5 RP -> PP 6.38 2.71 3.67 6.65 2.07 4.58
6 RP -> PP 6.52 3.52 3.00 6.73 2.92 3.81
7 RP -> PP 6.23 3.11 3.11 6.25 1.91 4.34
8 RP -> PP 6.60 2.42 4.18 6.60 3.03 3.56
9 RP -> PP 6.19 2.61 3.58 5.71 2.20 3.51
10 RP -> PP 5.83 2.12 3.71 5.72 2.56 3.16
11 RP -> PP 6.01 3.10 2.91 5.30 3.15 2.15
12 PP -> RP 6.20 2.66 3.54 5.82 2.55 3.27
13 PP -> RP 6.43 2.36 4.08 6.34 2.23 4.12
14 PP -> RP 6.35 2.08 4.27 6.46 2.77 3.68
15 PP -> RP 6.23 3.88 2.35 6.15 3.76 2.39
16 PP -> RP 6.64 2.27 4.37 6.48 2.59 3.88
17 PP -> RP 6.45 2.65 3.08 6.33 2.55 3.78
18 PP -> RP 6.08 2.92 3.16 5.77 1.72 4.05
19 PP -> RP 6.30 2.05 4.25 6.26 2.42 3.84
20 PP -> RP 4.92 1.52 3.40 4.04 1.96 2.08
21 PP -> RP 6.29 2.97 3.32 6.43 3.05 3.38
22 PP -> RP 6.43 2.61 3.82 6.72 2.77 3.95
Mean 6.21 2.58 3.64 6.15 2.60 3.55
SD Overall 0.36 0.54 0.51 0.61 0.49 0.66
N 22 22 22 22 22 22
Mean 6.22 2.61 3.60 6.23 2.63 3.60
SD RP -> PP 0.27 0.47 0.43 0.49 0.46 0.68
N 11 11 11 11 11 11
Mean 6.21 2.54 3.67 6.07 2.58 3.49
SD PP -> RP 0.45 0.61 0.60 0.73 0.54 0.68
N 11 11 11 11 11 11

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 5 of 17
Log Reduction Difference between Reference and Test Products

Difference
Volunteer RP PP
RP-PP
1 3.70 3.94 -0.24
2 3.87 3.01 0.87
3 3.75 3.48 0.27
4 4.16 4.08 0.08
5 3.67 4.58 -0.91
6 3.00 3.81 -0.81
7 3.11 4.34 -1.23
8 4.18 3.56 0.61
9 3.58 3.51 0.07
10 3.71 3.16 0.55
11 2.91 2.15 0.76
12 3.54 3.27 0.27
13 4.08 4.12 -0.04
14 4.27 3.68 0.59
15 2.35 2.39 -0.04
16 4.37 3.88 0.49
17 3.80 3.78 0.03
18 3.16 4.05 -0.89
19 4.25 3.84 0.42
20 3.40 2.08 1.32
21 3.32 3.38 -0.05
22 3.82 3.95 -0.13

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 6 of 17
Table B: Control tests and method validation for Table A

Validation Neutralizer toxicity Method validation

Test strain
suspension control control

E. coli K12 NV0: 1.40 x 102

B: 1.51 x 102 C: 1.10 x 102
NCTC 10538 NVB: 1.51 x 102

Acceptance criteria Results

Number of complete set of results 22

Overall mean of log prevalues of RP 6.21

Overall mean of log prevalues of PP 6.15

Number of log reductions of less than 3.0 in RP 3

Absolute mean differences of RP and PP in the ‘RP -> PP’ and ‘PP -> RP’ groups 0.18

Note

cfu: Colony forming units

VC: Number of cfu counted per 1.0 ml sample

x̅: Average VC1 and VC2 values

N: Number of cfu per ml in the test suspension

NV0: Number of cfu per ml in the mixture C at the beginning of the contact time

NVB: Number of cfu per ml in the mixture B at the beginning of the contact time

B: Number of cfu per ml in the neutralizer toxicity control

C: Number of cfu per ml in the dilution-neutralization method validation

RP: Reference hygienic handrub (60.00 % v/v propan-2-ol)

PP: Test product

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 7 of 17
Table C: Sorting of individual differences and computation for Hodges-Lehmann 97.5 %
upper confidence limits

Significance level (P): 0.025 (one-sided)

Number of volunteers: 22
Critical Value: 66
Inferiority margin: 0.6

Sorted Mean pairwise differences (di+dii) / 2

differences of
RP-PP
1.32 0.87 0.76 0.61 0.59 0.55 0.49 0.42 0.27 0.27 0.08
(descending
order)
1
1.32 1.32
2 9
0.87 1.09 0.87
3 10 13
0.76 1.04 0.81 0.76
4 14 19 29
0.61 0.97 0.74 0.68 0.61
5 15 22 30 33
0.59 0.95 0.73 0.67 0.60 0.59
6 16 23 34 37 39
0.55 0.93 0.71 0.65 0.58 0.57 0.55
7 20 28 38 41 42 47
0.49 0.90 0.68 0.62 0.55 0.54 0.52 0.49
8 25 32 45 46 48 51 58
0.42 0.87 0.64 0.59 0.51 0.50 0.48 0.45 0.42
11 35 43 53 55 62 66 73 91
0.27 0.80 0.57 0.52 0.44 0.43 0.41 0.38 0.34 0.27
12 36 44 54 56 63 67 74 92 94
0.27 0.80 0.57 0.52 0.44 0.43 0.41 0.38 0.34 0.27 0.27
17 49 57 72 76 81 87 102 118 120 135
0.08 0.70 0.47 0.42 0.34 0.33 0.31 0.28 0.25 0.18 0.18 0.08
18 50 59 75 77 82 89 103 121 122 136
0.07 0.70 0.47 0.41 0.34 0.33 0.31 0.28 0.24 0.17 0.17 0.07
21 52 65 78 83 84 98 108 124 125 139
0.03 0.67 0.45 0.39 0.32 0.31 0.29 0.26 0.22 0.15 0.15 0.05
24 60 69 85 90 99 106 113 128 129 142
-0.04 0.64 0.41 0.36 0.29 0.27 0.26 0.22 0.19 0.12 0.12 0.02
26 61 70 86 93 100 107 115 130 131 145
-0.04 0.64 0.41 0.36 0.28 0.27 0.25 0.22 0.19 0.12 0.11 0.02
27 64 71 88 95 101 110 116 132 133 146
-0.05 0.63 0.41 0.35 0.28 0.27 0.25 0.22 0.18 0.11 0.11 0.01
31 68 80 104 105 111 117 126 137 138 150
-0.13 0.59 0.37 0.31 0.24 0.23 0.21 0.18 0.14 0.07 0.07 -0.03
40 79 96 114 119 123 127 134 143 144 153
-0.24 0.54 0.32 0.26 0.19 0.18 0.16 0.12 0.09 0.02 0.02 -0.08
97 141 149 154 155 156 161 165 171 172 180
-0.81 0.26 0.03 -0.02 -0.10 -0.11 -0.13 -0.16 -0.20 -0.27 -0.27 -0.36
109 147 151 157 159 162 166 169 173 174 182
-0.89 0.22 -0.01 -0.06 -0.14 -0.15 -0.17 -0.20 -0.24 -0.31 -0.31 -0.41
112 148 152 158 160 163 167 170 176 177 184
-0.91 0.21 -0.02 -0.07 -0.15 -0.16 -0.18 -0.21 -0.25 -0.32 -0.32 -0.41
140 164 168 175 178 179 181 183 185 186 187
-1.23 0.04 -0.18 -0.24 -0.31 -0.32 -0.34 -0.37 -0.41 -0.48 -0.48 -0.58

From the critical values for Wilcoxon's matched-pairs signed-ranks test, for the entry of n = 22 and a one-sided P = 0.025 level
of significance, the critical value of 66 is found. Hence, c = 66 + 1 = 67. The pairwise differences are sorted in descending order.
The 67th value is 0.38. Hence, the Hodges-Lehmann upper one-sided 97.5 % confidence limit for the difference in lg Rs
between RP and PP is 0.38, which is below the agreed inferiority margin of 0.6.

Therefore, the hypothesis of inferiority of PP versus RP is rejected and it can be concluded that the test preparation PP is not
inferior to RP.

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 8 of 17
Afterworx - Hygiene Labs
3/85 Chipping Norton
Sydney, NSW 2170
Australia

Efficacy of A333B against Escherichia coli K12 NCTC 10538 on artificially contaminated hands according
to EN 1500:2013 (E)

EXPERT OPINION*

This expert opinion is based on the test report VX-TR-20-0432 dated 21 August 2020.

The bactericidal activity of the hand disinfectant A333B of Afterworx - Hygiene Labs against Escherichia coli K12 NCTC 10538
on the artificially contaminated hands of volunteers was investigated according to EN 1500:2013 (E).

According to this test, a hygienic handrub is considered as having bactericidal activity if the mean reduction of the release of the
test organism Escherichia coli K12 NCTC 10538 achieved by the test product is at least not inferior to that achieved by a
specific reference hygienic handrub (60.00 % v/v propan-2-ol).

3 ml of A333B was applied every 30 seconds using the standardized handrub procedure at the concentration of 100.00 % for
the total exposure time of 1 minute on hands artificially contaminated with Escherichia coli K12 NCTC 10538. After the exposure
time, the mean bacterial reduction of A333B was not inferior to that achieved by the reference hygienic handrub. Therefore, a
bactericidal activity against Escherichia coli K12 NCTC 10538 was measured as follows:

100.00 % 1 minute 3 ml every 30 seconds

Kuala Lumpur, 21 August 2020

Digitally signed
Digitally signed by
by SITI SYAZANI
PETER CHEONG BINTI SUHAIMI
CHIEW HING Date:
Date: 2020.08.28 2020.08.28
18:10:11 +08'00'
17:03:50 +08'00'

Dr Peter Cheong Dr Syazani Suhaimi

Head of Microbiological Testing Microbiologist

* Opinions and interpretations expressed here are outside the scope of SAMM (Laboratory Accreditation Scheme of Malaysia)
accreditation.

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 9 of 17
Appendix 1

QAU CERTIFICATE*

The results stated in test report VX-TR-20-0432 dated 21 August 2020 were compared to the raw data of the tests and
checked for correct transfer. No deviations were detected.

Kuala Lumpur, 21 August 2020

Digitally signed
by SITI SYAZANI
BINTI SUHAIMI
Date: 2020.08.28
18:10:31 +08'00'

Dr Syazani Suhaimi
Microbiologist

* Opinions and interpretations expressed here are outside the scope of SAMM (Laboratory Accreditation Scheme of Malaysia)
accreditation.

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 10 of 17
Appendix 2 Raw data

Test Method EN 1500:2013

Test Organism Escherichia coli K12 NCTC 10538
Incubation Temperature (°C) 36 Plating Method Pour plate
Reference Product (RP) 60 % Propan-2-ol Supplier / Batch No. R&M Chemicals / PGKP300919
Application Procedure (RP) Rub in 3 ml twice in 1 minute
60.00 g/L Tween 80, 60.00 g/L Saponin, 6.00 g/L Lecithin, 2.00 g/L Histidine, 2.00 g/L
Neutralizer (RP)
Cysteine in Tryptone Soya Broth
Test Product (PP) A333B Batch No. A001
Product Diluent (PP) Distilled water Lab No. VX-91-20-0001
Application Procedure (PP) Rub in 3 ml twice in 1 minute
60.00 g/L Tween 80, 60.00 g/L Saponin, 6.00 g/L Lecithin, 2.00 g/L Histidine, 2.00 g/L
Neutralizer (PP)
Cysteine in Tryptone Soya Broth
Test Date 14/08/2020 Analyzed By MIS Verified By ANE

Test and Validation Suspension

N VC1 VC2 x̅ wm = N = 4.30E+08 lg N = 8.63

Test Suspension
10-6 >330 >330 8.17 ≤ lg N ≤ 8.70
(N)
10 -7
45 41 Pass? Yes

Validation Suspension VC1 VC2 NV0 = 140.0 NV0 = NV/10

(NV) 141 139 30 ≤ NV0 ≤ 160 Pass? Yes

Validation Suspension VC1 VC2 NV0 = 150.5 NV0 = NVB/1000

(NVB) 151 150 30 ≤ NV0 ≤ 160 Pass? Yes

Validation and Control Procedures

Neutralizer Toxicity VC1 VC2 B = 150.5 Pass? Yes

Control (B) 148 153 B ≥ 0.5 x NVB/1000
VC1 VC2 C = 110.0 Pass? Yes
Method Validation (C)
112 108 C ≥ 0.5 x NV/10

Raw Data of Colony Count

N-6 N-7 NV NVB B C

VC1 >330 45 141 151 148 112
VC2 >330 41 139 150 153 108

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 11 of 17
Appendix 2 Raw data

Reference Product Procedure

Volunteer Colony Count (cfu/ml)

Prevalues Postvalues
No Sequence Hand -3 -4 -5 0
10 10 10 10 10-1 10-2
l >330 261* 29* >330 40 4
1 RP -> PP
r >330 177* 32* >330 50 4
l >330 206* 21* 180* 18* 3
2 RP -> PP
r >330 88 6 >330 18 3
l >330 62 5 104 10 1
3 RP -> PP
r >330 53 6 98 11 0
l >330 220* 20* >330 41 4
4 RP -> PP
r >330 280* 28* 72 7 0
l >330 263* 25* >330 39 1
5 RP -> PP
r >330 221* 22* >330 68 5
l >330 264* 30* >330 >330 71
6 RP -> PP
r >330 >330 41 >330 155* 15*
l >330 184* 17* >330 88 4
7 RP -> PP
r >330 150* 20* >330 189* 22*
l >330 >330 54 239* 27* 2
8 RP -> PP
r >330 >330 29 293* 25* 3
l >330 179* 17* >330 40 3
9 RP -> PP
r >330 132* 14* >330 41 4
l >330 139* 16* 79 7 0
10 RP -> PP
r >330 32 4 >330 22 2
l >330 >330 47 >330 131* 15*
11 RP -> PP
r >330 22 3 >330 115* 15*
l >330 184* 19* >330 58 4
12 PP -> RP
r >330 136* 14* >330 36 5
l >330 296* 27* 172* 17* 1
13 PP -> RP
r >330 252* 25* >330 30 0
l >330 171 11 42 4 2
14 PP -> RP
r >330 287* 31* >330 34 0
l >330 154 10 >330 >330 195
15 PP -> RP
r >330 190* 15* >330 292* 33*
l >330 >330 49 >330 31 3
16 PP -> RP
r >330 >330 39 112 11 1
l >330 244* 28* >330 35 2
17 PP -> RP
r >330 324* 28* >330 56 6
l >330 123* 14* >330 105 8
18 PP -> RP
r >330 115 12 >330 65 7
l >330 165* 16* >330 50 5
19 PP -> RP
r >330 246* 21* 25 1 0
l 31 5 1 27 4 0
20 PP -> RP
r >330 22 3 40 4 0
l >330 179* 18* >330 67 8
21 PP -> RP
r >330 210* 21* >330 123* 17*
l >330 160* 23* >330 107 11
22 PP -> RP
r >330 >330 43 153* 15* 1
Underlined: count used for further computation;
*Indicates adjacent dilutions used for computation;
**Strikethrough: values cannot be used for further computation

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 12 of 17
Appendix 2 Raw data

Test Product Procedure

Volunteer Colony Count (cfu/ml)

Prevalues Postvalues
No Sequence Hand -3 -4 -5 0
10 10 10 10 10-1 10-2
l >330 >330 38 253* 19* 1
1 RP -> PP
r >330 110 10 228 5 1
l >330 200* 30* >330 228* 32*
2 RP -> PP
r >330 218* 30* >330 186* 26*
l >330 108 10 >330 48 4
3 RP -> PP
r >330 127 12 >330 31 3
l >330 >330 59 >330 34 3
4 RP -> PP
r >330 >330 84 >330 101 10
l >330 >330 33 132 9 0
5 RP -> PP
r >330 >330 60 105 10 1
l >330 >330 45 >330 106 11
6 RP -> PP
r >330 >330 63 >330 65 7
l >330 142 11 79 7 1
7 RP -> PP
r >330 224* 26* 84 4 0
l >330 >330 54 >330 300* 20*
8 RP -> PP
r >330 >330 29 >330 40 3
l >330 55 7 187* 18* 2
9 RP -> PP
r >330 47 2 133 9 1
l >330 48 4 >330 28 3
10 RP -> PP
r >330 57 6 >330 47 2
l 242* 26* 2 >330 104 13
11 RP -> PP
r 162* 21* 2 >330 195 8
l >330 64 4 >330 23 4
12 PP -> RP
r >330 69 3 >330 56 7
l >330 160* 18* 89 5 0
13 PP -> RP
r >330 305* 25* >330 32 2
l >330 >330 34 >330 27 5
14 PP -> RP
r >330 >330 24 >330 130 10
l >330 138 10 >330 >330 148
15 PP -> RP
r >330 145* 14* >330 223* 22*
l >330 325* 23* >330 109 11
16 PP -> RP
r >330 284* 28* >330 14 1
l >330 202* 29* 324* 24* 3
17 PP -> RP
r >330 207* 29* >330 40 1
l >330 64 6 40 1 0
18 PP -> RP
r >330 55 5 70 2 0
l >330 150* 16* 160* 16* 2
19 PP -> RP
r >330 211* 25* >330 43 5
l 12 1 0 194 10 1
20 PP -> RP
r 10 1 0 43 6 0
l >330 271* 29* >330 111 11
21 PP -> RP
r >330 257* 31* >330 114 12
l >330 >330 33 >330 64 6
22 PP -> RP
r >330 >330 84 >330 54 3
Underlined: count used for further computation;
*Indicates adjacent dilutions used for computation;
**Strikethrough: values cannot be used for further computation

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 13 of 17
Note

cfu: Colony forming units

VC: Number of cfu counted per 1.0 ml sample

x̅: Average VC1 and VC2 values

N: Number of cfu per ml in the test suspension

N0: Number of cfu per ml at the beginning of the contact time

NV0: Number of cfu per ml in the mixtures A, B, and C at the beginning of the contact time

B: Number of cfu per ml in the neutralizer toxicity control

C: Number of cfu per ml in the dilution-neutralization method validation

RP: Reference hygienic handrub (60.00 % v/v propan-2-ol)

PP: Test product

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 14 of 17
Appendix 3 Summary of test description

1. Materials and reagents

1.1 Tryptone Soya Agar (TSA, Oxoid, catalogue no. CM0131)
1.2 Sodium deoxycholate (HiMedia, catalogue no. RM131)
1.3 Tryptone Soya Broth (TSB, Oxoid, catalogue no. CM0129)
1.4 Linseed oil (R&M Chemicals, catalogue no. 2707-00)
1.5 Potassium hydroxide (R&M Chemicals, catalogue no. 4608-80)
1.6 Ethanol (Copens Scientific, catalogue no. RP2246)
1.7 Magnesium chloride (MgCl2, Acros Organics, catalogue no. AC223211000)
1.8 Calcium chloride (CaCl2, R&M Chemicals, catalogue no. 9924-00)
1.9 Sodium bicarbonate (NaHCO3, Fisher Chemical, catalogue no. 10152780)
1.10 Neutralizer
1.10.1 Tween 80 (Fisher Chemical, catalogue no. 10498800)
1.10.2 Saponin (Nacalai Tesque, catalogue no. 30502-55)
1.10.3 Lecithin (Nacalai Tesque, catalogue no. 20335-65)
1.10.4 L-Histidine (Fisher Scientific, catalogue no. BP382-100)
1.10.5 L-Cysteine (Merck, catalogue no. 1.02838.0100)
1.10.6 Tryptone Soya Broth (TSB, Oxoid, catalogue no. CM0129)

2. Apparatus and glassware

2.1 Autoclave (TOMY, model SX500)
2.2 Water baths (Memmert, model WNB 29)
2.3 Incubator (Binder, model BD 260)
2.4 pH-meter (Ohaus, model 3100 Meter with ST310)
2.5 Vortex® mixer (Biosan model Biosan V-1 Plus)
2.6 Petri dishes (Wanpow Plastic)

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 15 of 17
3. Test procedure
3.1 Sampling of the test organisms before treatment (Prevalue)
3.1.1 Volunteers’ hands are prepared by washing for 1 minute with 5 ml of diluted soft soap without the use of a
brush.
3.1.2 After being rinsed with running tap water, they are thoroughly dried with paper towels for at least 30 seconds.
3.1.3 The contamination fluid is poured into a container and both hands are immersed up to the mid-metacarpals for 5
seconds with fingers spread apart.
3.1.4 Carefully allow surplus liquid to drain back into the container for a maximum of 30 seconds.
3.1.5 Allow the hands to dry in the air for 3 minutes, holding them in a horizontal position with the fingers spread out
and rotating them to and fro to avoid the formation of droplets.
3.1.6 Immediately after drying, rub the fingertips (including the thumb) for 1 minute on the base of a Petri dish
containing 10 ml of TSB as sampling fluid in order to assess the release of test organisms before the treatment
of the hands (prevalues). A separate Petri dish is used for each hand.
3.1.7 Dilutions of 10-2, 10-3 and 10-4 of these sampling fluids are prepared with the sampling fluid, i.e. TSB.
3.1.8 From each dilution, pipette 0.1 ml into separate Petri dish and add 15 to 20 ml of melted TSSA, cooled to 45 °C
± 1 °C to obtain 10-3, 10-4, and 10-5 dilutions. The interval between sampling and plating shall not exceed 30
minutes.
3.1.9 Incubate the plates for 20 to 24 hours at 36 °C ± 1 °C. Discard any plates which is not countable (for any
reason). Count the plates and determine the number of cfu. Incubate the plates for a further 20 to 24 hours.
3.1.10 Do not recount plates which no longer show well separated colonies. Recount the remaining plates. If the
number has increased, use only the higher number for further evaluation. Note the exact number of colonies for
each plate but record >330 for any counts higher than 300 and determine the V C-values.

3.2 Hygienic handrub procedure

3.2.1 After sampling for the prevalues, let the hands dry.
3.2.2 Immediately after drying and without recontaminating the hands, perform the handrub procedure in accordance
with either Sections 3.2.3 or 3.2.4, as applicable.
3.2.3 Reference hygienic handrub procedure (RP)
3.2.3.1 Pour 3 ml of 60 % propan-2-ol into the cupped dry hands of the volunteer and rub vigorously for 30
seconds onto the skin up to the wrists in accordance with the standard handrub procedure to ensure
total coverage of the hands.
3.2.3.2 As a first step, distribute the propan-2-ol all over the hands including the wrists palm to palm; continue
five times right palm over left dorsum and left palm over right dorsum.
3.2.3.3 Then continue with five strokes backwards and forwards, palm to palm with fingers interlaced.
3.2.3.4 Continue with five times rubbing the backs of fingers to opposing palms with fingers interlocked, then
five times rotational rubbing of right thumb clasped in left palm and left thumb clasped in right palm.
3.2.3.5 Then rub five times rotationally with clasped fingers of the right hand in the wet palm of the left hand
and clasped fingers of the left hand in the wet palm of the right hand.
3.2.3.6 Repeat the whole procedure with a further 3 ml of propan-2-ol to give a total rubbing time of 60
seconds.

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 16 of 17
3.2.3.7 Perform sampling according Sections 3.2.5 to 3.2.8.
3.2.4 Test hygienic handrub procedure (PP)
3.2.4.1 This procedure shall be performed according to the recommendation provided by the manufacturer,
which shall include volume of product, frequency of application, and the contact time (between 30 and
60 seconds).
3.2.4.2 In any case, the steps of the standard handrub procedure as described in Section 3.2.3 shall be
followed.
3.2.4.3 Perform sampling according to Sections 3.2.5 to 3.2.8.
3.2.5 Immediately after the handrub procedure, a similar sampling procedure is used as described for the prevalues
but the neutralizer is used as the sampling fluid and as diluent for the 10-1 dilution.
3.2.6 The regular time for rubbing on the base of the Petri dish is 1 minute.
3.2.7 Volumes of 1.0 ml and 0.1 ml of the undiluted sampling fluid and 0.1 ml from its 10 -1 dilution are plated out to
obtain 100, 10-1, and 10-2 dilutions. The interval between sampling and plating shall not exceed 30 minutes.
3.2.8 Incubate the plates for 20 to 24 hours at 36 °C ± 1 °C. Discard any plates which is not countable (for any
reason). Count the plates and determine the number of cfu. Incubate the plates for a further 20 to 24 hours.
3.2.9 Do not recount plates which no longer show well separated colonies. Recount the remaining plates. If the
number has increased, use only the higher number for further evaluation. Note the exact number of colonies for
each plate but record >330 for any counts higher than 300 and determine the V C-values.

3.3 Neutralizer control B – Verification of the absence of toxicity of the neutralizer

3.3.1 Pipette 9.0 ml of the neutralizer used in the test into a tube.
3.3.2 Add 1.0 ml of the validation suspension (NVB) containing 3.0 × 104 cfu/ml to 1.6 × 105 cfu/ml.
3.3.3 Start the stopwatch at the beginning of the addition and mix.
3.3.4 Transfer 0.5 ml of this mixture into a tube containing 4.5 ml of neutralizer to obtain 10 -1 dilution of NVB; repeat
this procedure to obtain 10-2 dilution of NVB.
3.3.5 Place the tubes of 10-2 dilution of NVB in a water bath controlled at 20 °C ± 1 °C for the neutralization time of 10
seconds ± 1 second.
3.3.6 Just before the end of this time, mix.
3.3.7 At the end of this time, take a sample of 1.0 ml of this mixture B (10 -2 dilution of NVB) in duplicate and inoculate
TSA plates using the pour plate or the spread plate technique.
3.3.8 Incubate the plates for 20 to 24 hours at 36 °C ± 1 °C.
3.3.9 Verify according to Section 3.5.

3.4 Method validation C – Dilution-neutralization validation

3.4.1 Pipette 2.0 ml of TSB into a tube.
3.4.2 Starting a stopwatch, add 8.0 ml of the product test solution. Mix, and place the tube in a water bath controlled
at 20 °C for t. Just before the end of t, mix again.
3.4.3 At the end of t, transfer 1.0 ml of the mixture into a tube containing 8.0 ml of neutralizer. Restart the stopwatch
at the beginning off the addition.

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.
 Description: Testing the efficacy of chemical disinfectants and antiseptics (EN 1500)
Lab No.: VX-91-20-0001 Client Name: Afterworx - Hygiene Labs
Test Period: 14 Aug 2020 Sample Name: A333B
Test Report No.: VX-TR-20-0432 Batch No.: A001
Report Date: 21 Aug 2020 Sample Receipt Date: 8 Jul 2020
Copy No.: 1

Page 17 of 17
3.4.4 Mix and place the tube in a water bath controlled at 20 °C ± 1 °C for 10 seconds ± 1 second (neutralization
time).
3.4.5 Add 1.0 ml of the validation suspension. Start the stopwatch at the beginning of the addition and mix.
3.4.6 Place the tube in a water bath controlled at 20 °C ± 1 °C for 30 minutes ± 1 minute.
3.4.7 Just before the end of this time, mix again.
3.4.8 At the end of this time, take a sample of 1.0 ml of the mixture C in duplicate and inoculate TSA plates using the
pour plate or the spread plate technique.
3.4.9 Incubate the plates for 20 to 24 hours at 36 °C ± 1 °C.
3.4.10 Verify according to Section 3.5.

3.5 Basic limits

3.5.1 N is between 1.5 x 108 and 5.0 x 108 (8.17 ≤ lg N ≤ 8.70)
3.5.2 NV0 is between 30 and 160 (3.0 x 101 and 1.6 x 102)
3.5.3 NV is between 3.0 x 102 and 1.6 x 103
3.5.4 NVB is between 3.0 x 104 and 1.6 x 105
3.5.5 B (dil.-neutr.) is equal to or greater than 0.0005 x NVB (half of one-thousandth)
3.5.6 C is equal to or greater than 0.5 x NV0
3.5.7 Control of weighted mean counts: quotient is not lower than 5 and not higher than 15
3.5.8 A complete set of results from at least 18 volunteers shall be available.
3.5.9 The overall means of the lg prevalues for RP and PP shall be both at least 5.00.
3.5.10 No more than three individual lg reductions less than 3.00 shall occur in RP.
3.5.11 The absolute difference of mean differences between lg reductions of RP and PP of groups ‘RP -> PP’ and ‘PP
-> RP’ shall be less than 2.00.

4. Literature
4.1 EN 1500:2013 (E): Chemical disinfectants and antiseptics – Hygienic handrub – Test method and requirements (phase
2, step 2)
4.2 EN 14885:2018 (E): Chemical disinfectants and antiseptics – Application of European Standards for chemical
disinfectants and antiseptics
4.3 EN 12353:2013 (E): Chemical disinfectants and antiseptics – Preservation of test organisms used for the determination
of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal (including bacteriophages)
activity

Test procedure accredited according to MS ISO/IEC 17025. The test report shall not be reproduced except in full without the written approval of
the laboratory. The test result relates only to the sample stated in the test report. The above analysis is based solely on the sample submitted by
the customer.