EN 13624:2013 EN 1275:2005 EN 1650:2008+A1:2013 EN 1657:2016

Scope Medical area (F1: hygienic handrub and handwash, F2: Food, industrial, domestic, and Food, industrial, domestic, Veterinary area (F1:
surgical handrub and handwash, F3: instrument institutional area and institutional area fungicidal activity, F2:
disinfection, and F4: surface disinfection) Medical area yeasticidal activity, and F3:
Veterinary area yeasticidal activity for teat
disinfectants
Test organism F1 & F2: C. albicans Fungicidal C. albicans Fungicidal C. albicans F1: C. albicans, A. niger
F3 & F4: C. albicans (fungicidal), A. niger (yeasticidal) A. niger A. niger F2 & F3: C. albicans
Yeasticidal C. albicans Yeasticidal C. albicans
Test temperature F1 & F2: 20 °C 20 °C 20 °C F1 & F2: 10 °C ± 1 °C
F3: 20 °C - 70 °C F3: 30 °C ± 1 °C
F4: 4 °C - 30 °C
Contact time F1: 30 s – 60 s 5 minutes 15 minutes 30 minutes 5 minutes
F2: 1 min – 5 min
F3: 60 min
F4: 5 min or 60 min
Interfering Clean 0.3 g/L BSA 0.3 g/L BSA F1 & F2: 3.0 g/L BSA
substance F3: 10 g/L skimmed milk
(IS) Dirty 3.0 g/L BSA + 3.0 g/L erythrocytes 3.0 g/L BSA F1 & F2: 10.0 g/L BSA + 10.0
g/L yeast extract
F3: 10 g/L skimmed milk
Test C. 1. Take 10 mL of diluent 1. Take 10 mL of diluent
suspensio albicans 2. Transfer loopfuls of working culture 2. Transfer loopfuls of working culture
n (N) 3. Shake using shaker for 3 min 3. Shake using shaker for 3 min
4. Aspirate and transfer 4. Aspirate and transfer
5. Adjust cfu to 1.5 – 5.0 x 107 (modified method: 1.5 – 5.0 5. Adjust cfu to 1.5 – 5.0 x 107
x 108) 6. Maintain N at 20°C and use within 2 h
6. Maintain N at 20°C and use within 2 h 7. For counting prepare 10-5 and 10-6, pour plate: ½ of each 1 mL into separate petri dishes
7. For counting prepare 10-5 and 10-6 (modified method: (MEA), spread plate: ¼ of each 1 mL on appropriate number of MEA
10-6 and 10-7), pour plate: ½ of each 1 mL into separate
petri dishes (MEA), spread plate: ¼ of each 1 mL on
appropriate number of MEA
A. niger 1. Suspend conidiospores from working culture in 10 mL 1. Suspend conidiospores from working culture in 10 mL 0.05% polysorbate 80 in dH2O
0.05% polysorbate 80 in dH2O 2. Detach conidiospores using rod or spatula
2. Detach conidiospores using rod or spatula 3. Transfer suspension
3. Transfer suspension 4. Shake gently for 1 min with glass beads
4. Shake gently for 1 min with glass beads 5. Filter suspension through fritted filter
5. Filter suspension through fritted filter 6. Use within 4 h
5. Microscopic observation (x400) 5. Microscopic observation (x400)
6. Adjust cfu/mL to 1.5-5.0 x 107(modified method: 1.5 – 6. Adjust cfu/mL to 1.5-5.0 x 107
5.0 x 108) 7. For counting prepare 10-5 and 10-6
 7. Use within 4 h
8. For counting prepare 10-5 and 10-6 (modified method:
10-6 and 10-7)

Validation 1. Dilute N w/ diluent to 3.0 x 102 – 1.6 x 103 cfu/mL (1+3 1. Dilute N w/ diluent to 3.0 x 102 – 1.6 x 103 cfu/mL (1+3 of the 10-4 dilution)
suspension (NV and of the 10-4 dilution) 2. For counting: 10-1. Mix. Take 1.0 mL x2. Inoculate
NVB) 2. NVB: dilute N w/ diluent to 3.0 x 104 – 1.6 x 105 cfu/mL
(1+3 of the 10-2 dilution)
3. For counting: 10-1 (modified method: 10-2, NVB: 10-3). Mix.
Take 1.0 mL x2. Inoculate
Colonies count Moulds: <165
Yeasts: <330
Product test solutions -Concentration: 1.25x desired concentration (diluted to -Concentration: 1.25x desired concentration (diluted to 80%), prepared in hard H2O
80%), prepared in hard H2O - Solid products: dissolve 1.0 g ± 10 mg in H2O
-Ready-to-use: dilute to 97% in H2O - Liquid products: dilute in hard H2O
-Handwash: dilute to 62.5% in hard H2O - Prepared freshly and used within 2 h
- Solid products: dissolve 1.0 g ± 10 mg in hard H2O
- Liquid products: dilute in hard H2O
- Prepared freshly and used within 2 h
Procedure Dilution-neutralization Modified Dilution-neutralization
Test Na
1. Pipette 1.0 part IS 1. Pipette 0.2 part 1. Pipette 1.0 part H2O
2. Add 1.0 part TS concentrated IS (5x) 2. Add 1.0 part TS
3. Start stopwatch 2. Add 0.1 part 3. Start stopwatch
4. Mix concentrated TS (10x) 4. Mix
5. Place in H2O bath (θ, 2 3. Start stopwatch 5. Place in H2O bath (θ, 2 min ± 10 s)
min ± 10 s) 4. Mix 6. Add 8.0 parts product
6. Add 8.0 parts product 5. Place in H2O bath (θ, 2 7. Restart stopwatch
7. Restart stopwatch min ± 10 s) 8. Mix
8. Mix 6. Add 9.7 parts undiluted 9. Place in H2O bath (θ, t)
9. Place in H2O bath (θ, t) product 10. Before t ends, mix
10. Before t ends, mix 7. Restart stopwatch 11. Take 1.0 part mixture Na
11. Take 1.0 part mixture 8. Mix 12. Transfer to tube contains 8.0 parts neutralizer + 1.0 part H2O
Na 9. Place in H2O bath (θ, t) 13. Mix
12. Transfer to tube 10. Mix before t ends 14. Place in H2O bath (20 °C ± 1 °C, 5 min ± 10 s)
contains 8.0 parts 11. Take 1.0 part mixture Na 15. Mix
neutralizer + 1.0 part H2O 12. Transfer to tube 16. Take 1.0 part mixture Na (x2)
13. Mix contains 8.0 parts 17. Inoculate- pour plate 1.0 part sample or spread plate 1.0 part divided into 2 plates
14. Place in H2O bath (20 neutralizer + 1.0 part H2O
°C ± 1 °C, 5 min ± 10 s) 13. Mix
15. Mix 14. Place in H2O bath (20
16. Take 1.0 part mixture °C ± 1 °C, 5 min ± 10 s)
Na (x2) 15. Mix
17. Inoculate- pour plate 16. Take 1.0 part mixture Na
1.0 part sample or spread (x2)
plate 1.0 part divided into 2 17. Inoculate- pour plate 1.0
plates part sample or spread plate
18. Transfer 0.5 part 1.0 part divided into 2
mixture to tube contains plates
4.5 parts neutralizer (10-1), 18. Transfer 0.5 part
mix and dilute to obtain 10-2 mixture to tube contains 4.5
19. Take 1.0 part each & parts neutralizer (10-1), mix
inoculate and dilute to obtain 10-2
19. Take 1.0 part each &
inoculate
Control A
1. Pipette 1.0 part IS 1.Pipette 0.2 part IS 1. Pipette 1.0 part H2O 1. Pipette 1.0 part IS
2. Add 1.0 part Nv 2. Add 0.1 part Nv (10x) 2. Add 1.0 part Nv 2. Add 1.0 part Nv
3. Start stopwatch 3. Start stopwatch 3. Start stopwatch 3. Start stopwatch
4. Mix 4. Mix 4. Mix 4. Mix
5. Place in H2O bath (θ, 2 5. Place in H2O bath (θ, 2 5. Place in H2O bath (θ, 2 min 5. Place in H2O bath (θ, 2 min ± 10 s)
min ± 10 s) min ± 10 s) ± 10 s) 6. Add 8.0 parts hard H2O (ready-to-use use water)
6. Add 8.0 parts hard H2O 6. Add 9.7 parts H2O 6. Add 8.0 parts H2O 7. Restart stopwatch
(ready-to-use use water) 7. Restart stopwatch 7. Restart stopwatch 8. Mix
7. Restart stopwatch 8. Mix 8. Mix 9. Place in H2O bath (θ, t)
8. Mix 9. Place in H2O bath (θ, t) 9. Place in H2O bath (θ, t) 10. Mix before t ends
9. Place in H2O bath (θ, t) 10. Mix before t ends 10. Mix before t ends 11. Take 1.0 part mixture A (x2)
10. Mix before t ends 11. Take 1.0 part mixture A 11. Take 1.0 part mixture A 12. Inoculate
11. Take 1.0 part mixture A (x2) (x2)
(x2) 12. Inoculate 12. Inoculate
12. Inoculate
Control B
1. Pipette 9.0 parts neutralizer 1. Pipette 8.0 parts neutralizer + 1.0 part H2O
2. Add 1.0 part NVB 2. Add 1.0 part Nv
3. Start stopwatch 3. Start stopwatch
4. Mix 4. Mix
5. Transfer 0.5 part mixture to tube contains 4.5 parts 5. Place in H2O bath (20 °C ± 1 °C, 5 min ± 10 s)
neutralizer (10-1), mix and dilute to obtain 10-2 of NVB 7. Mix before time ends
6. Place 10-2 NVB tube (mixture B) in H2O bath (20 °C ± 1 8. Take 1.0 part of mixture B (x2)
°C, 5 min ± 10 s) 9. Inoculate
7. Mix before time ends
8. Take 1.0 part of mixture B (x2)
9. Inoculate
Validation C
 1.Pipette 1.0 part IS
2. Add 1.0 part diluent
3. Start stopwatch
4. Add 8.0 parts product
(Chighest)
5. Mix
6. Place in H2O bath (θ, t)
7. Mix before t ends
8. Transfer 1.0 part mixture
to tube contains 8.0 parts
neutralizer
9. Restart stopwatch
10. Mix
11. Place in H2O bath (20
°C ± 1 °C, 5 min ± 10 s)
12. Add 1.0 part Nv
13. Start stopwatch
14. Mix
15. Place in H2O bath (20
°C ± 1 °C, 30 min ± 1 min)
16. Mix before t ends
17. Take 1.0 part mixture C
(x2)
18. Inoculate
Calculation
VC values = no. of cfu counted per 1 mL sample
c
N=
( n+0.1 n2 ) 10−6
No = number of cells/mL in test mixture at end of contact time
1.Pipette 0.2 part IS
2. Add 0.1 part diluent
3. Start stopwatch
4. Add 9.7 parts product
5. Mix
6. Place in H2O bath (θ, t)
7. Mix before t ends
8. Transfer 1.1 part mixture
to tube contains 8.8 parts
neutralizer
9. Restart stopwatch
10. Mix
11. Place in H2O bath (20
°C ± 1 °C, 5 min ± 10 s)
12. Add 0.1 part
concentrated Nv(10x)
13. Start stopwatch
14. Mix
15. Place in H2O bath (20
°C ± 1 °C, 30 min ± 1 min)
16. Mix before t ends
17. Take 1.0 part mixture C
(x2)
18. Inoculate
1.Pipette 1.0 part H2O
2. Add 1.0 part diluent
3. Start stopwatch
4. Add 8.0 parts product (Chighest)
5. Mix
6. Place in H2O bath (θ, t)
7. Mix before t ends
8. Transfer 1.0 part mixture to tube contains 8.0 parts neutralizer
9. Restart stopwatch
10. Mix
11. Place in H2O bath (20 °C ± 1 °C, 5 min ± 10 s)
12. Add 1.0 part Nv
13. Start stopwatch
14. Mix
15. Place in H2O bath (20 °C ± 1 °C, 30 min ± 1 min)
16. Mix before t ends
17. Take 1.0 part mixture C (x2)
18. Inoculate
Determination of VC values
Calculation of N and No
Calculation of Na
Na = number of survivors per mL in test mixture at end of contact time and before neutralization

Mean for each dilution step Na0, Na-1, Na-2 = 10 c / n

Calculation of NV, NVB, NV0 Calculation of NV, NV0

Nv = number of cells/mL in validation suspension Nv = number of cells/mL in validation suspension

NV0 = number of cells/mL in mixtures A, B, and C at NV0 = number of cells/mL in mixtures A, B, and C at beginning of contact time (t0)
beginning of contact time (t0)
Formula: NV = 10 c / n
Formula: NV = 10 c / n
NV0 = c / n
NVB = 1000 c / n

NV0 = c / n

Calculation of A, B, and C
A, B, and C = numbers of survivors in experimental conditions control A, neutralizer control B or filtration control & method validation C at end of contact time
t (A) or defined times 5 min (B), and 30 min (C)

Formula: A, B, C = c / n
Reduction
Reduction, R = N0 / Na

lg R = lg N0 – lg Na